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188 Advances in Environmental Biology, 5(1): 188-193, 2011 ISSN 1995-0756 This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE Chemical Composition, Brine Shrimp Toxicity and Free-radical Scavenging Activity of Leaf Essential Oil of Acalypha Ornata (Hochst) Patricia A. Onocha, Ganiyat K. Oloyede and Gbenga S. Olasunkanmi 1 Natural products/Medicinal Chemistry Unit, Department of Chemistry, University of Ibadan, Nigeria. Patricia A. Onocha, Ganiyat K. Oloyede and Gbenga S. Olasunkanmi: Chemical Composition, Brine Shrimp Toxicity and Free-radical Scavenging Activity of Leaf Essential Oil of Acalypha Ornata (Hochst) ABSTRACT Essential oil from the leaves of Nigerian specie of Acalypha ornata of the family Euphorbiaceae was obtained by distillation using a hydro-distiller (all-glass clavenger apparatus) and was found to contain 100 compounds out of which 89 was successfully identified representing 54.64% of the total oil composition when analysed by GC/GC-MS. Alcohols, aldehydes, esters and terpenoids are the dominating group of compounds. The principal constituents are isopulegly acetate, valenchi, vividiflorene, α –muurolene, 2-hexyne, 6-methyl – α – ionone, γ-elemene, (E) -2-methyl-4- undecene, ledol, cis-3hexynyl benzoate, 2-methyl -1- octadecene, apiole, oplopanone and γ-endesmol. Brine shrimp lethality test was carried out to know the toxicity of the oils to living organisms (shrimps). LC50 value (µg/ml) of 116.6 at 95% confidence level obtained showed that the essential oil of A. ornata leaves was toxic. The antioxidant property of essential oil was investigated by using the UV/Visible spectrophotometer. The essential oil of A. ornata scavenged 2, 2–diphenyl–1–picrylhydrazyl radical (DPPH) less effectively than ascorbic acid and butylatedhydroxylanisole (BHA) but better than α-tocopherol at 517 nm. A. ornata oil has weak ability to donate hydrogen. The absorption is however concetration dependent. Key words: Free-radical, toxicity, 2, 2–diphenyl–1–picrylhydrazyl radical, ascorbic acid, butylatedhydroxylanisole, α-tocopherol, Gas Chromatography/ Mass Spectroscopy. Introduction Naturally occurring antioxidants are substances from plants or animals sources that can delay or inhibit oxidation processes of free radicals. Antioxidants like Vitamin C, Vitamin E, carotenes, phenolic acids, polyphenols and fl a v onoids scavenge free radica l s l i k e hydroperoxide, peroxide, hydroxyl or lipid peroxyl and thereby inhibit the oxidative mechanisms that lead to degenerative diseases [3,4,27,20,23]. Search for new antioxidant agents especially from plants is therefore desirable. Essential oil is any concentrated hydrophobic containing volatile aroma compounds from plants. Essential oils volatize on exposure to air at normal temperature and find application in food, confectioneries, perfumery industries and classical medicine. Myrrh (Commiphora myrrha) containing 62% sesquiterpenes is used by Arabians for skin conditions such as wrinkled, chapped and cracked skin. Eugenol has antibacterial action; thymol is used in mouth wash for its antiseptic principle, chanlmoogra oil has curative action on leprosy amongst others [8,37,38,6]. Acalypha ornata Hochst is a shrub which is widespread across tropical Africa. It is claimed to have several medicinal uses even though not much is known about its chemistry and pharmacology [5,34,3317]. Several species of the family, A. indica, A. fruticosa, A. guatemalensis, A. wilkesiana, A. siamensis, A. communis, A. marginata have however been widely investigated. They are found to contain condensed and Corresponding Author: Patricia A. Onocha, Natural products/Medicinal Chemistry Unit, Department of Chemistry, University of Ibadan, Nigeria. E-mail: [email protected] Telephone: +234 703 6015339 189 Adv. Environ. Biol., 5(1): 188-193, 2011 hydrolysable tannins as well flavonoid glycosides [12,7, 2,31,18]. Antimicrobial activities and anticancer potential of some Acalypha species have also been reported [30,11]. The aim of this research work is to determine the volatile oil constituent of the leaves of Acalypha ornata which will be obtained by h y d r o d i s t i llation a nd a na lyse d b y G a s