DETERMINATION OF LETHAL CONCENTRATION FIFTY (LC50) OF WHOLE PLANT ETHANOLIC EXTRACT OF AMARANTHUS VIRIDIS, CYNODON DACTYLON & AERVA SANGUINOLENTA ON ZEBRAFISH (DANIO RERIO) EMBRYOS

DETERMINATION OF LETHAL CONCENTRATION FIFTY (LC₅₀) OF WHOLE PLANT ETHANOLIC EXTRACT OF AMARANTHUS VIRIDIS, CYNODON DACTYLON & AERVA SANGUINOLENTA ON ZEBRAFISH (DANIO RERIO) EMBRYOS

Shabnam Zahir ^{* 1}, Tamal Kanti Pal ², Abhijit Sengupta ³, Shibendu Biswas ⁴, Shyamal Bar ⁵ and Samrat Bose ³

Department of Pedodontics and Preventive Dentistry ¹, Department of Oral and Dental Sciences ², Department of Pharmacy ³, Department of Microbiology ⁴, Gurunanak Institute of Dental Science & Research, Kolkata - 700114, West Bengal, India.

Guru Nanak Institute of Pharmaceutical Sciences & Research ¹, Kolkata - 700110, West Bengal, India.

Department of Orthodontics ⁵, Burdhaman Dental College and Hospital, Bardhaman - 713101, West Bengal, India.

ABSTRACT: Introduction: Most potent toxic elements are derived from nature only. So safety and toxicological evaluation of plant extracts which are otherwise thought to be very safe, are mandatory. The zebrafish embryo has become an important model organism for toxicological research due to its small size, genetic similarity to humans, etc. The Objective: The objective of the present study is to determine acute toxicity that is lethal concentration fifty (LC₅₀) of whole plant ethanolic extract of Amaranthus viridis (A1), Aerva sanguinolenta (A2) & Cynodon dactylon (A3) on Zebrafish (Danio rerio) embryos at a different time interval. Material and Method: Acute toxicity testing of the three whole plant ethanolic extracts A1, A2, A3 with six different concentrations each were performed according to OECD guideline 236 on Zebrafish embryos. Embryo development was monitored after 24 h interval for 120 h. After incubation, the living and dead embryos were evaluated using Light Microscope and images obtained with an optical camera. Statistical Analysis: LC₅₀ values were calculated using the Probit method in the SPSS package. Graphs were plotted using Sigma Plot. Result: Amaranthus viridis (A1), Aerva sanguinolenta (A2), Cynodon dactylon (A3) whole plant ethanolic extract have mean LC₅₀ of 316.22080 µg/ml, 575.42800 µg/ml, 35486.000 µg/ml respectively at a time interval of 24 hours on Zebrafish embryos. Conclusion: LC₅₀ values of three whole plant ethanolic extracts A1, A2, A3 with six different concentrations each on Zebrafish embryos will aid in determining the therapeutic dose of the plant extracts.

Toxicological study, Plant extracts, Zebra fish embryo

INTRODUCTION: Amaranthus viridis, Aerva sanguinolenta & Cynodon dactylon are the three medicinal plants with many therapeutic virtues Table 1. However, like medicinal plants, Amaranthus viridis, Aerva sanguinolenta & Cynodon dactylon are not exempt from the risks of intoxication.

The work of Ugwah-Oguejifor CJ et al., revealed that some plant species have hepatotoxic effects ¹. Thus studies on the safety and effectiveness of medicinal plant extract and extract-derived phytoconstituents have become one of the main concerns to guarantee their use for mankind ².

Rats and mice are traditionally used for the estimation of acute toxicity of substances for mammals. But they are expensive and difficult to manipulate and assess during the embryonic stage. Within the past decade (since 1950), the zebrafish has become an important model organism for toxicological research ³.

