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Phytochemical profiling of Turbinaria ornata and its antioxidant and anti-proliferative effects

Objectives To analyse the phytochemicals and evaluate the antioxidant and anti-proliferative ability of Turbinaria ornata (Turner) J. Agardh, 1848. Methods A phytochemical analysis of the T. ornata-hexane extract (To-HE) and T. ornata-aqueous extract (To-AE) was performed. T. ornata extracts were analysed by gas chromatography-mass spectrometry (GC-MS), Fourier transform infrared spectroscopy (FTIR) and high-performance liquid chromatography (HPLC). The antioxidant properties of To-HE and To-AE were determined by 2,2-diphenyl-1-picrylhydrazyl radical scavenging (DPPH) and ferric ion reducing power (FRAP) assays. In addition, the in vitro anti-proliferative properties of To-HE and To-AE were assessed in kidney epithelial cells from the African green monkey (Vero) and in adenocarcinomic human alveolar basal epithelial cells (A549) using the MTT (3-(4,5-dimethylthiazol- 2yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole) assay. Results The phytochemical screening of T. ornata revealed the presence of saponin, alkaloids, amino acids, fixed oil and fat and phenolic compounds (tannins, flavonoids and total phenol). A higher antioxidant ability was found in To-HE than in To-AE. The anti-proliferative efficacy values (μg/mL) of To-HE and To-AE for A549 and Vero cells were 62.91 and 93.00 and 72.64 and 106.6, respectively. The FTIR analysis revealed the presence of functional groups such as alcohols, amides, aromatics, amines, alkyl halides, alkynes, alkanes and carboxylic acids. The GC-MS analysis of To-HE revealed the presence of 13 active compounds. Conclusion Owing to its recorded anti-proliferative effect, further pharmaceutical studies on the development of this anticancer drug are merited.


Abstract
Objectives: To analyse the phytochemicals and evaluate the antioxidant and anti-proliferative ability of Turbinaria ornata (Turner) J. Agardh, 1848.
Methods: A phytochemical analysis of the T. ornatahexane extract (To-HE) and T. ornata-aqueous extract (To-AE) was performed. T. ornata extracts were analysed by gas chromatography-mass spectrometry (GC-MS), Fourier transform infrared spectroscopy (FTIR) and high-performance liquid chromatography (HPLC). The antioxidant properties of To-HE and To-AE were determined by 2,2-diphenyl-1-picrylhydrazyl radical scavenging (DPPH) and ferric ion reducing power (FRAP) assays. In addition, the in vitro anti-proliferative properties of To-HE and To-AE were assessed in kidney epithelial cells from the African green monkey (Vero) and in adenocarcinomic human alveolar basal epithelial cells (A549) using the MTT (3-(4,5-dimethylthiazol-2yl)-2,5diphenyltetrazolium bromide, a yellow tetrazole) assay.
Results: The phytochemical screening of T. ornata revealed the presence of saponin, alkaloids, amino acids, fixed oil and fat and phenolic compounds (tannins, flavonoids and total phenol). A higher antioxidant ability was found in To-HE than in To-AE. The antiproliferative efficacy values (mg/mL) of To-HE and To-AE for A549 and Vero cells were 62.91 and 93.00 and 72.64 and 106.6, respectively. The FTIR analysis revealed

Introduction
Cancer is a group of diseases characterized by the uncontrolled growth of cells that can lead to death. Despite many therapeutic treatments available for cancer, the survival rate and disease curative percentage are very low. It has become an increasing public health problem that accounts for 6 million cases every year throughout the world. 1 Of the various types of cancer, lung cancer is the second most important cause of death worldwide that accounts for 75e80% of the deaths. 2 Current cancer chemotherapy can damage or kill the rapidly dividing and healthy cells, which causes serious side effects such as anaemia, hair loss, and nausea. In addition, the costs of chemotherapeutic drugs are high compared to those of the natural compounds derived from medicinal plants. 3 Therefore, the use of natural products could be an alternative method to control and eradicate cancer. 4 Natural sources such as plants, microorganisms and marine organisms are potential bioresources for anticancer compounds. 5 Earlier studies reported that the seaweeds Undaria pinnatifida, 6 Gracilaria edulis, 7 Turbinaria conoides, 8 and Sargassum pallidum 9 exhibit antiproliferative effects against human lung cancer cells (A549).
Turbinaria ornata (Turner) J. Agardh, 1848 is an extensive species of marine green alga belonging to the family Phaeophyceae and is rich in fucoids and sulphated polysaccharides. 10 It is widely distributed on the southeast coast of Tamil Nadu (India) and is reported to be used as an animal food, food ingredient and fertilizer. This alga is widely distributed in tropical and subtropical areas of the central and western Pacific and in the Indian Ocean. 11 A wide range of biological properties of this seaweed, including antibacterial, 12 anti-coagulant, 13 anti-inflammatory 14 and antioxidant properties, have been reported. 15 In addition, studies on fucoidan isolated from Turbinaria spp. have shown therapeutic benefits such as the prevention of myocardial injury, 16 along with hepatoprotective, 17 anticancer 9 and neuroprotective activities. 18 In view of these findings, the present study aims to evaluate the in vitro antioxidant and anti-proliferative effects of T. ornata-hexane extract (To-HE) and T. ornata-aqueous extract (To-AE) against A549 and Vero.

