INTRODUCTION:

Nannochloropsis oculata is a genus of alga comprising known species. The genus in the current taxonomic classification was first termed by^[1]. The necessary leap in productivity could be reached by integrating both technological and genetic improvements to the current oleaginous strains. Nannochloropsis (also called marine chlorella) is a small green microalgae genus, which is well known in aquaculture due to its nutritional value and potency to produce valuable materials. They have been extensively used in the aquaculture industry for growing small zooplanktons such as rotifers and fish hatcheries and for producing green water^[2].

The algae of the genus Nannochloropsis differ from other related microalgae in that they have Chlorophyll a and completely lack Chlorophyll B and Chlorophyll c. Microalgae, which comprise of all the eukaryotic photosynthetic microorganisms, have been used by the indigenous populations for centuries. In recent years, they have been studied for numerous commercial applications, ranging from animals and human nutrition to cosmetic products. In fact, algae are a diverse group of organisms with valuable ingredients like proteins, carbohydrates, lipids, vitamins, pigments, polyunsaturated fatty acids, and fibres^[3]. Some microalgal strains are recognised as excellent sources of proteins, carbohydrates, lipids, and vitamins, to be used and feed additives, for more than 40 years^[4-5]. However, the importance of microalgae in aquaculture is large because they start the food chain. The nutritional value related to the biochemical composition, makes Nannochloropsis oculata well appreciated for feeding rotifers and fish hatcheries. Nannochloropsis oculata, which includes marine, fresh, and brackish water algal species, is a potential resource for bio fuel production. Several bioactive metabolites produced by cyanobacteria and algae have been discovered by screening programs, employing target organisms quite unrelated to those for which the metabolites evolved. Nowadays microalgae are explored for nutraceutical and bioactive compounds that promote health.

In recent years, genotoxicity testing has become more and more important in the process of early screening for potential development compounds. The Salmonella strains used in the test have different mutations in various genes in the histidine operon; each of these mutations is designed to be responsive to mutagens that act via different mechanisms. The Salmonella mutagenicity test was specifically designed to detect chemically induced mutagenesis^[6]. The test is used world-wide as an initial screen to determine the mutagenic potential of new chemicals and drugs because there is a high predictive value for rodent carcinogenicity when a mutagenic response is obtained. The Ames Salmonella microsome mutagenicity assay evolved over the years from the initial screening of a number of histidine mutants which led to the selection of mutants that were highly sensitive to reversion by a variety of chemical mutagens^[7,8]. The purpose of the in vitro chromosome aberration test is to identify agents that cause structural chromosome aberrations in cultured mammalian cells^[9]. At the present time, the available data suggest that it is important to consider the p53 status, genetic (karyotype) stability, DNA repair capacity and origin (rodent versus human) of the cells chosen for testing. The exogenous metabolic activation system does not entirely mimic in vivo conditions. Care should also be taken to avoid conditions that would lead to artefactual positive results which do not reflect intrinsic mutagenicity, and may arise from such factors as marked changes in pH or osmolality, or by high levels of cytotoxicity^[10]. This test is used to detect chromosomal aberrations which may result from clastogenic events. To analyse the induction of chromosomal aberrations, it is essential that mitosis has occurred in both treated and untreated cultures.

There are number of clinically efficacious antibiotics becoming less effective due to the development of antibiotic resistant microorganisms^[11-12]. It becomes a greater problem to treat many diseases caused by resistant pathogenic microorganisms worldwide. In addition, decreased activity of commonly used antibiotic and resistance of pathogens to such antibiotics have anticipated the development of new alternatives^[13]. Marine planktons especially algae are rich source of many interesting bioactive molecules including lipid which may be useful for the development of antimicrobial drugs^[14]. Marine microalgae have been an unique source of chemical compounds of pharmaceuticals, aquaculture, cosmetics, anticancer agents, enzymes, pigments, antioxidants, polyunsaturated fatty acids, dietary supplements, agrochemicals and biofuel^[15]. There are many reports related to antimicrobial activity of crude extracts of marine macro and microalgae^[16].

However, despite the therapeutic use of natural sources, it is quite often essential to evaluate the toxicity and antigenotoxicity of medicinal plants and extracts to prove them as safe, nontoxic and potential bioactive agent. This has become inevitable as they are explored for use in long term treatment against various disorders. Hence, the aims of this study were to determine the antigenotoxicity testing, cytogenetic assay and antibacterial activity of methanol extract of Nannochloropsis oculata.