Zebrafish (Danio rerio), are vertebrate organisms that are an excellent alternative model due to their small size, rapid external development, optical transparency during early development, permeability to small molecules, genetic similarity to humans, and great fecundity. Zebrafish embryos develop most of the major organ systems present in mammals, including the cardiovascular, nervous, and digestive systems in less than a week ^{4, 5}. The median lethal concentration, LC₅₀ is the amount of a material given all at once, which causes the death of 50% of a group of test animals. The LC₅₀ is a way that measures the short-term poisoning potential (acute toxicity) of a material ⁶.

The present study is performed to determine acute toxicity that is lethal concentration fifty (LC₅₀) of whole plant ethanolic extract of Amaranthus viridis (A1), Cynodon dactylon (A2) & Aerva sanguinolenta (A3) at different time intervals on Zebrafish (Danio rerio) embryos.

The study is also targeted to determine the percentage mortality of Zebrafish (Danio rerio) embryos on exposure to different concentrations of whole plant ethanolic extract of Amaranthus viridis (A1), Cynodon dactylon (A2) & Aerva sanguinolenta (A3) at different time intervals.

TABLE 1: CHARACTERIZATION OF PLANT SPECIMEN (STUDY SAMPLE)

MATERIALS AND METHODS:

Ethical Clearance: Requisite ethical clearance (GNIDSR/IEC/18-18) & permission to undertake the study was obtained from the institutional ethical committee of Guru Nanak Institute of Dental Sciences and Research, Kolkata.

Collection, Authentication & Pre-treatment of Plant Sample: Whole plant of Amaranthus viridis (A1), Aerva sanguinolanta (A2) & Cynodon dactylon (A3) were collected from medicinal garden of Ramkrishna mission ashram, Narendrapur, Kolkata, West Bengal during the month of December 2018. Herbarium were made and the specific plants were authenticated from Botanical survey of India, Howrah, West Bengal -711103. The voucher specimen (GNIDSR 002, 003 & 004) were deposited in College Herbarium.

The plant samples were washed, shade dried on paper sheets, chopped into pieces, made into a coarse powder in a mixer grinder (Philips HL), and finally the coarse powder was stored in a sealed, labelled polythene packet in the refrigerator at 4 degree centigrade.

Preparation of Plant Extracts: The coarse powder of Amaranthus viridis (A1)  (60.8 gm), Aerva sanguinolanta (A2) (53.3gm) & Cynodon dactylon (A3)  (50.29 gm ) were subjected to continuous hot extraction with 350 ml, 300ml, and 300 ml ethanol (Mercks) respectively by Soxhlet apparatus (Borosil) with a yield of (26.001gm) (A1), (23.5462g) (A2), (20.546gm) extract by weight. Extracts with solvent were concentrated by distillation using a rotary evaporator (RE100PR0 MFGD silicogex/USA Takashi) with a yield of (14.001gm) (A1), (12.1162g) (A2), (11.006gm) (A3) extract by weight which was stored in a sterilized glass beaker in a refrigerator at 4-degree centigrade. Preparation of plant extracts was performed in the Guru Nanak Institute of Pharmaceutical Science and Technology, Kolkata.

Preparation of Different Concentration of Plant Extracts: Plant extracts were measured on an electronic weighing scale (Model CAS –ME–310). The premeasured amount of the three stored plant extracts (100 microgram) were mixed with a fixed volume (1 ml) of DMSO (Mercks) to prepare the stock solution. Electrical stirrer (Remi Laboratory Instruments) was used for 30 min for proper mixing of plant extract and solvent. The prepared 100 µg/ml concentration mix was kept as a stock solution. For each plant extract six different concentration of solution (100 micron gram/ millilitre, 200 micron gram/millilitre, 300 micron gram/millilitre, 400 micron gram/millilitre, 500 micron gram/millilitre, 600 micron gram/millilitre) with Dimethyl sulfoxide (DMSO, Himedia) were prepared Fig 1.