Collection of seaweed material and processing
Algal samples of T. ornata ( Figure 1) were collected from Mandapam, Ramanathapuram District, Tamil Nadu, southeastern India (9 22 0 N, 78 52 0 E). The samples were washed thoroughly with tap water and then distilled water to remove the associated biota and salt debris and then shade dried for 2e3 weeks. Finally, the seaweed was powdered using a kitchen electric blender. The seaweeds were identified based on standard keys 19

Preparation of To-HE
Approximately 10 g of powdered seaweed material was initially soaked in 50 mL of hexane for three days with mild shaking. Then, the extract was filtered through Whatman filter paper and concentrated in a Rotary evaporator. This extract was stored in refrigerator until use.

Preparation of To-AE
Aqueous extract was prepared by mixing 10 g of dry seaweed powder in 100 mL of sterile double distilled water and boiling at 60 C for 30 min. Finally, the extract was filtered with Whatman no. 1 filter paper and stored in a refrigerator until use.

Phytochemical screening
The samples (To-HE and To-AE) were subjected to preliminary phytochemical screening as described by Harborne (1973). 20 The FTIR analysis of To-HE and To-AE was recorded in the mid-IR region (4000e400 cm À1 ) at a resolution of 4 cm À1 using an FTIR spectrometer coupled with a TGS (Tri-glycine sulphate) detector. In brief, 1 mg of each dried sample was mixed with 100 mg of potassium bromide (KBr) and then compressed to prepare salt-discs (3 mm dia). These discs were analysed with a Fourier transform infrared spectrophotometer (Bruker, D8, Germany model). 8 High-performance liquid chromatography (HPLC) The HPLC analysis of To-HE and To-AE was performed following the methodology of Eahamban et al. Approximately 1 mg of sample was dissolved in 1 mL of methanol, and 20 mL was injected. Methanol:water (50:50) was used as the mobile phase. The experiment was performed in a Shimadzu LC solution 20 AD, Japan and SPD 20 A, an instrument equipped with a UV detector (254 nm) in order to determine the peak purity. An LCGC C18 column was used for isocratic resolution using the mobile phase at a flow rate of 1.0 mL/min. 21

Gas chromatography-mass spectrometry (GC-MS)
GC analysis of To-HE was performed by injecting 1 mL of sample on a 5MS column of a GC-MS instrument (Perkin Elmer, Massachusetts, USA) with helium as the mobile phase. The qualitative and quantitative analysis of To-HE was carried out using a CP 3800 Saturn 2200 Gas Chromatography-Mass Spectrometer. The temperature programme was 80e350 C at the rate of 3 C/min. The ion temperature was 200 C, and the scan range was 20e500 AMU (Atomic Mass Unit). The identification of components was based on comparison of their mass spectra with those from the Wiley library. 22 Antioxidant activity DPPH radical scavenging assay DPPH (1,1-diphenyl-2-picryl-hydrazyl) radical scavenging activity of To-HE and To-AE was measured according to the method of Adeosun et al. In brief, 2-mL (1 mg/ mL) samples and standards at varying concentrations (20, 40, 60, 80 and 100 mg/mL) were mixed with 1 mL of 0.1 mM DPPH in methanol. The mixture was instantly shaken and incubated for 30 min in the dark. The absorbance of the samples and control was measured at 517 nm. 23 Ferric ion reducing power (FRAP) assay FRAP assay of To-HE and To-AE was performed according to the method of Adedayo et al. The FRAP stock reagent was prepared with 300 mM acetate buffer (pH-3.6), 10 mM/L TPTZ (2,4,6-tri[2-pyridyl]-s-triazine) and 20 mM ferric chloride hexahydrate solution. The working solution was prepared with 30 mL of acetate buffer, 4 mL of TPTZ solution and 4 mL of ferric chloride hexahydrate solution. In brief, 500-mL (1 mg/mL) aliquots of varying concentrations (6.25e100 mg/mL) of samples and standard were prepared, and methanol was added to 2.5 mL of FRAP reagent and incubated in the dark for 30 min. The absorbance of the sample and control was measured at 593 nm. All the experiments were performed in triplicate. Ascorbic acid was used as a standard. 24

Statistical analysis
The in vitro antioxidant activities of the samples were calculated using the following formula.
where A 0 is absorbance of the control, A 1 is absorbance of the sample.
MTT (100 mL) was added in each well and incubated for 4 h and then dissolved in 100 mL of dimethyl sulphoxide (DMSO). Finally, reaction mixtures were recorded at 620 nm in a multi-well ELISA plate reader. Optical density (OD) was used to determine the percentage of cell viability by using the following formula. 25 where T is the OD of the sample, C is the OD of the control, T 0 is the OD at time zero. The activity of marine seaweed was expressed by the inhibitory concentration (IC 50 ) values that were calculated using Graph pad prism version 5.

