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An in Vitro Study on The Antimicrobial Activity and Antioxidant Activities of The Extract of A Seaweed, Enteromorpha Intestinalis Against Certain Pathogens


 Seaweeds are potential bio resources of marine ecosystem and they are the producers of marine ecological energy chain and also possess many bioactive compounds with them. The seaweed Enteromorpha intestinalis is the plant material chosen for the study and was collected from the Pulicat estuary. The collected seaweeds were processed to synthesise nanoparticles out of them and the synthesised silver nanoparticle’s in vitro antimicrobial and antioxidant activity was evaluated. The antibacterial activity was determined by the action against Salmonella typhi, Pseudomonas aeruginosa, Staphylococcus aureus, Vibrio parahaemolyticus and Micrococcus luteus. Similarly the in vitro antifungal effect was also explored against Candida albicans, Trichoderma viridae, Aspergillus niger, Rizhopus sp and Penicillium notatum. Among them, Staphylococcus aureus and Aspergillus niger were found to be highly susceptible to the synthesised nanoparticles. When the antioxidant efficacy of synthesized nanoparticle was tested using different methods such as DPPH and ABTS it was observed that they possessed appreciable antioxidant property whose efficiency was correlated with their respective positive standards. These investigations illustrated the potential effects of the seaweed E. intestinalis for using them as an active antimicrobial and antioxidant agent.


Introduction
In India, seaweed resources are exclusively utilized for the production of commercially and industrially important phycocolloids (Reddy et al., 2014).Seaweeds are a diverse and large group of macro algae which are broadly classi ed into various types based on the presence of photosynthetic pigments as rhodophyta (red algae), chlorophyta (green algae), and phaeophyta (brown algae) (Hemasudha et al., 2019). Seaweed extracts contains major and minor nutrients, amino acids, vitamins, cytokinins, auxin and abscisic acid like growth promoting substances (Mooney and Staden, 1986). Agar, carrageenan and alginate are popular examples of seaweeds these have been used as food for human beings, fertilizers for plants and source of various chemicals (Shelar et al., 2012).
Antioxidant compounds play an important role against various diseases (e.g., chronic in ammation, atherosclerosis, cancer and cardiovascular disorders) and ageing processes (Kohen and Nyska, 2002). . In this present study the seaweed E. Intestinalis was collected from the Pulicat estuary and the nanoparticles of the same were tested for various bioactivities to illustrate its potential utilizations.

Enteromorpha intestinalis
The marine seaweed Enteromorpha intestinalis was collected from the Pulicat estuary at the Thiruvallur district of Tamil Nadu and were processed for extraction using distilled water as solvent to obtain the aqueous extract by following standard methods (Rajeshkumar et al., 2016).
Synthesis of silver nanoparticles Silver nanoparticles were synthesized by adding standard amounts of silver nitrate solution to the prepared aqueous extract (Rajeshkumar et al., 2016). The reaction was characterized by the colour change and monitored by UV spectroscopy.

Characterization
The morphology of the synthesised silver nanoparticle was identi ed and con rmed using different characterisation techniques such as Fourier Transform Infrared spectroscopy (FTIR), UV-VIS Spectroscopy and Scanning Electron Microscopy (SEM). Antioxidant Activity DPPH (1, 1-diphenyl-2-picrylhydrazyl) assay DPPH (1, 1-diphenyl-2-picrylhydrazyl) assay was performed to evaluate the radical scavenging activity of the synthesised silver nanoparticles (Molyneux, 2004). BHT was taken as the standard for the evaluation. About 100 µl of respective samples were added to all tubes marked as tests except one which received BHT. 200 µl of DPPH reagent was added to all the test tubes including blank and all the test tubes were incubated at room temperature in dark condition for 30 min. The absorbance were read at 517 nm and the anti-oxidant activity was determined using the given formula ABTS assay The radical scavenging activity was determined as described by Re et al. (1999).The decolourisation assay involves the generation of the ABTS + chromophore by the oxidation of ABTS with ammonium persulphate. The scavenging activity of the plant extracts on ABTS radical action were measured at 734 nm. Seaweed samples were diluted to produce 20, 40, 60, 80, 100µg/ml.