Chromatography/Gas Chromatography–Mass Spectrometry (GC/GC-MS), to carry out in-vitro antioxidant assays by determining the free radical scavenging effect on 2,2-diphenyl-1-picrylhydrazyl radical (DPPH). DPPH radical gives strong absorption at 517 nm (deep violet colour) in visible spectroscopy. The absorption vanishes or is decolorized as the electron becomes paired off in the presence of a free radical scavenger. Studies have revealed that essential oils serve as powerful antioxidants that produce adverse environment for damaging free radicals thus preventing mutations and oxidants in cells [30,27]. A simple bench top bioassay for the determination of elementary toxicity; Brine shrimp lethality test (BST) was used to determine the toxicity of the oil[9,8]. Materials and methods Plant Materials: Fresh samples of the A. ornata leaves (300 g) were collected in August 2009 at the Botanical Gardens, University of Ibadan. Specimens were identified at the Botany and Microbiology department, University of Ibadan, Oyo State, Nigeria. The volatile oil was immediately collected from the fresh plant material by hydrodistillation. Reagents: Hexane and methanol (BDH chemicals), ascorbic acid, butylatedhydroxyanisole (BHA), αtocopherol and 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) were obtained from Sigma Chemical Co (Germany). accordance with the British pharmacopoeia specifications [8]. The essential oil was collected and stored at 4 0C until analysis. The oil yield was calculated relative to the dry matter. Analysis of the Essential Oils: Gas Chromatography: GC-MS analyses of the essential oil was carried out on an Agilent Technologies 7890A GC system coupled to a 5975C VLMSD mass spectrometer with an injector 7683B series device. An Agilent (9091)-413:325 0C HP-5 column (30 m x 320 µm x 0.25µm) was used with helium as carrier gas at a flow rate of 3.3245ml/min. The GC oven temperature was initially programmed at 50 0C (hold for 1min) and finally at 300 0C (hold for 5min) at a rate of 800C/min while the trial temperature was 37.25 0C. The column heater was set at 250 0C and was a split less mode while the pressure was 10.153 psi with an average velocity of 66.45 cm/sec and a hold-up time of 0.75245 min was recorded. Mass spectrometry was run in the electron impact mode (EI) at 70eV. The percentage compositions were obtained from electronic integration measurements using flame ionization detector (FID) set at 250 oC. The peak numbers and relative percentages of the characterized components are given in Table 1. Gas Chromatography–mass Spectrometry: The essential oil was analysed by GC-MS on an Agilent Technologies 7890A GC system coupled to a 5975C VLMSD mass spectrometer with an injector 7683B series device. An Agilent (9091)-413:325 0C HP-5 column (30 m x 320 µm x 0.25 µm) was used with helium as carrier gas at a flow rate of 3.3245 ml/min. GC oven temperature and conditions were as described above. The injector temperature was at 250 oC. Mass spectra were recorded at 70 eV. Mass range was from m/z 30 to 500. Identification of Components: UV-Visible Spectrophotometer (Unico1200 & Perkin Elmer lambda 25 models), GC-Mass spectrophotometer (Agilent Technologies), Hydro distiller - Clavenger apparatus. The individual constituents of the oil were identified on the basis of their retention indices determined with a reference to a homologous series of n-alkanes and by comparison of their mass spectral fragmentation patterns (NIST 08.L database/chemstation data system) with data previously reported in literature[1,14,19] Isolation of Essential Oils: Brine Shrimp Lethality Test: The oil was obtained by hydro distillation on a Clavenger type apparatus for 4 hours in The brine shrimp lethality test (BST) was used to predict the toxicity of the oils. The Major Equipments Used: Adv. Environ. Biol., 5(1): 188-193, 2011 shrimp’s eggs were hatched in sea water for 48h at room temperature. The nauplii (harvested shrimps) were attracted to one side of the vials with a light source. Solutions of the extracts were made in DMSO, at varying concentrations (10000, 1000 and 100 ppm) and incubated in triplicate vials with the brine shrimp larvae. Ten brine shrimp larvae were placed in each of