Phytochemical Analysis, In Vitro Antioxidant and Wound Healing Activities of Turbinaria ornata (Turner) J. Agardh from Gulf of Mannar, India

Turbinaria ornata, tropical brown algae found in the South pacic and Indian Ocean ecosystems. In accordance with recent studies, Turbinaria ornata J. has potent anti-inammatory effects. Therefore, this study is aimed to explore the biological activities of ethanolic extract of Turbinaria ornata J by analyzing the presence of phytochemical components, antioxidant property, antimicrobial activity and the wound healing activity. From the results, phytochemical analysis of ethanolic extract of T. ornata showed the presence of alkaloids, saponins, oils, total phenolic and total avanoid content were estimated to be 0.683 Abs and 0.433 Abs respectively. Anti-oxidant activity of the ethanolic extract of T. ornata extract showed remarkable DPPH radical scavenging activity of about 58.8% at 200μg/ml and total anti-oxidant activity of 0.257 absorbance at 100μg/ml concentration, as compared to that of their respective controls. The ethanolic extract of T. ornata exhibited the maximum zone of inhibition against the clinical pathogens like Pseudomonas aeuruginosa, Escherichia coli, Staphylococcus aureus, Candida albicans and Methicillin-resistant Staphylococcus aureus with their potent anti-microbial activity. Wound healing effects of the ethanolic extract of T. ornata was analysed by using Zebra sh model. The results showed the rapidand signicant regeneration of the wounded caudal n on day 14. Therefore, the preliminary results of this study strongly supports the ethanolic extract of T. ornata may be effective in wound healing and regeneration of the wounded tissues.


Introduction
Wound healing is the disruption of the normal biological organs and tissues. It regenerate healing in a dynamic, regulated and molecular mechanisms of cells to the wounded areas [1]. Emerging trends in healthy economy amongst the innovations with in the eld of wound healing is growing expeditiously [2].
Many researchers widely use natural products for the design of therapeutic drugs and its shows potential wound healing compounds [3]. Zebra sh exhibits the same wound repair property as that of mammalian wound repairing steps [4]. Seaweeds play an emerging trend in the eld of wound healing property against Zebra sh. Earlier studies by researchers have showed that most of the biological therapeutics such as immunomodulatory, stress tolerance, wound healing [5] anti-in ammatory, antiviral, antitumor, antithrombotic, anticoagulant and antioxidant bioactivities have been reported in seaweeds [6].
Turbinaria ornata (Turner) J. Agardh, is a marine green algae belongs to the family phaeophyceae which is rich in fucoids and sulphated polysaccharides and are commonly distributed on southeast coast of Tamilnadu, India, tropical and subtropical areas of Central and Western Paci c and also in Indian Ocean ( Fig. 1a., 1b.). The algae is commonly used in animal food, in fertilizers and also used as food ingredients [7].
T. ornata possess wide range of biological properties such as anti-coagulant, antioxidant [8], antiin ammatory [9], antibacterial [10], and wound healing [11] properties have been reported. The present study focus on the invitro antioxidant and wound healing effects of T. ornata ethanol extract against zebra sh model.

Total Phenolic Content
The ethanolic extract of Turbinaria ornata was soaked in different solvents such as hexane, chloroform, ethyl acetate, methanol and water were kept in the orbital shaker for 24 hrs. The residues were then ltered and the ltrate was evaporated. The different extracts of plant material were then centrifuged at 10,000 rpm for 15 min at 4°C. Twenty µL of extracts was prepared using the supernatant and made up to 3 mL of distilled water. Then, 0.5 mL of Folin-Ciocalteu's phenol reagent was added to all the tubes. The tubes were then placed in the incubator for 3 min at 45°C. After 3 min, 2 mL of 20% Na 2 CO 3 was added to all the tubes and kept for incubation after which, its absorbance was measured at 650 nm [16].

Total Flavonoids
Flavonoid contents were determined by slightly modi ed spectrophotometry method of Karadenizet al. (2005). One gram Ethanolic extract of Turbinaria ornata was weighed and ground with 200 mL of 80 % aqueous methanol in a mortar and pestle. The ground sample was ltered and a clear ltrate was obtained. The aliquot of the sample (0.5 mL) was taken in a test tube add 3 mL of distilled water and 0.3 mL of 5% sodium nitrite were added. The solution was vortexed and allowed to stand at room temperature for 5 min and 0.6 mL of 10% aluminium chloride was added to the solution. After 6 min, 2 mL of 1 M sodium hydroxide was added to the test tube. The solution was made up to 10 mL with distilled water. The absorbance was read at 510 nm [17].