Antifungal Activity Assay
The antifungal activity of the synthesised silver nanoparticles was determined by disc diffusion method on Sabouraud Dextrose agar (SDA) medium. The fungal pathogens like Candida albicans, Trichoderma viridae, Aspergillus niger, Rizhopus sp and Penicillium notatum were tested using amphotericin-B is taken as positive control. 20 µl of respective concentrations of test samples and positive control were added in SDA plates. The plates were incubated at 28ºC for 24 h. The antifungal activity was determined by measuring the diameter of zone of inhibition (Bauer et al., 1966).

Results
The seaweed Enteromoprha intestinalis (Fig. 1.) was processed and the silver nanoparticles were synthesized, which were identi ed and con rmed by following characterisation methods. The FTIR peak values indicated the presence of characteristic bioactive compounds (Fig. 2.). UV visible absorption spectroscopy through UV light was absorbed by the molecule and the range of peak value was 380 to 480 nm. The synthesized silver nanoparticle was monitored at the peak value of 420 nm (Fig. 3). The size of the nanoparticles synthesized from the collected seaweed varied from 57.87 and 82.09 nm in diameter (Fig. 3).
The silver nanoparticles synthesized from the seaweed Enteromorpha intestinalis was evaluated for its radical scavenging activity using DPPH assay and the maximum antioxidant activity was found was 40.86% at 100 µg/ml.
The results of the ABTS assay showed maximum activity of 23.04% at 100 µg/ml and minimum of 2.38% at 20 µg/ml concentrations. The overall e cacy of synthesized nanoparticles was found to approximately similar to the standard (Fig. 4).
The in vitro antibacterial activity of E. intestinalis seaweeds using silver nanoparticle showed maximum activity (Zone of inhibition -8.66 ± 1.15 mm) against Staphylococcus aureus at 1000 mg/ml (Fig. 7.) ( Table 1). On evaluation, in vitro antifungal activity of E. intestinalis silver nanoparticles showed highest inhibitory action on Aspergillus niger at (10 ± 1 mm of inhibitory zone) 1000 mg/ml (Fig. 8)

Discussion
Seaweed extracts contains major and minor nutrients, amino acids, vitamins, cytokinins, auxin and abscisic acid like growth promoting substances (Mooney and Staden, 1986). The secondary metabolites derived from marine macro-algae have been associated with a broad range of biological activities such as antibacterial, antiviral, antifungal, antifouling and anti-in ammatory effects as well as cytotoxic and antimitotic activities (Mayer et al., 2009). Several silver-based compounds have been utilized effectively as antimicrobial specialists (Nomiya et al., 2004). The compounds of silver are also used in the medical eld to treat burnt wounds and various other types of infections. Nanoparticles of silver have aptly been investigated for their antibacterial property because the silver nanoparticles have high speci c area than their volume, which will lead to excellent antimicrobial activity as compared with bulk silver metal In the present study was in vitro antibacterial activity of Enteromorpha intestinalis seaweeds using silver nanoparticle showed good maximum activity against Staphylococcus aureus species of pathogen at 1000 mg/ml due to the presence of phytochemicals like avonoids, tannins, phenols and proteins which may act as an antimicrobial agent (Priyadarshni and Mahalingam, 2017)  . Seaweeds possess various and abundant secondary metabolites which has good radical scavenging activity and therefore could be utilised as antioxidant source (Diplock, 1997). The present study explored the antioxidant e cacy of synthesized nanoparticle from Enteromorpha intestinalis using DPPH and ABTS methods. Silver nanoparticle of Enteromorpha intestinalis showed antibacterial, antifungal and antioxidant activity, thereby could be effectively used as a therapeutic agent.

Declarations AUTHOR'S CONTRIBUTIONS
This work was carried out in collaboration among all authors. Author JG and LJ carried out the assays and experiments of the study. Author JJ designed the experimental setup for this study. Author MGR supervised the whole research work and corrected the manuscript draft. All authors read and approved the nal manuscript.

DECLARATION OF COMPETING INTERESTS
Authors have declared that no competing interests exist.

FUNDING SOURCE
Not applicable    Antibacterial activity by disc diffusion method Antifungal activity by disc diffusion method