Acute Toxicity Study and Antidiabetic Activity of Marine alga-Halimeda gracilis Chooranam (HGC) in Freshwater Zebrafish Model

1MGMCRI (Department of Pharmacology), Puducherry. India. 2Melaka Manipal Medical College (Manipal Campus) (Department of Pharmacology), MAHE, Manipal-576104, Karnataka, India. 3MVJ Medical College and Research Hospital (Department of Pharmacology), Hoskote. Karnataka, India. 4Animal Biotechnologist, MGMCRI (Department of Pharmacology), Puducherry. India. 5MGMCRI (Department of Pharmacology), Puducherry. India. *Corresponding Author E-mail: syed.mushraf@manipal.edu

Diabetes Mellitus is one of the heterogeneous metabolic disorder causing both microvascular & macro vascular complications which is characterized by hyperglycaemia due to alterations in the storage & mobilization of metabolic compounds, including catabolism & anabolism of carbohydrates, lipids & proteins, resulting from defects in insulin synthesis & secretion, resistance to action or both 1 .
The long-term effects of diabetes lead to the development of specific complications like retinopathy, nephropathy, neuropathy, cardiovascular diseases, and co-morbidities 2 . Hence, it is vital to diagnose the undiagnosed people with diabetes or pre-diabetic state and impart proper care for them as early as possible. Regular medications are utilized to treat diabetes by enhancing insulin sensitivity, increasing insulin production, and reducing blood glucose levels. There is always a drawback in drug treatment, in regulating the blood glucose levels due to certain adverse effects like GIT disturbance, tiredness, weight gain., etc.
In modern medicine, no satisfactory effective therapy is yet available. Marine algae (or) seaweed compounds have contributed to the global search for novel medicinal agents. In the current time, a noticeable number of novel metabolites with potent pharmacological properties have been distinguished from the marine organism which is one of the abundant source of structurally diverse natural products [3][4][5] . These algae are the most essential part of the diet of many eastern countries, and their use as food is well documented. Marine algae have a wide variety of compounds which have promising health benefits, and some have exhibited chelating property on heavy metals 6 . They are an abundant natural resource for many bioactive substances like polyunsaturated fatty acids, sterols, proteins, polysaccharides, antioxidants, colours, and trace elements in a focus a lot higher than in earthly plants 7 and offer a wide range of secondary metabolites, which shows different pharmacological actions like anti-cancer, antimicrobial, antifungal, anti-inflammatory, antioxidants, anti-fouling, and anti-diabetic activity, etc [8][9] .
Hence, in this study, we have tested the acute toxicity and antidiabetic activity of Halimeda gracilis as various studies on other macro algae have potentially proven to have anti-diabetic action in animal models & patients and it's being used in alternate medicines to treat diabetes mellitus.

Collection of Halimeda gracilis
The fresh marine green alga Halimeda gracilis was collected from Rameswaram coastal area, Tamil Nadu, India, and was carried to the lab in plastic bags with seawater. The alga was processed for a thorough wash with seawater & then tap water to remove epiphytes, salts, and other extraneous materials. The seaweeds were identified and authenticated as Halimeda gracilis (Halimeda gracilis Harvey ex. J Agarah 1887) by Dr. S. Bragdeeswaran, Associate Professor, Centre of Advanced Study (CAS) in Marine biology, Annamalai University, Parangipettai, Tamil Nadu, India & also prepared herbarium & museum specimens for the repository. The sample was then shade dried at 37°C and ground to a fine powder. The powder was then stored in the refrigerator for further use 10 .

Preparation of extracts
The dried Halimeda gracilis was made into a coarse powder in a mechanical grinder and it was subjected to maceration at 24-25°C in 95% methanol for 72 hours. The methanolic extract was derived after the process of distillation, evaporation, and drying under reduced pressure as per the standard procedure 11 .

Acute toxicity study
Zebrafish were housed in a home tank at the population density of 2 fish per litre. The aquarium was filled with dechlorinated purified tap water and reverse osmosis water in the ratio of 1:2. The water was analysed and adjusted for the optimum pH, conductivity, salinity, and hardness. The aquarium was equipped with an aquarium filter and aerator units (aquarium aerator and pump). The fish was quarantined for a minimum period of 12 days in the laboratory test room before starting the experiment and the fish was fed with commercial fish food pellets two times per day. The temperature was maintained in the range of 27±2ÚC. The fish was provided with a photoperiod of 12 hours of artificial light and 12 hours of darkness throughout the experiment. They were acclimatized in the test tanks for 4 days before the study and the feeding was stopped 24 hours before the initiation of the drug dose.

Procedure
As per OECD guideline 203 12 , the limit test at 100 mg/L of the test compound demonstrated that the LC 50 is greater than this concentration. The maximum concentration of the study was fixed as 100mg/L. The spacing factor of the concentration range was 2. The volume of the exposure medium was 2.5 litres per tank (not exceeding the maximum load of 1g of fish per litre). Exposure medium, maintained at the optimum pH, temperature, dissolved oxygen, and 12 hours of photoperiod was maintained throughout the study.

Grouping and dosing
As the test compound was sparingly soluble in water, the stock solution was prepared by dissolving the 500 mg of the test compound in 1ml of methanol. The stock solution is aliquoted into different tubes based on the desired concentrations as mentioned ( Table 1). The desired concentrations of exposure medium were prepared by adding the aliquoted quantity of the stock solution in the aquarium habitat water of known quantity. During the test period of 96 hours, all experimental fish were treated with MEHGC except normal control fish and observed for mortality, morbidity, and other behavioural changes in Zebrafish.