FIG. 1: PREPARATION OF DIFFERENT CONCEN-TRATIONS OF PLANT EXTRACT

Acute Toxicity Testing of Whole Plant Ethanolic Extract of Amaranthus viridis, Cynodon dactylon & Aerva sanguinolanta: According to OECD  guideline 236 (Fish  Embryo Toxicity Test) acute toxicity that is  lethal concentration (LC₅₀) of whole plant ethanolic extract Amaranthus viridis, Cynodon dactylon & Aerva sanguinolanta on Zebrafish (Danio rerio) embryos was determined ⁷.

Procedure for Acute Toxicity Testing in Zebrafish Embryos: Zebrafish were procured from a local supplier and were maintained in 1 ft aquaria with 12 h light: 12 h dark cycle at ±28ºC and fed twice daily with dry flake food. Egg production, collection of embryos were obtained from spawning zebrafish adults in breeding tanks with a sex ratio 3 male: 5 female. Spawning was induced in the morning, and after 2 h, embryos were collected and washed using embryo medium. Fertilized and dead embryos were separated according to the descriptions of Kimmel ⁷.

FIG. 2: EXPOSURE OF ZEBRAFISH EMBRYO TO SPECIFIC CONCENTRATION OF PLANT EXTRACTS

Embryos were divided into six different groups with 10 embryos per group. For each of the three plant extracts Amaranthus viridis, Cynodon dactylon & Aerva sanguinolenta, six different concentration of a solution (100-micron gram/ milliliter, 200-micron gram/milliliter, 300-micron gram/milliliter, 400-micron gram/milliliter, 600-micron gram/milliliter, 800-micron gram/milliliter) with Dimethyl sulfoxide (DMSO, Himedia)  were prepared, and the embryos were exposed to test compounds Fig. 2 for a period of 120 h, at a temperature of 28 ºC and twelve dark and twelve light exposure. Plates were sealed with paraffin to minimize evaporation. Embryo development was monitored after 24 h interval for 120 h. After incubation, the living and dead embryos were placed on glass slides, and morphological characteristics were evaluated using Light Microscope and images obtained with an optical camera Fig.  3, 4, 5, 6.

Experiments were conducted in triplicates. The maximum concentration at which 50% of the Zebrafish embryos were killed was recorded. The percentage of Zebrafish embryos' mortality for every six concentrations of the three test compounds each was recorded after 24, 48, 72, 96, and 120 h. Embryos were scored as dead if the tissues had changed from a transparent to an opaque appearance and if they were no longer moving Fig. 5, 6.

Calculation of LC₅₀: LC₅₀, median lethal concentration (LC₅₀) were statistically analyzed by Graph Pad PrismVersion 6 software. LC₅₀ value is determined based on probit analysis which is a graphical method for calculating the LC50. In this Log dose or Log concentration on the X-axis vs. Probits of mortality on the Y-axis were plotted for a varied range of doses.

Statistical Analysis: Statistical analysis was performed using the SPSS program with one-way ANOVA and Tukey test. A significant difference was considered as p<0.05 - Significant, p<0.001 - Highly significant

RESULT: Graph 1,3,5 reveal the percentage of mortality of Zebrafish embryos treated with 6 different concentrations (100-micron gram/ml, 200-micron gram/ml, 300-micron gram/ml , 400-micron gram/ml, 600-micron gram /ml, 800-micron gram/ml) of Amaranthus viridis (A1), Aerva sanguinolanta (A2) & Cynodon dactylon (A3) plant extracts respectively each at a time interval of 24 hours, 48 h, 72 h, 96 h, and 120 h. For all the three plant extracts the lowest (100µg/ml)  concentration exposure to Zebrafish embryos produced Percentage of mortality of zebrafish embryo 0% after a time interval of 24 h, but after 120 h plant extract A1, A2, A3 produced Percentage of mortality of 20%, 10%, and 0% respectively. For the three plant extracts, mortality rate of embryos increased as their concentrations increased.