Phytochemical screening
Flavonoids, saponins, alkaloids and fixed oils were found in both of the extracts (To-HE and To-AE) ( Table 1). Carbohydrates, glycosides, proteins and amino acids were absent. In contrast, tannin was found only in the To-HE extract.

Fourier transform infrared spectroscopy
The FTIR analysis of To-HE revealed twelve major peaks (Figure 2a) Table 2 and 3.

High performance liquid chromatography
The HPLC profile of To-HE and To-AE displayed a sharp intense peak at retention times 5.297 and 2.385,  respectively. In addition, To-AE displayed some minor peaks (Figure 3a and b).

Gas chromatography-mass spectrometry
The GC-MS analysis of To-HE indicated the presence of 13 compounds ( Figure 4 and Table 4). The major phytochemical constituents present in the To-HE were 1,2benzenedicarboxylic acid, butyl 2-methylpropyl ester (MW: 278, MF: C 16 Table 2.

Discussion
Naturally occurring marine algae are receiving much attention from researchers because they are rich sources of    biologically active secondary metabolites. Several studies have shown that seaweed is an excellent source for compounds such as polysaccharides, tannins, flavonoids, phenolic acids, bromophenols and carotenoids that have been studied for their various biological activities. 26 Several studies have been carried out using seaweed extracts that exhibited various biological activities such as antibacterial, antioxidant, anti-coagulant, anti-inflammatory, larvicidal, and anti-proliferative effects. 27 Hence, the focus of the present study was to evaluate the antioxidant and antiproliferative effects of hexane and aqueous extracts of T. ornata. The biological activity of any bioorganic compound may be influenced by its functional groups. Thus, the FTIR analysis of the extract reveals the physicochemical properties of a compound. The FTIR analysis of To-HE and To-AE indicated that the various functional groups such as alkanes, alkynes and alkyl halides are similar to those found in an earlier study of Meenakshi et al. (2011). 28 In both types of assays of antioxidant properties (DPPH and FRAP), the To-HE extracts displayed greater levels of inhibition than those of To-AE. These findings are comparable to those observed in a previous study by Chanthini et al. 29 The research of Vijayabaskar et al. revealed that the DPPH scavenging effect of T. ornata was 84.27% and is comparable to the values found in the present study. 13 The activity against cancer cell lines is one of the most important specificities of marine algae, and many algae have shown cytotoxic and anti-proliferative activities. 30 The results of this study are consistent with those reported by Marudhupandi et al. who found strong inhibitory potential of T. conoides-based fucoidan at the concentrations (mg/mL) of 45 and 325, respectively, on A549 and Vero cell lines. 9 The anti-proliferative effect of the ethyl acetate extract of G. edulis on A549 has been found to show the maximum inhibition at a concentration of 24.5 mg/mL. 8 In another study, the polysaccharides extracted from the seaweed Sargassum pallidum inhibited A549 cells by 64.8% at the concentration of 1 mg/mL. 10 To identify the phytocomponent responsible for the recorded inhibitory effect on free radicals and cancer cells, To-HE was analysed by GC-MS. Among the compounds identified, tetradecanoic acid was found to have both antioxidant and cancer-preventive properties. 31 The previous research on the GC-MS analysis of methanolic extracts of Cassia italic leaf and the endophytic fungi Alternaria sp. revealed the presence of some of these compounds such as 1,2benzenedicarboxylic acid, butyl 2-methylpropyl ester, 1,2benzenedicarboxylic acid, mono (2-ethylhexyl) ester and nhexadecanoic acid that had anticancer, cytotoxic and antiinflammatory effects such as those in our present study. 32,33 In this study, hentriacontane was a major component in the hexane extract of T. ornata that was previously isolated from Oldenlandia diffusa, a natural source used for the treatment of cancer in Asia. 34 Finally, the recorded anti-proliferative effect of To-HE could be due to the hentriacontane compound.

Study limitations
The GC-MS analysis of the To-HE-crude extract confirmed the presence of hentriacontane as a major compound, and it has previously been reported to be a cancer preventive agent. 34 Therefore, future study on the isolation and purification of hentriacontane from To-HE along with its structural confirmation is warranted.

Conclusion
The present study revealed that the hexane extract of T. ornata (To-HE) was more effective than the aqueous extract (To-AE) due to the presence of hentriacontane. In addition, To-HE has shown maximum inhibition effects during the antioxidant and anti-proliferative studies. Therefore, the findings of the present study could form a basis for the development of new pharmaceutical formulations for the treatment of lung cancer.

Recommendation
Based on the present research work, the use of food/diet supplement of T. ornata is recommended.

Conflicts of interest statement
The authors have no conflict of interest to declare.

Authors' contribution
PD designed, performed and wrote the article. RS performed the antioxidant studies. The interpretation of HPLC, FTIR and GC-MS studies was conducted by GB. PP supervised the work and performed the careful revision of this article, and all the authors have read and approved the final copy of this article.