Induction of diabetes in Zebrafish
All experimental fish received STZ (0.35 mg/g body weight, i.p.) single dose for 5 groups for 19 days, except normal control based on its body weight ( Figure 1). The fishes were kept on fasting for 12 hours on 20 th day and on Day 21, blood samples (1.0 to 2.0 µl/fish) were collected through caudal fin and analysed for blood glucose level as a baseline. Diabetic animals were randomized into 6 groups based on an acceptable range of glucose level ± 20% mean between the groups (Table 2). It was ensured to have a minimum of 8 animals with disease induction in each group at the start of drug treatment 13 .

dose formulation
The test drug was freshly prepared before administration & the respective doses were given orally for each group based on the body weight of the fish for 7 consecutive days 14 . (Figure 2)

Blood Collection
After induction of diabetes on day 21 as baseline values and after treatment on day 28 of the experiment the blood was collected from caudal fin puncture and analysed for blood glucose level in all the test groups 13 .

Necropsy, organ collection, and pathology
At the end of the experiment, fishes were euthanized with ms-222, for collecting pancreas & intestinal tissue. The collected tissues were fixed in 10% neutral buffered formalin for 48 hours, processed, embedded in paraffin, sectioned on a microtome, and stained with H & E. statistical analysis SPSS was used for statistical analysis and the data expressed as the mean ± standard error (S.E). The differences in blood glucose level (fasting and postprandial) obtained between the diabetic control group, test group, and the positive group was compared by using Student's t-test and the difference was considered to be statistically significant when the p-value was <0.05.

Acute toxicity study
The result provides evidence that the treatment of MEHG doesn't cause mortality up to the maximum concentration, 100 mg/L as there were no morbidity or mortality observed in experimental fish throughout the study. MEHG didn't cause any remarkable behavioural changes with a different range of concentrations within the period of the test. No abnormal behavioural changes were observed in experimental fish in the entire study, as all the fishes were found to be normal [ Table 3-7].

Antidiabetic activity of MehGC
In experiment groups, group 4 Zebrafish treated with a high dose of MEHG showed significant reduction [p <0.001] in both the fasting and postprandial blood glucose levels compared to MEHG treated group 2, 3 & untreated group 5 -positive control [ Table 8].
Fasting and postprandial glucose levels are expressed as mean ±SEM, P<0.05 when compared to control and group 6.

Histopathological examination
The pancreatic cells in group 1 fishes exhibited normal morphology, whereas, in group 2 and group 3 fishes, vacuolation in the exocrine  pancreas was observed. In group 4, most of the fishes exhibited regenerative morphology and exhibited normal pancreatic architecture. Few fishes exhibited vacuolation in the exocrine pancreas. In the untreated group 5, high pancreatic damages were observed like pancreatic degeneration and vacuolation in islets. Group 6 fishes showed normal pancreatic cellular architecture [ Figure 3]. Hence from obtained histopathological examination analysis group 4 exhibited almost normal pancreatic cellular morphology compared to MEHG treated group 2, 3 & untreated group 5.

disCussion
Seaweeds or marine algae are crude nonflowering plants without true root stem and leaves 10 . They are the sustainable living sources of food, fodder, and fertilizer in many parts of the world. Macro algae are found to have potentially highly active secondary metabolites with diverse activities in various researches reports which have been screened widely to isolate life-saving drugs or biologically active substances worldwide. Marine algae are a rich source of ingredients such as polyunsaturated acids, â-carotene, and their pigment carotenoids, sulphated polysaccharide, and sterol 15 . They also contain secondary components that have the potential to be developed in various fields such as pharmaceuticals, cosmetics, and other industrial purposes such as biomass, biofuels, bio-oil, biodiesel, etc., and the waste can be used as fertilizer and fodder for animal (or) fish 16 .
In the present study, HGC used in acute toxicity study didn't show any abnormal behavioural changes, mortality, and morbidity in the Zebrafish even after 96 hours at maximum concentration 100mg/L.
The MEHGC showed quite significant antidiabetic activity at higher doses (500µg/g body weight) by reducing blood glucose levels (p<0.001) showing better efficacy in comparison with other doses. It also maintained the normal morphology of pancreatic cells & regenerative property.
We would like to conclude that Halimeda gracilis has better efficacy as antidiabetic, by maintaining the normal morphology of pancreatic cells & also has regenerative property without causing any mortality & morbidity in the acute toxicity test. It can be included in the diet of diabetic patients for the prevention and treatment of their condition.

ACknowledGMent
Dr. S. Bragdeeswaran, Associate Professor CAS in Marine biology, Annamalai University, Parangipettai, Tamil Nadu, India for identifying and authenticating the species and preparing the herbarium.

Ethical Approval
Not applicable (Ethical approval for Zebra fish study is not required as there are no ethical committees formed for the fish studies).

Competing interests
The authors have no competing interest to declare.

Funding
Not applicable

Ethical clearance
Not applicable

Code availability
Not applicable Author's contribution SAJV-Initiated and conducted experiments. SRN-wrote manuscript. UNS-analysed the data. SV -is an animal biotechnologist -helped in animal studies. MK -Drafted the manuscript. SM-Conceived and designed the research. All authors read and approved the manuscript and all data were generated in-house and that no paper mill was used.