For all the three plant extracts, the highest (800µg/ml) concentration exposure to Zebrafish embryos produced Percentage of mortality of zebrafish embryo 100% after a time interval of 120 h, but after 24 h plant extract A1, A2, A3 produced Percentage of mortality of 100%, 80%, and 10% respectively. On comparing the three plant extracts Cynodon dactylon (A3) produced the lowest, Aerva sanguinolanta (A2) produced moderate and Amaranthus viridis (A1), which produced highest mortality rate of embryos at both highest and lowest concentrations and at shortest and longest time durations. Graphs 2, 4, 6 shows median lethal concentration (LC₅₀) of ethanolic whole plant extracts of Amaranthus viridis (A1), Aerva sanguinolanta (A2) & Cynodon dactylon (A3) after 24 h, 48 h, 72 h, 96 h and 120 h time interval. Table 1, 2, 3, 4, 5 shows a comparison of LC50 values at different time intervals in terms of {Mean (SD)} among all the groups using ANOVA test. At a time interval of 24 h, 48 h, 72 h, 96 h and 120 h  there was a statistically significant difference observed between the three groups (p value<0.001). Further using Tukey’s post hoc analysis, significant diffe-rence was observed between all the three groups.

The lower the LC₅₀ value the more toxic the substance. Amaranthus viridis (A1), Aerva sanguinolanta (A2), Cynodon dactylon (A3) have mean LC₅₀ of 316.22080 µg/ml, 575.42800 µg/ml, 35486.000 µg/ml respectively at a time interval of 24 h on Zebrafish embryos. Among the three plant extracts Amaranthus viridis (A1) has the highest, Aerva sanguinolanta (A2) has medium, and Cynodon dactylon (A3) has the lowest toxicity on Zebrafish embryos.

GRAPH 1: PERCENTAGE OF MORTALITY OF ZEBRA FISH EMBRYO FOLLOWING EXPOSURE TO SIX DIFFERENT CONCENTRATIONS OF AMARANTHUS VIRIDIS (A1) AFTER 24 h, 48 h, 72 h, 96 h AND 120 h TIME INTERVAL

GRAPH 2: LC₅₀ OF ETHANOLIC WHOLE PLANT EXTRACTS OF AMARANTHUS VIRIDIS (A1) AFTER 24 h, 48 h, 72 h, 96 h AND 120 h TIME INTERVAL

GRAPH 3: PERCENTAGE OF MORTALITY OF ZEBRA FISH EMBRYO FOLLOWING EXPOSURE TO SIX DIFFERENT CONCENTRATION OF AERVASAN GUINOLENTA (A2) AFTER 24 h, 48 h, 72 h, 96 h AND 120 h TIME INTERVAL

GRAPH 4: LC₅₀ OF ETHANOLIC WHOLE PLANT EXTRACTS OF AERVASAN GUINOLENTA (A2) AFTER 24 h, 48 h, 72 h, 96 h AND 120 h TIME INTERVAL

GRAPH 5: PERCENTAGE OF MORTALITY OF ZEBRA FISH EMBRYO FOLLOWING EXPOSURE TO SIX DIFFERENT CONCENTRATION OF CYNODON DACTYLON (A3) AFTER 24 h, 48 h, 72 h, 96 h AND 120 h TIME INTERVAL

GRAPH 6: LC₅₀ OF ETHANOLIC WHOLE PLANT EXTRACTS OF CYNODON DACTYLON (A3) AFTER 24 h, 48 h, 72 h, 96 h AND 120 h TIME INTERVAL

TABLE 2: COMPARISON OF LC₅₀ VALUES (µgm/ml) AT 24 h TIME INTERVALS IN TERMS OF {MEAN (SD)} AMONG THE THREE GROUPS A1, A2, A3 USING ANOVA TEST (AT 24 h)

(p <0.05 - Significant*, p < 0.001 - Highly significant**)

(Tukey’s post hoc analysis)

