Chemical composition, antioxidant activity and cytotoxicity of the essential oils of the leaves and stem of Tarchonanthus camphoratus

1 Department of Chemistry, University of Zululand, Private Bag X1001, KwaDlangewa, 3886. South Africa. 2 Department of Bio-Chemistry and Microbiology, University of Zululand, Private Bag X1001, KwaDlangewa, 3886. South Africa. 3 Department of Agriculture, Cape Peninsula University of Technology, Private Bag X8, Wellington, 7654.South Africa. 4 Department of Chemistry and Chemical Technology, Walter Sisulu University, Private Bag X1, Mthatha, 5099. South Africa. 5 Department of Biochemistry, University of KwaZulu-Natal, Westville Campus, Durban, 4000. South Africa


INTRODUCTION
In living systems, free radicals are constantly generated and when in excess they can cause extensive damage to tissues and biomolecules leading to various disease conditions, especially degenerative diseases, and extensive lysis (Halliwell and Gutteridge, 1998).The interest in antioxidants has been increasing because of their high capacity in scavenging free radicals which are related to various diseases (Silva et al., 2007).The most commonly used synthetic antioxidants; butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propylgallate *Corresponding author.E-mail: snanyonga1@yahoo.com.
(PG) and test butylatedhydroquinone have been reported to cause liver damage and carcinogenesis (Sherwin et al., 1990).There is growing interest in natural antioxidants present in medicinal plants that might help attenuate oxidative damage (Silva et al., 2005;Muhammad et al., 2012).The health promoting effects of plants were found to be due to bioactive substances such as essential oils, flavonoids and phenolic compounds which have antioxidant activity (Liu, 2003;Komal et al., 2012).
Tarchonanathus campharatus L., (family Asteraceae) is a shrub of rarely more than six meters in height with a greyish appearance and occurs in a wide range of habitats (van Wyk et al., 1997).The strongly scented tree of T. campharatus has many medicinal applications in traditional healing mainly by smoking from burning leaves or by drinking infusions or decoctions.Traditionally, infusions and tinctures of the leaves are used for stomach trouble, headache, toothache, asthma, bronchitis, inflammation, rheumatism, venereal diseases, indigestion, heartburn, coughs, paralysis and cerebral haemorrhage (Hutchings et al., 1994;Anthony, 1999).The plant also shows powerful insect repellent action (Omolo et al., 2004;Essential oil newsletter, 2005).
In this study, the chemical composition, antioxidant potential and the cytotoxicity of the essential oils of the leaves and stem of T. camphoratus were investigated in order to find out their suitability as raw materials in food, pharmaceutical and industrial products.

Plant material
Fresh materials of T. camphoratus were collected from Sangoyana in the northern part of Kwa-Zulu Natal province, South Africa during the month of March, 2010.The plant was identified by the local people during the time of collection and further identified by Mrs N.R Ntuli in the Department of Botany, University of Zululand.A Voucher specimen, (NSKN 1), was deposited at the University of Zululand herbarium.The fresh plant material was separated into leaves and the other part with leaves still attached to the stem was dried at room temperature.

Extraction of the essential oil
The fresh leaves, dry leaves and the dry stem were subjected to hydro-distillation using a Clevenger-type apparatus.The essential oils were collected 4 h after boiling, weighed and kept at 4 o C in sealed glass vials before analysis and bioassay.

Gas chromatography-mass spectrometry analysis
The GC-MS analysis was carried out using an Agilent 6890 GC with an Agilent 5973 mass selective detector [MSD, operated in the EI mode (electron energy = 70 eV), scan range = 45 to 400 amu, and scan rate = 3.99 scans/sec], and an Agilent ChemStation data system.The GC was equipped with a fused silica capillary HP-5 MS column of an internal diameter of 0.25 mm, film thickness 0.25 µm and a length of 30 m.The initial temperature of the column was 70°C and was heated to 240°C at a rate of 5°C/min.Helium was used as the carrier gas at a flow rate of 1 ml/min.The split ratio was 1:25.Scan time was 50 min with a scanning range of 35 to 450 amu.A 1%, w/v, solution of the samples in hexane was prepared and 1 µL was injected using a splitless injection technique.