MATERIALS AND METHODS:

Preparation of Extract:

The methanolic extract was prepared by suspending the dry algal biomass in methanol in the ratio of 1:5 wt/volume. The suspension is mixed well and incubated for 24 hours. After incubation, the suspension is filtered and the filterate is evaporated to dryness and collected in clean bottle which is stored at 15˚C for further studies.

IN VITRO GENOTOXICITY TESTING:

Ames test:

Cells were treated with various concentrations of methanolic extract of Nannochloropsis oculata. Tester strains used were Salmonella histidine auxotrophs TA98, (Ames et al., 1975). Tester strains were exposed to the test sample via the plate incorporation methodology described by Maron and Ames (1983). Mutagenicity was assessed by the pre-incubation assay as described by^[17]. Briefly, 100µL of overnight cultures (1.2×108cfu/ml) of strain TA98 were treated separately for 30 mins at 37˚C with various concentrations of the test extract in the absence of s9 mix. For the Ames test, the control and microalgae of Nannochloropsis oculata in methanol extract -treated cells were mixed with 2ml of sterile top agar (0.6% agar and 0.5% NaCl containing 0.5nM histidine and 0.5nM biotin) and poured onto minimal glucose agar plates (1×Vogel-Bonner salts (0.2g/L magnesium sulfate, 2g/L citric acid monohydrate, 10g/l dipotassium hydrogen phosphate, 3.5g/L sodium ammonium phosphate, 2% glucose and 1.5% agar). The plates were then incubated at 37˚C for 48 hours, after which revertant experiments were conducted and each experiment consisted of three replicate plates for each treatment. Sodium azide at a concentration of 1.5µg/100µl is used as inducer.

Plating procedures:

These procedures were used in the dose range-finding and mutagenicity assays. Each plate was labeled with the test item, test phase, tester stain, activation condition, and dose. Treatments in the absence of S9 were performed by adding 100µl tester strain and 100µl test or control to 2.5ml molten diluted top overlaid onto the surface of bottom agar dishes. After the overlay solidifies the plates were inverted and incubated for 72hrs at 37±2˚C. After incubation, the plates were evaluated for the condition of the background lawn for the evidence of cytotoxicity and test item precipitate in comparison with the control and the plates were evaluated for the number of revertant colonies.

IN VITRO CYTOGENETIC ASSAY:

HUMAN LYMPHOCYTES CULTURE AND CHROMOSAL ABERRATION STUDIES:

Heparinized blood sample (0.5ml) was collected from healthy individuals and were placed in sterile culture flasks with 0.7mk of RPMI1640 supplemented with fetal bovine serum (1.5ml), antibiotic-antimycotic mixture (1.0ml) and phyto hemagglutinin (0.1ml). The cultures are placed in incubator at 37˚C for 48hours. After 48 hours incubation, test compound (varied concentration of Nannochloropsis oculata in methanol extract)/positive mutagen of known concentration prepared in DMSO will be added to the culture at a volume of 0.1ml to achieve desired final concentration after all other constituents are added. Negative control cultures received DMSO alone at a volume of 0.1ml while Mitomycin C is used as positive control. Negative control culture without metabolic activation will receive only 0.5ml of phosphate buffer. The vials/centrifuge tube/flasks will be transferred to co₂ incubator. The culture will be incubated at 37˚C and 5% CO₂ for 3-6hours. Flasks were transferred to labeled sterile 15ml centrifuge tubes, centrifuged at room temperature at 1600rpm for 5-10 min, supernatant will be aspirated gently and to the pellet a freshly made working growth medium RPMI 1640 without PHA-M were added. The total volume of the culture will be made up to 10ml using the culture medium. The centrifuge tubes were transferred to CO₂incubator. The culture will be incubated at 37±1˚C and 5% CO₂ for 18-21 hour. 100µl of 10µg/ml of colchicines is added and incubated for additional 2 hours. The entire content of the flask was transferred to a sterile centrifuge tube and centrifuged at 800-1000rpm for 10 minutes. The supernatant was discarded and the pellet was suspended in 5ml of hypotonic 0.075M KCl solution and incubated in a water bath at 37˚C for 15-20 minutes. The equal amount of freshly prepared ice cold fixative were added (Acetic acid: methanol 1:3 parts).The fixative was removed by centrifugation and this process is repeated twice. The slides were prepared and they were stained with 3% Giemsa stain solution in phosphate buffer (pH 6.8) for 15 min. Atleast 300 metaphases were scored in each slide to examine different types of abnormality according to standard protocol of Savage^[18]. Mitotic index (MI) was calculated by using formula, MI = of dividing cells/total number of cells×100, where MI, Mitotic index.