TABLE 3: COMPARISON OF LC₅₀ VALUES (µgm/ml) AT 48 h TIME INTERVALS IN TERMS OF {MEAN (SD)} AMONG THE THREE GROUPS A1, A2, A3 USING ANOVA TEST (AT 48 h)

(p <0.05 - Significant*, p < 0.001 - Highly significant**)

(Tukey’s post hoc analysis)

TABLE 4: COMPARISON OF LC₅₀ VALUES (µgm/ml) AT 72 h TIME INTERVALS IN TERMS OF {MEAN (SD)} AMONG THE THREE GROUPS A1, A2, A3 USING ANOVA TEST (AT 72 h)

(p <0.05 - Significant*, p < 0.001 - Highly significant**)

(Tukey’s post hoc analysis)

TABLE 5: COMPARISON OF LC₅₀ VALUES (µgm/ml) AT 96 h TIME INTERVALS IN TERMS OF {MEAN (SD)} AMONG THE THREE GROUPS A1, A2, A3 USING ANOVA TEST (AT 96 h)

(p <0.05 - Significant*, p < 0.001 - Highly significant**)

(Tukey’s post hoc analysis)

TABLE 6: COMPARISON OF LC₅₀ VALUES (µgm/ml) AT 120 h TIME INTERVALS IN TERMS OF {MEAN (SD)} AMONG THE THREE GROUPS A1, A2, A3 USING ANOVA TEST (AT 120 h)

(p <0.05 - Significant*, p < 0.001 - Highly significant**)

(Tukey’s post hoc analysis)

DISCUSSION: Most plant extracts are generally recognized as safe, but many a time it is a misleading perception, and adverse events sometimes occur after consuming herbal products. An adverse effect may cause from several factors including the side effects of active compounds, contamination or substitution with toxic herbs, heavy metal contamination, etc. leading to liver, kidney and lung failure, high blood pressure, heart attack and stroke have been reported ^{8, 9, 10, 11, 12}. Plant phytochemicals have a significant role in the plant's defense mechanism and also important for their unambiguous physiological action in the human body. Bioactive compounds are many times toxic in high doses. Pharmacology is basically toxicology at a lower dose, and toxicology is just pharmacology at a higher dose ¹³.

In the present study, among the three plant extracts Amaranthus viridis ethanolic extract showed the highest toxicity in Zebrafish embryos with a mean LC₅₀ of 316.22080 µg/ml. Arsirapol S et al., found out the 24 h LC₅₀ value of the methanolic extract of Amaranthus viridis on guppy fish to be ca.947mgL(^-1)(r²=0.95). No mortality is observed at higher concentrations for thirty seconds ¹⁴. K.J. Umar et al., found out that feeding the albino rats with 75% spiny Amaranthus leaves decreased their body weight when compared to the control group. The results showed that spiny Amaranthus leaves have a relatively low or no toxicity ¹⁵. In the present study, Amaranthus viridis ethanolic extract showed no mortality at 100µg/ml but showed 100% mortality Zebrafish (Danio rerio) embryos with 800µg/ml when exposed for 24 h.

Similarly, Affy Mataphouet Emmanuel in a toxicological study on rats, found out that the dose 200 and 400 mg/kg of methanolic extract of Amaranthus viridisis safe; in contrast, a dose of 600 mg/kg, revealed signs of toxicity when administered for 12 days ¹⁶. In an earlier study by the present author's qualitative phytochemical evaluation of ethanolic whole plant extract of Amaranthus viridis (A1) revealed the presence of Alkaloids as phytoconstituent ¹⁷. The toxic effect of a high dose of Amaranthus viridis (A1) extract on zebrafish embryo could be attributed to its alkaloid content. Alkaloids are among the largest groups of secondary metabolites and are low-molecular-weight nitrogen-containing compounds. It is widely accepted that the main role of alkaloids in plants is toxicity against predators and pathogens. The same toxic properties observed in the plant defense scenario can often be used in prospection for new drugs ¹⁸.