Total Proteins
Add an equal volume of 1 M NaOH to 100µg sample and vortex. Add NaOH to standards as well if this option is used. Add 5 ml dye reagent and measure the absorbance at 595 nm [18].

Total Lipids
The ethanolic extract of Turbianria ornata (2 gm) was placed in a porous thimble of a Soxhlet extractor with cotton plug at its mouthed and thimble was placed in an extraction chamber which was suspended to previously weighed ask containing methanol, methanol-chloroform or petroleum ether. The whole assembly was adjusted and ask was heated using heating mental for 2 hrs to extract crude lipid. After the extraction, thimble was removed from the Soxhlet apparatus and the solvent was removed under reduced pressure to afford crude lipid. Furthermore, the ask containing lipid was placed in oven at 100°C for 30 minutes to remove residual solvent, cooled in a desiccator and weighed. The amount of crude lipid was calculated and expressed as percentage crude lipid content (AOAC. 1990) [19].

Total Carbohydrates
To 1 mL of the sample, 1 ml of phenol and 5 ml of concentrated Sulphuric acid was added and the mixture was mixed thoroughly. The solution is allowed to stand for 15 min in a boiling water bath and the OD for the solution was read at 490 nm. The amount of total carbohydrates was calculated using standard graph prepared by D-glucose and the values are expressed as µg/Ml [19].
Antioxidant Activity

DPPH Assay
The percentage of antioxidant activity of each substance was assessed by DPPH free radical assay. The measurement of the DPPH radical scavenging activity was performed according to methodology

Total Antioxidant Assay
Extracts in different concentration ranging from 10 to 100 µg/mL were added to each test tube individually containing 1 ml of distilled water and 1 ml of Molybdate reagent solution, 1mL of Sodium phosphate and 1 mL of Sulphuric acid were added separately. These tubes were kept incubated at 95°C for 90 min. After incubation, these tubes were normalized to room temperature for 20-30 min and the absorbance of the reaction mixture was measured at 695 nm. The values were recorded [21].

Antimicrobial Activity
Antimicrobial assay of different samples was performed by agar well diffusion method in Mueller Hinton Agar (MHA) plates. The test organisms were inoculated in Nutrient broth and incubated overnight at 37°C to adjust the turbidity to 0.5 McFarland standards giving a nal inoculum of 1.5 × 108 CFU/ml. MHA plates was cultured with standardized microbial culture broth. Each well was lled with varying concentrations from 100, 125, 150 µg/ml of the samples with positive control as streptomycin 25 mcg and negative/solvent control as DMSO, respectively. The plate was allowed to diffuse for about 30 minutes at room temperature and incubated for 18-24 hours at 37°C. After incubation, plates were observed for the formation of a clear zone around the well which corresponds to the antimicrobial activity of the tested samples. The zone of inhibition (ZOI) was observed and measured in mm [22].
Wound Healing Activity 6-12 month old wild-type sh of the TL/Ek strain was used for adult wounding experiments. Puncture wounds in embryonic median ns were manually introduced with a sterilized blade with a diameter of approximately 2 mm (Richardson et al., 2013).The treated and control shes were weighed, measured before the puncture. The ns were carefully collected and stored in 10% formalin for further study. The shes were fed with the feed combined with extracts for the regeneration study. They were watched carefully and after 14 days of treatment, the grown ns were again measured and collected for histochemical parameters [23].