the triplicate vials. Control brine shrimp larvae were placed in a mixture of sea water and DMSO only. After 24h the vials were examined against a lighted background and the average number of larvae that survived in each vial was determined. The concentration killing fifty percent of the larvae (LC50) was determined using the Finney computer programme [21,10,16]. Free Radical Scavenging Activity: Scavenging Effect on DPPH: A solution of 0.5 mM of 2, 2–diphenyl–1–picrylhydrazyl radical (DPPH) in methanol was prepared and 3ml of this solution was mixed with 1ml of the oil sample in methanol. The free radical scavenging activity of the oil at 10 and 20 µg/ml was determined in the visible region of a UV-Visible spectrophotometer. The decrease in absorption at 517nm of DPPH was measured after 10min of incubation. The actual decrease in absorption was measured against that of the control. The same experiment was carried out on ascorbic acid, butylatedhydroxylanisole (BHA) and α-tocopherol which are known antioxidant agents. All test and analysis were run in triplicates and the result obtained was averaged [15,16,20,24]. The activities were determined as a function of their %Inhibition which was calculated using the formula; % Inhibition A2 A1 100 A2 1 Where A1 is the absorbance sample and A2 is the absorbance of control. Results and discussion The yield of the 300 g hydrodistilled A. ornata leaves oil was 0.60% (w/w). The essential oil, colourless, with characteristic smell was analyzed by GC and GC/MS systems using a polar column, resulting in the identification of 89 constituents in the hydrodistilled sample, representing 54.64% of the total essential oil. The compounds present and their percentage composition are reported in Table 1. Majority of the compounds are less than 1-2% of the total composition except the following compounds isopulegyl acetate, valenche, viridiflorene, αmuurolene, 2-hexyne, 6-methyl-α-ionone, γ-elemene, 190 (E)-2-methyl-4-undecene, ledol, cis-3-hexenyl benzoate, 2-methyl-1-octadecene, apiole, oplopamone and γ-eudesmol. Monoterpenes common in the essential oil of plants are found present though in trace amount in the essential oil of A. ornata. The occurrence of different secondary metabolites suggests a wide range of biological application of the plant [35,36,29]. The result of brine shrimp lethality test as analyzed by Finney probit computer programme showed that the oil from the leaves of A.ornata was found to be toxic having an LC50 value of 111.6 mg/ml with lower and upper confidence limits of 26.38 μg/ml and 239.45 μg/ml respectively therefore its use at higher concentrations should be monitored. The high toxicity can also be beneficial in the therapy of some ailments involving cell or tumour growth. It has been found out that medicinally active natural products are most times toxic to Artemia silina nauplii [26]. The DPPH assay method is used to evaluate antioxidant potential of plant extracts, foods and pure compounds [16]. The free radical scavenging activity of the plant was evaluated by the decrease in absorption of the stable radical 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) at 517 nm. A ornata essential oil decolorized DPPH due to its hydrogen donating ability. The activity of the essential oil of leaves of A. ornata on the stable radical DPPH is concentration dependent and possessed weak scavenging activity as shown in Table 3. The free radical scavenging activity was compared with the activities of known antioxidants; ascorbic acid, butylated hydroxyanisole (BHA) and α –tocopherol. At 10 and 20 µg/ml, the oil gave a % inhibition of 20.5 and 14.8 respectively which were less than that of ascorbic acid and BHA but higher than α- tocopherol. Hence the oil of this plant has weak ability in scavenging free radicals. The absorption is stoichiometric with respect to the number of electron taken up. DPPH is known to be a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule [32]. A growing number of diseases are reported to be caused or influenced by free radicals. Free radicals are the toxic agents behind ageing and cellular damage [4]. The results obtained from this study are in agreement with other species of Acalypha species [22,28]. 