Algal inhibitory efficiency of secondary metabolites of Tamarindusindica and Azadirachtaindica – A comparative pilot scale study

— This study includes isolation of oil producing algae (Anabaena, Nostoc. Spirulina, Diatom, Volvox, Spirogyra) and subjecting the mixture of algae to the sun dried pulp extract of Tamarindusindica to observe inhibitory effect of secondary metabolites on algal growth. The comparative analysis of inhibiting efficiency was done between the extracts of Tamarindusindica and Azadirachtaindica which proved that the secondary metabolites of Azadirachtaindica are more efficient than the secondary metabolites of Tamarindusindicain inhibiting the growth of algae. 100% and 75% concentration of the crude extracts were used to evaluate the inhibitory effect. Contamination from bacteria and fungi was prevented by maintaining the pH at 8.5. The extract of the sun dried pulp of Tamarindusindica showed inhibitory activity against the above mentioned species of algae.

INTRODUCTION Accelerated eutrophication has been one of the most widespread environmental problems (UNEP). Eutrophication is the enrichment of water by nutrient salts (Nitrate, Phosphate, Potassium) that causes structural changes to the ecosystem such as increased production of algae and aquatic plants, depletion of fish species, general deterioration of water quality and other effects that reduce and preclude use (OECD, 2005). All water bodies are subjected to slow eutrophication but in recent years eutrophication has been accelerated due to anthropogenic activities (Hu et al. 2008). Human development and associated increasing population growth in the watershed area underlie many of the environmental problems occurring in fresh water, transitional (e.g. estuaries and lagoons) and coastal ecosystems (OECD, 2005). Nutrient enrichment (N, P, and K) is one of the most prominent consequences directly related to human activities (Paerl 2006). The nutrient composition has been one of the main factors in excessive proliferation of algae in aquatic ecosystems (Paerl& Huisman 2009). Algal growth promoted by these salts can clog the gills of fish, in addition to anoxic water conditions and death of aquatic life forms (Najemet al. 2011). The risk to water quality deterioration is aggravated by the co-dominance of bloom-forming members of the green algae (Chia et al. 2016). Algae like Chlorophytes on their own are not considered being a nuisance, however, nutrient-enriched conditions favor the excessive proliferation of members of this group (Paerlet al. 2001). Many types of researches were carried out to control the algal growth by mechanical, physical and chemical methods in addition to bio manipulation (Tessonet al. 2014, Zhao et al. 2018). All these approaches were unsatisfactory and hence extracts of bioactive compounds from plants that inhibit or prevent algal growth have been in use (Ghorbanianet al. 2008). The secondary metabolites of these plants are known to contain antimicrobial properties (Wallace. 2004). Most of the phytochemicals from plant sources such as polyphenols and flavonoids have been reported to have a positive impact on health and cancer prevention (Venugopalet al. 2012). The excessive production of oxygen radicals during algal metabolism is known, especially when they are exposed to stress conditions (Zhang et al. 2013). The presence of bioactive secondary metabolites in plants induce high production of compounds like nitric oxide and H2O2, which have the potential to inhibit antioxidant enzyme activity (Clark et al. 2000;Qiaoet al. 2014). The mechanism of action of these phytochemical extracts may be via lysing the cell, increasing permeability of the cell wall and membrane, inhibition of protein and DNA synthesis and/or by inhibiting the transport of nutrient across the cell wall or membrane (Stewart et al. 1979). Tamarindusindica is a medicinal plant belonging to the family Fabaceae. It has been used as a medicinal plant for centuries; its fruits being the most valuable part. It contains majorly flavonoids, alkaloids and polyphenols (Arranzet al. 2010) and has exhibited an inhibitory effect against various organisms (Okohet al. 2017). Many studies have been done to prevent the growth of algae using the secondary metabolites of various medicinal plants in addition to various physical as well as chemical methods. However, these chemical methods have been causing harmful effects on the environment. This study explores the potential of Tamarindusindica extract to inhibit the proliferation of algae in an environmentally friendly way and compare its efficiency of inhibition with Azadirachtaindicaleaf extract.

II.
MATERIALS AND METHODS SAMPLE -Algae (lake water), Tamarindusindica(fruit), Azadirachtaindica(leaves). GLASSWARE REQUIRED -Petri plates, conical flasks, beakers, glass slides, pipettes. Instruments / apparatus -Compound microscope, autoclave, centrifuge, colorimeter, Soxhlet apparatus, cork borer, micropipettes. Chemicals used -Methylene blue, Safranin, 1N NaOH, Ethanol, 0.1 N HCl, acetone, Triple distilled water, conc.HCl, conc. H2SO4. Other Requirements -Forceps, needles, pH paper, centrifuge tubes, plastic trays, sample bottles, spatula, blotting paper, filter paper, cotton, muslin cloth. Sampling of Algae The water sample was collected from Kempabudi Lake, Chamarajpet around the month of August 2018. Isolation and growth of algae The collected sample was inoculated into Algae Culture Broth. After a significant amount of growth, the algae mixture was sub cultured on agar plates in the same Algae Culture media.

Diatom
Identification of algae The algal isolates that were obtained did not represent the whole algae in the collected samples in this study. Some algae need typical media with typical environmental factors to be grown which were different from those utilized in this study (Abedin and Taha, 2008).  Control of contaminants Growth of contaminating bacteria and fungi were prevented by maintaining the pH of the media at 8 -8.5.