Results And Discussion
Phytochemical Screening The phytochemical screening of Turbinaria ornata revealed the presence of Alkaloids, Saponins and xed oils. Also revealed the absence of carbohydrates, glycosides, proteins, aminoacids, phenols and terpenoids (Table 1). Three types of extracts such as hexane, acetone and methanol was subjected to T. ornata and the results showed that the presence of alkaloids, terpenoids, avonoids, polyphenols and quinones in hexane extract whereas alkaloids, terpenoids and avonoids were found to be absent in both acetone and methanol extracts [24]. The ethanolic extract of Turbinaria ornata contains the highest amount of phenols and avanoids of 0.684nm and 0.434nm, whereas the amount of carbohydrates and proteins were found to be 0.274nm and 0.242nm respectively and total lipids was found to be in meager amount as 0.01mg/g. Earlier work have supported our data that among the two extracts used for the analysis, aqueous extract contained the highest amount of phenol and avanoid compounds of about 1.187 and 1.020. The difference in the results obtained might possibly be due to the different method of extraction and solvents polarities [25]. The DPPH radical scavenging activity of the ethanol extract of Turbinaria ornata showed dose-dependent with maximum percentage of inhibition of 58.80 µg/mL at 200 µg/mL concentration and minimum percentage of inhibition of 17.30% at 100 µg/mL concentration (Fig. 2) when compared with that of standard quercetin. The half maximal inhibitory concentration (IC 50 ) of the concentration is 175.98μg/ml.
Studies have reported that acetone extract showed signi cant scavenging ability on DPPH (65%) at a concentration of 1000 μg/mL when compared with that of a standard BHT (97%). However, none of the extracts exhibited higher activity than BHT at the same concentration [7].

Total Antioxidant Activity
The total antioxidant activity in the ethanol extract of Turbinaria ornata is presented in Fig. 3 showed that maximum absorbance of 0.257 at 100μg/ml and minimum of 0.028 at 10μg/ml concentration when compared with that of standard ascorbic acid. The reducing capacity of various concentrations of ethanol extract at different concentrations along with the standard (Ascorbic acid) showed signi cant phosphomolybnedic activity presented in Fig. 3. The reducing capacity of the extract was found to be increased with the concentration of the sample. Work on CSP of T. ornata has the total antioxidant activity of 22.21 0.88 mg of ascorbic acid equivalents per g of the sample [26].

Antimicrobial Activity
The antimicrobial activity of ethanolic extract of Turbinaria ornata showed maximum zone of inhibition against Pseudomonas aeruginosa and Methicillin Resistant Staphylococcus aureus (MRSA) strains used and moderate inhibition of organism against Staphylococcus aureus. The least level of zone inhibition was observed in the organisms Escherichia coli and Candida albicans ( Table 5, Fig. 4). According to the earlier reports the inhibition zone of 19mm, 23mm and 24mm was observed for hexane, acetonic and methanolic extracts, respectively. The positive control (amoxicillin) produced 21-24mm zone of inhibition.
The negative controls (hexane, acetone and methanol) did not show any inhibition for marine algae [24]. The n growth measurement was observed on days 3, 7, 10 and 14 were compared with that of control. The shes were grouped in two groups, with each group consisting of 3 shes. First group was treated with ethanolic extract of T. ornata and the second group was treated as control. First group showed better regeneration of n compared to those of control shes (Fig. 5). The shes were treated with 500 µg of the ethanol extract of T. ornata showed maximum n regeneration on day 14 when compared to the control shes. Tissue regeneration was observed by histochemical analysis where, the control shes recruited less number of neutrophils compared to the ones treated with the ethanol extract (Fig. 6). Earlier work reveals that the seaweeds of Turbinaria ornata, Gracillaria crassa and Laurencia papillosa, collected from the Tuticorin coast of the Southeast coast of India and selected based on preliminary screening, were extracted with acetone and evaluated for antiulcer, wound healing and hepatoprotective activities among the seaweeds studied Laurencia papillosa showed the greatest activity [11].

Conclusion
There is a constant endeavor amongst scientist to acquire new therapeutic agents from the marine algae. The present work is devoted to the determination of the yield, chemical composition, antioxidant, radical scavenging properties and antibacterial effect of ethanolic extract of Turbinaria ornata.
Phytochemical screening revealed the richness in alkaloids, saponins and xed oils. In addition, the quantitative analysis revealed signi cant levels of phenols and avanoids are presented and the subsequent minimal levels of carbohydrates, proteins and lipids are revealed.
With regards to the results the total antioxidant activity revealed the tremendously increased antioxidant activity to the concentration of the sample on the one hand and on the another hand it shows the highest free radical scavenging abilities on DPPH which is very powerful, close to the positive control respectively.
Antimicrobial tests show the highest inhibition against the Pseudomonas aureus and Methicillin-resistant Staphylococcus aureus. Whereas, it shows strongest activity against the antibiotic-resistant bacterial strain. So we further proceed for the drug development, and furthermost, it reveals the immense response on the wound healing and also better regeneration of the wounded tissues on zebra sh.      Histochemical parameters of regenerative tissues of Zebra sh