191 Adv. Environ. Biol., 5(1): 188-193, 2011 Table 1: Chemical Composition of the volatile oil from A. ornata leaves by GC and GC/ MS analysis* Peak No Constituents RT (sec) % 1 2- hexenal 211.14 0.549 2 methyl tiglate 225.06 0.544 3 propyl butyrate 261.54 0.566 4 α – fenchene 335.10 0.553 menth -2-en-1-ol, (cis-para-) 559.08 0.761 6 3-octanyl acetate 690.42 0.532 7 p- cresyl acetate 797.64 0.430 8 4- Terpineol 820.92 0.468 9 Z-Ocimenone 960.66 0.433 10 Linalyl acetate 1024.38 0.509 11 Isopulegyl acetate 1066.44 1.275 12 Perilla alcohol 1129.02 0.704 13 δ-Elemene 1240.86 0.549 14 Benzyl butyrate 1257.00 0.999 15 Eugenol 1285.68 0.537 16 cis-Cariyl acetate 1297.26 0.655 17 Neryl acetate 1301.04 0.442 18 α – Ylangene 1321.2 0.712 19 α – copaene 1334.88 0.483 21 trans- methyl cinnamate 1340.28 0.636 23 Longifolene 1409.46 0.695 24 Caryophyllene 1444.5 0.478 25 α – Santalene 1445.7 0.608 26 Isobutyl salicylate 1452.3 0.874 27 Nopyl acetate 1456.26 0.438 31 β-Humulene 1494.54 0.532 32 trans-Cinnamyl acetate 1500.12 0.618 34 ethyl-Vanillin 1523.82 0.648 35 Geranyl acetate 1524.60 0.858 36 α –Humulene 1527.18 0.580 37 Cis-methyl isoenzenol 1530.6 0.691 38 (z), β-santalene 1547.52 0.439 39 Trans-ethyl cinnamate 1548.60 0.878 41 Furfuryl heptanoate 1560.48 0.997 42 γ – Gurjunene 1574.66 0.543 43 6-methyl-γ-Ionone 1590 0.558 45 β-selinene 1609.32 0.917 50 Valenche 1622.46 1.468 52 Viridiflorene 1628.22 2.658 53 Germacrene B 1632.84 0.608 54 α-Muurolene / 7-hexadecyne 1643.58 1.357 56 Cuparene 1650.18 0.605 57 2-hexyne 1679.88 1.770 58 6-methyl-α-Ionone 1686.24 0.747 61 Myristicin 1692.6 0.626 66 β-Sesquiphellandrene 1705.56 0.799 68 2-ethyl-1,1-dimethyl cyclopentane 1711.56 0.818 69 Trans-γ-bisabolene 1722.3 0.602 72 Thymo hydroquinine 1770.12 2.276 73 Elemicin 1772.34 0.790 74 γ-Elemene 1776.72 1.305 75 (E)-2-methyl-4-undecene 1780.68 1.506 77 Ledol 1798.02 1.610 78 cis-3-Hexenyl benzoate 1810.02 1.225 79 2-methyl-1-octadecene 1811.40 1.142 81 E,z-3, 13-octadecadien-1-ol, acetate 1820.16 1.325 86 cis-2-methyl-7-octadecene 1898.28 1.159 87 (Z)-2-methyl-4-undecene 1904.46 1.467 95 Apiole 2066.10 1.360 98 Oplopanone 2182.26 1.414 70 cis-Nerolidol 1728.90 1.074 89 γ-Eudesmol 1952.46 1.240 Total composition % 54.64% *RT = Retention Time, T.P. = Total Percentage, (-)= Not quoted DBS 0214 0228 0263 03375 0559 0690 0795 0820 09.57 1023 1067 1127 1236 1251 1279 1296 1303 1322 1334 1342 1404 1442 1447 1451 1455 1494 1500 1522 1525 1527 1532 1547 1548 1560 1575 1591 1808 1624 1628 1632 1643 1652 1688 1691 1702 1721 1770 1772 1777 1798 1809 2065 2183 1724 1951 192 Adv. Environ. Biol., 5(1): 188-193, 2011 Table 2: Brine shrimp lethality test of A. ornata leaves Essential Oils* Sample Concentration ----------------------------------------------------------------------------------------------------------------------10000ppm 1000ppm 100ppm Control Survivor 0 6 10 30 Dead 30 24 20 0 111.6 LC50(μg/ml) *Upper confidence limit 239.45 Lower confidence limit 26.38 Table 3: Scavenging Effect of Essential Oils of A. ornata leaves at Absorbance517nm* CONC. (μg/ml) OIL SAMPLE ASCORBIC ACID BHA 10 0.785±0.016 0.0843±0.010 0.0370±0.006 20 0.842±0.031 0.2893±0.128 0.0460±0.008 *Absorbance of essential oil of A. ornata leaves, ascorbic Acid, BHA and α- Tocopherol at 517nm. α- TOCOPHEROL 0.6800±0.029 0.7040±0.003 Table 4: %Inhibition of A. ornata Leaves Essential Oil and known Antioxidants on 2,2-dipheynl-1-picrylhydrazyl (DPPH)* CONC. (μg/ml) OIL SAMPLE ASCORBIC ACID BHA α- TOCOPHEROL 10 20.50 90.90 95.42 15.4 20 14.80 78.71 94.31 12.4 * % inhibition of essential oil, ascorbic acid, BHA and α- Tocopherol measured at 517nm. Absorbance of control = 0.988 ± 0.001 Conclusion: The essential oil composition of the leaves of A. ornata investigated revealed the presence of 89 compounds as determined by GC and GC/MS analysis constituting 54.64% of the total oil composition. Alcohols, aldehydes, esters and terpenoids are the dominating group of compounds. Brine shrimp lethality test was carried out to know the toxicity of the oils to living organisms (shrimps). LC50 value (µg/ml) of 111.6 obtained showed that the essential oil of leaves of A. ornata is toxic. 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