Extraction of Tamarindusindicapulp
The fruit of the Tamarindusindica was collected from a nearby botanical garden and was sun dried. The dried pulp was administered into the Soxhlet apparatus and the extract was obtained using solvent extraction method by using ethanol.
The extract was subjected to drying to evaporate the ethanol. Evaluation of the inhibitory effect of the pulp extract Wells were cut into the algae cultured plate and the extract was administered into the wells. Inhibitory effects of Tamarindusindica pulp extract was tested against the mixture of algae depending on the diameters (mm) of inhibition zones through the agar-well diffusion method. Extraction of Azadirachtaindica leaves The leaves of the Azadirachtaindica were collected from a nearby botanical garden and were sun-dried. The dried leaves were administered into the Soxhlet apparatus and the extract was obtained using solvent extraction method by using ethanol.
The extract was subjected to drying to evaporate the ethanol. Evaluation of the inhibitory effect of the leaf extract Wells were cut into the algae cultured plate and the extract was administered into the wells. Inhibitory effects of Azadirachtaindica leaf extract was tested against the mixture of algae depending on the diameters (mm) of inhibition zones through the agar-well diffusion method.

III. RESULTS
The zone of inhibition was observed on day 2 of administration. The diameter of the zone of inhibition continued to increase until the fifth day. After the fifth day, there was no increase in the diameter. On reducing the concentration of the extract administered, the diameter decreased in direct proportion.
On comparison with Azadirachtaindica leaf extract, the diameter (in mm) observed was larger for Azadirachtaindica than Tamarindusindica.

IV. DISCUSSION
This study brought out that there is an initial lag phase in algal growth; the duration of the lag phase decreased on further sub-culturing. It was also noted that algae grow only when the substratum is not completely solid (less agar content) along with the presence of moisture. Tamarindusindica is known to exhibit antimicrobial activity as it contains flavonoids, alkaloids and polyphenols (Arranzet al. 2010). Alkaloids are shown to possess some level of allelopathy on plants (Macias et al. 2007).

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This study showed that Tamarindusindica extracts effectively inhibited the growth of algae. In the case of the 100% crude extract, it has been observed that the zone of inhibition was 11 mm in diameter) on day 2. The magnitude of the zone of inhibition increased on the fourth day (12 mm) and was found to remain constant after that. In the case of 75% of crude extract, the diameter of the inhibition zone was found to be 8 mm on day 2 and the magnitude increased successively up to day 5 (8.5 mm). The diameter of inhibition zones increased with the increased extract concentration. Research has shown that Azadirachtaindica has the ability to prevent algal growth (Chia et al. 2016).
In contrast with Azadirachtaindica leaf extract, the diameter (in mm) observed was larger in case of Azadirachtaindica.
The zone of inhibition with 100% of crude leaf extract of Azadirachtaindica was 12 mm on day 2, while in case of Tamarindusindica it was 11 mm. The zone of inhibition with 75% of crude leaf extract of Azadirachtaindica was 9 mm on day 2, while in case of Tamarindusindica it was 8 mm. The inhibition zone in case of both remained constant after day 5. Ethanol has been used as a control sample to eliminate experimental error (since the vaporization of ethanol hasn't been done in an ideal method). Ethanol has exhibited a smaller inhibition zone compared to Azadirachtaindica and Tamarindusindica. The diameter of the zone of inhibition with absolute ethanol was found to be 10 mm on day 2 and increased to 11 mm on day 5. The diameter of zone of inhibition with 75% ethanol was to be 7 mm on day 2 and 7.3 on day 5. The observed result suggests that fruit extract of Tamarindusindicacan be used as an alternative way to prevent algal bloom.
V. CONCLUSION Solvent extract of Tamarindusindica is a cheap and effective alternative for the prevention of excessive algal growth which is causing disruption in the aquatic ecosystem. Its inhibitory effect was observed on day 2 and increased upto day 5. On comparison, the leaf extract of Azadirachtaindica proved to be more effective than Tamarindusindica fruit extract. The comparison was done because both are excellent medicinal plants with effective secondary metabolites. Even without the isolation of specific secondary metabolite of the Tamarindusindicafruit, the solvent extract proved to be very efficient. More efficiency of inhibition of algal growth might be achieved by scrutinizing the secondary metabolites of Tamarindusindicaand administering them specifically. Further studies and investigation need to be done on the effects of the extract on other organisms.

ACKNOWLEDGEMENT
This project would not have been possible without the support of our parents, teachers and friends. We extend our heartfelt gratitude to the Research and Development Cell for providing us with this opportunity to do research at the undergraduate level. We are deeply and sincerely grateful to our research guide Prof. Mathews P Raj for his constant and immense support and guidance throughout our work. It is our honour to thank Dr. Asha Rajiv, Director, Jain University, School of Sciences for giving us the wonderful opportunity to conduct research and for her constant support and help. We would like to thank Prof. Anitha A Abraham, Head of Department of Life Sciences for her constant advice and facilitating with the equipment and infrastructural support we needed to complete our work. We extend our gratitude to all the faculty members of the Jain University School of Sciences for being supportive. We are very thankful to the non-teaching lab expertise for assisting and helping us in all the possible ways. Finally, we thank the administration and management of the Jain University for the financial and infrastructural support.