Identification of components
The identification of the oil constituents was based on their retention indices determined by reference to a homologous series of n-alkanes (C 8 -C 30 ), and by comparison of their mass spectral fragmentation patterns with those reported in the literature (Joulain and Koenig, 1998;Adams, 2007)

Nitric oxide radical inhibition assay
The reaction mixture containing 2 ml sodium nitroprusside (10 mM), 0.5 ml phosphate buffer saline (pH 7.4) and 0.5 ml of different concentrations of the essential oil (50 to 250, µg/ml) or standard solution (ascorbic acid, 0.5 ml) was incubated at 25ºC for 150 min.Then 1 ml of sulfanilic acid reagent (0.33% sulfanilic acid in 20% glacial acetic acid) was added to 0.5 ml of reaction mixture for 5 min to complete diazotization and 1 ml naphthyl ethylene diamine dihydrochloride was added and allowed to stand for 30 min at 25ºC.The absorbances of these solutions were measured at 540 nm (Badami et al., 2005).

Total reducing power
The reducing power was determined according to the method of Oyaizu (1986).Different concentrations, (25 to 250 µg/ml), of the essential oils in methanol (2.5 ml) were mixed with 2.5 ml of 0.2 M sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide.The mixture was incubated at 50°C for 20 min followed by addition of 2.5 ml of 10% trichloroacetic acid (TCA) and centrifuged at 1000 rpm for 10 min.2.5 ml of the mixture was mixed with 2.5 ml of deionised water and 0.5 ml of 0.1% ferric chloride and its absorbance measured at 700 nm against a blank.Ascorbic acid was used as the reference standard.To determine reducing power, 2.5 ml of the mixture was mixed with 2.5 ml of deionised water and 0.5 ml of 0.1% ferric chloride and its absorbance measured at 700 nm.Ascorbic acid was used as the reference standard.Higher absorbance of the reaction mixture indicates greater reducing power.

Brine shrimp cytotoxicity assay
The brine shrimps were hatched in sea water for 48 h at room temperature.The nauplii (harvested shrimps) were attracted to one side of the vessel with a light source.The essential oil were prepared at 1000, 500, 100 and 10 μg/ml (each test in triplicates) in 0.02% Tween 80.The essential oil (0.5 ml) was introduced in a testtube and sea water (4 ml) added.Ten shrimps per test tube were added for each concentration and made up to 5 ml with sea water.Potassium dichromate was used as positive control.The negative control was 0.02% Tween 80 (5 ml).The surviving larvae were counted after 24 h and the percent deaths at each dose and positive control were determined.

Cytotoxicity analysis by the MTT assay
The MTT assay was done using two cell lines, human embryonic kidney cells and human hepatocellular carcinoma cells.The cells were grown to confluenecy in 25 cm 3 flasks.This was then trypsinised and plated into 48 well plates at specific seeding densities.Cells were incubated overnight at 37°C.The medium was removed and fresh medium (MEM + Glutmax + antibiotics) was added.Extracts (50 to 100 µg) were added in triplicate and incubated for 4 h.The medium was again removed and replaced by a complete medium (MEM + Glutmax + antibiotics + 10% Fetal bovine serum).After 48 h the cells were subjected to MTT [3-(4,5dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide] assay.Briefly, the medium was removed from the cells and 200 µl of 5 mg/ml MTT in phosphate buffered saline (PBS) as well as 200 µl of medium were added to each well containing cells.The multiwell plate was incubated for 4 h and thereafter the medium and MTT were removed and 200 µl of DMSO was added to each well and incubated at 37°C for 10 min.Absorbance of the dissolved solutions were read using a Mindray Plate Reader at 570 nm.The cytotoxicity was calculated after comparing with the control.The control consisted of cells without the extract.

Statistical analysis
Results of antioxidant activity are presented as means ± SD of three measurements.Data were evaluated through regression analysis using QED statistics program and IC 50 values, where applicable, were determined by linear regression.Means between treatments were compared by Tukey's Studentized Range Test using one way ANOVA.

DPPH radical scavenging assay
The radical scavenging activity of the essential oils of T. camphoratus was determined from the reduction in absorbance at 517 nm due to scavenging of the stable DPPH radical.DPPH is a stable nitrogen-centred free radical the colour of which changes from violet to yellow upon reduction by either the process of hydrogen or electron donation.The oils showed a relatively weak dose dependent inhibition of DPPH activity, with high LC 50 of 12578.89,9942.08 and 7010.03μg/mL for fresh leaves, dry leaves and dry stem respectively (Table 2).The LC 50 values of the oils were not comparable to that of the standard BHT at p ≤ 0.05.

ABTS •+ radical scavenging assay
The ABTS radical cation is reactive towards most antioxidants and the decolorization of the ABTS •+ radical reflects the capacity of an antioxidant species to donate electrons or hydrogen atoms to inactivate this radical species (Re et al., 1999).The results for percent scavenging at different concentrations and LC 50 values for the oils are shown in Table 3.There was a significant difference in the means of the oils and that of BHT at p ≤ 0.05.The high LC 50 values of the oils suggest poor ABTS •+ radical scavenging activity.