ANTIBACTERIAL ACTIVITY:

Agar well-diffusion method was followed to determine the antimicrobial activity. Nutrient agar (NA) plates were swabbed (sterile cotton swabs) with 8 hour old - broth culture of E.coli, S. aureus and B. subtilis. Wells (10mm diameter and about 2 cm a part) were made in each of these plates using sterile cork borer. Stock solution [1mg/ml] of methanol extract of Nannochloropsis oculata was prepared and diluted to different concentration. About 25μl of methanol solvent extracts of different concentration ranging from 25-100 μg/ml were added using sterile syringe into the wells and allowed to diffuse at room temperature for 2hrs. The plates were incubated at 37°C for 18-24hrs for bacterial pathogens. Methanol (100%) without Nannochloropsis oculata extract was used as negative control and Streptomycin disc (50μg) was used as the positive control. The diameter of the inhibition zone (mm) was measured. The experiment was repeated thrice, for each replicate the readings were taken in three different fixed directions and the average values were recorded^[19].

STATISTICAL ANALYSIS:

The Mean and standard deviation was calculated for each parameter. The data was analyzed by SPSS 17.0 software. Two ways ANOVA was performed to determine significance of treatment. The mean separation was performed according to Duncan’s new multiple range test (p<0.05).

RESULTS AND DISCUSSION:

The prior study, Reactive oxygen species (ROS) are involved in various serious diseases including cancer. Zeocin is a well-known oxidative stress inducer, stimulating MDA and H₂O₂ production and as result induction of single- and double- strand breaks, and DNA base loss resulting in apurinic/apyrimidinic (AP) sites. The removal of potentially deleterious ROS through antioxidants has been suggested to be an important mediator for the protection of cells. A significant interest arises to find and investigate natural substances possessing anti-mutagenic and anti-oxidant activities against ROS inducers and to replace the synthetic compounds in food applications. At first, it is essentially to study the safety of the natural compounds and extracts at doses of pharmacological range. Many authors report data about cytotoxic and genotoxic potential of different natural plant extracts using various endpoints^[20-23]. A previous study investigated Poppy extract widely applied in folk medicine against many diseases, using markers for cytotoxicity and genotoxicity in two types of test-systems – barley and human lymphocytes in vitro. Different sensitivity to poppy extract was observed depending on the test-systems. Poppy extract has no, or weak cytotoxic and genotoxic effects in Hordeum vulgare depending on the concentrations. Human lymphocytes are more sensitive than barley. Clearly expressed cytotoxic and genotoxic effects were observed depending on the concentration. In our study the lowest concentrations used in both test-systems have no harmful effects. Our results are in confirmity with the finding of other studies^[24]. Chromosome abnormalities are considered to be one of the most important cytogenetic parameters for the manifestation of Genotoxicity. The genotoxicity potential of several substances may be detected and quantified using fish as the experimental model, since these animals, similar to mammals, may activate the enzymatic system from cytochrome P450. Further more spontaneous formation of micronuclei is normally low and nearly uniform among species. Thus, the action of any genotoxic agent may give rise to an increase in micronucleus frequency ^[25].

Our work aimed at conducting Genotoxicity studies by Ames test for the screening of mutagenicity and to prove this as precursors of drug. The mutagens (positive controls) treated without metabolic activation system showed a 3 fold increase of average revertant colonies per plate when compared with that of concurrent vehicle controls, thus exhibiting the ability to identify the mutagen by the tester strains. The mutagenicity assay was performed with three dose levels (0.312, 1.25 and 5.00 mg/ml) in the absence of metabolic activation system. Inhibition of background growth of non revertant bacteria was not found at any of the three dose levels.

The study data represented in (Table 1) showed no significant increase of His⁺revertant colonies when exposed to the methanol extract of Nannochloropsis oculata the dose levels incubated with any of the tester strains, without S9 addition when compared to the respective controls in the mutagenicity assay. The average revertant colonies per plate treated with the control in the absence of metabolic activation system were found to be within the acceptance limits of the spontaneous revertant control values of respective salmonella strains. The results showed no significant increase in the His⁺revertant colonies following exposure to the samples at any tested concentration in any of the tester strains without S9 when compared with the negative control of each tester strain. Based on the above results, it is conduced that the synthesised the microalgae of Nannochloropsis oculata were non-mutagenic at the dose levels ranging from 0.313 to 5000µg/plate by the Ames bacterial reverse mutation assay in the absence of S9 mix, under the conditions of the test employed.

Table 1 Mean Colony Count - Strain TA 98 spontaneous mutation

Test Item	Test Concentration (µg/plate) / (mg/ml)	Histidine Revertant Colonies (CFU/Plate)
Positive Control	Sodium azide (1.5 µg/plate)	432
S3 (without S9)	5	107
	1.25	72
	0.312	53

Fig 1 Normal, Positive control, DMSO for TA 98 and Methanol control

When human leukocytes were treated with methanol extract from the microalgae Nannochloropsis oculata alone at different doses; the incidence of cells having aberrant (including gap) in percentage of chromosomal aberration frequency maximum concentration of dose of 1.25 and 5mg/ml no increase in number of CA were observed when compared to untreated. In contrast, the incidence of aberrant cells in each positive control (mitomycin c) group increased greatly as compared with each solvent group (p<0.001) (Table 2).

Present study, showed no significant increase of His⁺ revertant colonies following exposure to the Nannochloropsis oculata methanol extract at any of the dose levels incubated with any of the tester strains, without S9 addition, when compared to the respective controls in the mutagenicity assay. It is well known the cyclophosphamide could cause damage in cultured mammalian cells. With the majority of chemical mutagens, induced aberrations are of the chromatid type, but chromosome type aberrations also occur. The in vitro chromosomal aberration test may employ cultures of established cell lines, cell strains or primary cell cultures. Chromosomal aberrations are the cause of many human genetic diseases and there is substantial evidence that chromosomal damage and related events causing alterations in oncogenes and tumor suppressor genes of somatic cells are involved in cancer induction in humans and experimental animals.

Table 2 Cytogenetic Assay Measuring Chromosomal Aberration without S9

Dose (mg/ml culture)	Total number of cells scored	Percent numerical aberration	Mean of structural aberration	Total number of aberration	Total no. of cells with aberration	No. of aberration per cell	Aberration frequency (%)
Negative control (DMSO)	100	0	0	0	0	0	0
0.312	100	0	0	0	0	0	0
0.312	100	0	0	0	0	0	0
1.25	100	0	0	52	4	13	4.5
1.25	100	0	0	39	5	7.8	4.5
5	100	0	0	50	8	6.25	13.5
5	100	0	0	57	11	5.18	13.5
Positive control	50	0	0	176	30	5.87	50
Mitomycin C	50	0	0	165	40	4.13	50

CONCLUSION:

Among the avialble marine resources, the microalgal species also constitute various bioactive compounds responsible for the pharmacological activity. The present study proved the antigenotoxicity and antimicrobial activity of methanolic extract of Nannochloropsis oculate. Thus, the evidence for non toxic nature of natural sources and experimental study of bioactivity pave way to further explore these species for potential medicinal applications.

REFERENCES

1. Hibberd. Notes on the taxonomy and nomenclature of the algal classes Eustigmatophyceae and Tribophyceae (Synonym Xanthophyceae). Botanical Journal of the Linnean Society. 1981; 82: 93–119.

2. Pfuhler S, Fellows M, Van Benthem J, Corvi R, Curren R, Dearfield K, Fowler P, Frötschl R, Elhajouji A, Le Hégarat L, Kasamatsu T, Kojima H, Ouédraogo G, Scott A and Speit G. In vitro genotoxicity test approaches with better predictivity: Summary of an IWGT workshop. Mutation Research. 2011; 723: 101-107.

3. Sugimura T, Sato S, Nagao M, Yahagi T, Matsushima T, Seino Y. Overlapping of carcinogens and mutagens. In: Fundamental of Cancer Prevention, pp. 191-215; 1976. University Park Press, Baltimore

4. Ames BN, McCann J and Yamasaki E. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutation Research 1975; 31: 347–364.

5. Brusick, D. Genotoxic effects in cultured mammalian cells produced by low pH treatment conditions and increased ion concentrations, Environ. Mutagen 1986; 8: 789-886.

6. Ames BN, Lee FD and Durston WE. An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proceedings of the National Academy of Science U.S.A. 1973; 70: 2281-2285.

7. McCann J, Choi E, Yamasaki E and Ames BN. Detection of carcinogens in the Salmonella/microsome test. Assay of 300 chemicals. Proceedings of the National Academy of Science U.S.A. 1975; 72: 5135–5139.

8. Ames BN. The detection of chemical mutagens with enteric bacteria. In: Chemical Mutagens, Principles and Methods for Their Detection vol. 1 (ed. A. Hollaender), pp. 267-282. Plenum, New York; 1971.