Nitric oxide (NO) assay
In this assay, the ability of the essential oils to counteract the oxidation of nitric oxide with oxygen and reduce the production of nitrite ions which act as free radicals was investigated.
Table 4 shows the %inhibition of nitric oxide generation by the essential oils and of the standard ascorbic acid.The activity of the standard ascorbic acid was more pronounced with LC 50 value of 210.50 μg/ml when compared to LC 50 values of the essential oils at p ≤ 0.05.

Total reducing power
In the reducing power assay, the presence of antioxidants in the samples results in the reduction of Fe 3+ to Fe 2+ by donating an electron.The method evaluates the ability of plant extracts to reduce potassium ferricyanide solution which is monitored by measuring the formation of Perl's Prussian blue at 700 nm.Increasing absorbance at 700 nm indicates an increase in reductive ability.Figure 1 shows dose-response curves for the reducing powers of the essential oil.It was found that the reducing power

Cytotoxicity assay
The LC 50 values of the essential oil of the fresh leaves, dry leaves and dry stem were 889.0, 676.8, 442.9 µg/ml respectively and for the standard, potassium dichromate, 3.44 µg/ml (Table 5).There was a significant difference in the means of percent mortality of the essential oils and of the standard, potassium dichromate (p ≤ 0.05).There was no significant difference in the activity of the essential oils against the brine shrimps (p ≤ 0.05).The MTT assay is a well established method to assess mitochondrial competence (Freshney, 2000).Using this assay, we assessed the ability of the essential oils of the fresh leaf, dry leaf and dry stem to suppress mitochondrial respiration in human embryonic kidney cells and human hepatocellular carcinoma cells.The LC 50 values of the essential oils used in this study in both cell lines were above 100 µg/ml (Table 6).The results revealed that there was no significant difference, (p ≥ 0.05), in the action of the essential oils of the dry stem, fresh and dry leaves on each of the human cells used.

Antioxidant
In all the antioxidant assays carried out in this study, the essential oils of the dry leaves, fresh leaves and dry stem of T. camphoratus showed poor antioxidant activity.A good correlation between the phenolic content in plants and their antioxidant activity has been reported (Tawaha et al., 2007;Othman et al., 2007;Nadeem et al., 2012).Essential oils rich in monoterpene hydrocarbons have also been reported to have high antioxidant activity (Tepe et al., 2005).The poor antioxidant activity of these essential oils, probably, is due to their lack of phenolic contents and low concentrations of monoterpene hydrocarbons.However, the low values of antioxidant and reducing power may not imply low medicinal value.Emerging trends in antioxidant research point to the fact that low levels of phenolics and other phytochemicals plus low value of antioxidant indices in plants do not translate to poor medical properties (Makari et al., 2008;Nasir et al., 2011).

Cytotoxicity
One indicator of a toxicity of a substance is LC 50 which is the amount of a substance that kills 50% of the test organisms.All the essential oils investigated in this study, were found to have LC 50 values > 30 µg/ml.According to the American National Cancer Institute, the LC 50 limit to consider for a crude extract promising for further purification to isolate biologically active (toxic) compounds should be lower than 30 µg/ml (Suffness and Pezzuto, 1990).Other authors suggest that oils and extracts from plants presenting LC 50 values below 1000 µg/ml are known to contain physiologically active principles (Meyer et al., 1982).The essential oils investigated showed low + antioxidant activities but do have some physiologically active principles.
oil of the dry stem, fresh and dry leaves of T. camphoratus was much lower than that of the standards, ascorbic acid and BHT.

Figure 1 .
Figure 1.Total reducing power of the essential oils of the fresh leaves, dry leaves and dry stem of Tarchonanthus camphoratus.Ascorbic acid and BHT were used as the positive control.

Table 1 .
Chemical constituents of the essential oils of the fresh leaves, dry leaves and dry stem of T. camphoratus.

Table 2 .
DPPH radical scavenging assay of the essential oils from the fresh leaves, dry leaves and dry stem of Tarchonanthus camphoratus.

Table 3 .
ABTS •+ radical scavenging assay of the essential oils from the fresh leaves, dry leaves and dry stem of Tarchonanthus camphoratus.

Table 4 .
Nitric oxide scavenging activity of the essential oils from the fresh leaves, dry leaves and dry stem of Tarchonanthus camphoratus. d

Table 5 .
Inhibitory effects of the essential oils from the fresh leaves, dry leaves and dry stem of Tarchonanthus camphoratus on brine shrimps. d

Table 6 .
Cytotoxicity of the essential oils from the fresh leaves, dry leaves and dry stem of Tarchonanthus camphoratus on human embryonic kidney cells and hepatocellular carcinoma cells.