9. Maron D and Ames BN. 1983. Revised methods for the Salmonella mutagenicity test. Mutation Research. 1983; 113: 173–215

10. Zeiger E. The Salmonella mutagenicity assay for identification of presumptive carcinogens. In: Handbook of Carcinogen Testing (ed. H.A. Milman, E.K. Weisburger), pp. 83-99. Noyes Publishers, Park Ridge, NJ; 1985.

11. Monnet DL, Archibald LK, Phillips L, Tenover FC, McGowan JE Jr and Gaynes RP. Antimicrobial use and resistance in eight US hospitals: complexities of analysis and modeling. Infect Control Hospital Epidemiology. 1998; 19: 388-394.

12. Monnet DL Sakthivelkumar S, Rajendran K and Janarthanan S. Screening of selected marine algae from the coastal Tamil Nadu, South India for antibacterial activity. Asian Pacific Journal of Tropical Biomedical. 2012; 2: S140-S146.

13. Omar HH, Shiekh HM, Gumgumjee NM, El-Kazan MM and El-Gendy AM. Antibacterial activity of extracts of marine algae from the Red Sea of Jeddah, Saudi Arabia. African Journal of Biotechnology. 2012; 11(71): 13576-13585.

14. Lazarus S and Bhimba V. Antibacterial activity of marine microalgae against multidrug resistant human pathogens. International Journal of Applied Bioengineering. 2008; 2: 32-34.

15. Srinivasakumar KP and Rajashekhar M. In vitro studies on bactericidal activity and sensitivity pattern of isolated marine microalgae against selective human bacterial pathogens. Indian Journal of Science and Technology. 2009; 2: 16-23.

16. Tuney I, Cadirci BH, Unal D and Sukatar A. Antimicrobial activities of the extracts of marine algae from the Coast of Urla (Izmir, Turkey). Turkish Journal of Biology. 2006; 30: 171-175.

17. Maron D, Ames BN. Revised methods for the Salmonella mutagenicity test. Mutation Research. 1983; 113: 173–215.

18. Mendes RL. Supercritical fluid extraction of active compounds from algae. In Martinez, J. L. (Eds). Supercritical fluid extraction of nutraceuticals and bioactive compounds, p. 189-213. United States: CRC Press; 2007.

19. Vijay DSM, Sirajunnisa AR, Subramaniyan T, Shellomith AS and Tamilselvam K. A green synthesis of antimicrobial compounds from marine microalgae Nannochloropsis oculata. Journal of Coastal Life Medicine.2014; 2: 859-863.

20. Velioglu YS, Mazza G, Gao L and Oomah BD. Antioxidant activity and total phenolics in selected fruits, vegetables and grain products. Journal of Agricultural Food Chemistry. 1998; 46: 4113-4117.

21. Balasubramanian A, Ramalingam K, Krishana S and Christina AJM. Anti-inflammatory activity of Morus indica Linn. Iran Journal of Pharmacology and Therapeutics. 2005; 4: 13-15.

22. Hudecova A, Hasplova K, Miadokova E, Magdolenova Z, Rinna A, Galova E, Sevcovicova A, Vaculcikova D, Gregan F and Dusinska M. Cytotoxic and genotoxic effect of methanolic flower extract from Gentiana asclepiadea on COS 1 cells. Neuro Endocrinology Letter. 2010; 31: 21-25.

23. Mothana RAA, Abdo SAA, Hasson S, Althawab FMN, Alaghbari SAZ and Lindequist U. Antimicrobial, antioxidant and cytotoxic activities and phytochemical screening of some Yemeni medicinal plants. eCAM. 2010; 7: 323-330.

24. Hasplova K, Hudecova A, Miadokova E, Magdolenova Z, Galova E, Vaculcikova L, Gregan F and Dusinska M.. Biological activity of plant extract isolated from Papaver rhoeas on human lymphoblastoid cell line. Neoplasma 2011; 58: 386-391.

25. Giiez CM, Emily Waczuk P, Pereira KB, Querol MVM, Rocha JBT and Oliveira LS. In vivo and in vitro Genotoxicity studies of aqueous extract of xanthium spinosum. Brazilian Journal of Pharmaceutical Science. 2012; 48.

26. Deb DD, Kapoor P, Dighe RP, Padmaja R, Anand MS, D'Souza P Deepak M, Murali B, Agarwal A. In Vitro Safety Evaluation and Anticlastogenic Effect of BacoMind on Human Lymphocytes. Biomedical and Environmental Science. 2008; 21: 7-23.

Received on 25.08.2019 Modified on 02.10.2019

Research J. Pharm. and Tech 2020; 13(5): 2297-2302.

DOI: 10.5958/0974-360X.2020.00414.X