Among the three plant extracts Aerva sanguinolanta (A2) showed moderate toxicity with a mean LC₅₀ 575.42800 µg/mlat a time interval of 24 hours on Zebrafish (Danio rerio) embryos. A. Mangala Gunatilake et al., in their study, using rat animal model, demonstrated that the administration of dried Aervasan guinolenta at a concentration of 25g/200 ml and 100g/200ml for a period of one month did not have a negative impact on the renal function of rats ¹⁹. B. Kayode S. et al., found out the LD₅₀ of aqueous extract of Aervasan guinolenta on albino rats for oral and intraperitoneal acute toxicity tests were 22.62 g/kg and 0.432 g/kg respectively ²⁰. In the present study Aervasan guinolenta (A2) ethanolic extract caused no mortality at 100µg/ml but showed 80% mortality of Zebrafish (Danio rerio) embryos with 800µg/ml when exposed for 24 h.

Annie Shirwaikar et al., found out that the ethanol extract of the entire plant of Aerva sanguinolenta possesses significant nephroprotective activity and minimal toxicity in cisplatin- and gentamicin-induced acute renal injury in albino rats of either sex ²¹. In an earlier study by the present author, qualitative phytochemical evaluation of ethanolic whole plant extract of Aerva sanguinolanta (A2) revealed presence of Flavonoid as phytoconstituent ¹⁷. Flavonoids are widely-distributed polyphenolic secondary metabolites with diverse biological activities in plants and benefit human health as protective dietary agents ²². Flavonoids have the capacity to act as anti-oxidants ²³. The minimum toxic effect of a high dose of Aerva sanguinolanta (A2) extract on a zebrafish embryo could be attributed to its flavonoid content.

In the present study Cynodon dactylon (A3) ethanolic extract caused no mortality of Zebrafish (Danio rerio) embryos at both 100µg/ml (lowest concentration) and 800 µg/ml (highest concentration) when exposed for 24 h. Heikham Chandani Devi et al., in 2017 studied the hepatoprotective activity of aqueous extract of Cynodon dactylon at 200 mg/kg body weight and 400 mg/kg body weight against paracetamol-induced liver damaged albino rats and found out that there is a significant reduction in the values of SGOT, SGPT, and ALP ²⁴.

Arun K. Yadav, Purobi Nath in an in-vitro assay, tested Cynodondactylon whole plant extract at 10, 20, and 40 mg/ml concentrations against adult Hymenolepis diminuta (hymenolepididae), a zoonotic tapeworm. They found out that the LD₅₀ of the extract was estimated to be greater than 2000 mg/kg ²⁵. Whereas in the present study LC₅₀ value for zebrafish embryo when exposed to ethanolic whole plant extracts of Cynodon dactylon (A3) after 24 h time interval is found to be 35486.000 µg/ml, which is the highest among all the three plant extract thereby with least toxicity on Zebrafish (Danio rerio) embryos. In an earlier study by the present author, qualitative phytochemical evaluation of ethanolic whole plant extract of Cynodon dactylon (A3) revealed the presence of Alkaloids, Flavonoids, Saponin Glycoside, Carbohydrate and Protein as phytoconstituents ¹⁷. The least toxicity induced by the extract to the fish embryos may be attributed to an individual or synergistic action of phytochemicals found in this plant over the period of exposure ²⁶.

Current safety and toxicology testing for preclinical studies comprise of in-vitro and in-vivo models. Typically, these tests are time-consuming, expensive, and required a large number of animals. Potentially, zebrafish (Danio rerio), an emerging model in the early drug discovery and toxicological screening offers several advantages on physiological, biological, and molecular alteration. Its low maintenance, high fertility, ex utero development, and transparent eggs offer clear visualization in all stages of organogenesis monitoring ^{27, 28, 29}.

CONCLUSION: Amaranthus viridis (A1), Aerva sanguinolenta (A2), Cynodon dactylon (A3) have LC₅₀ of 316.22 µg/ml, 575.43 µg/ml, 35481µg/ml respectively at a time interval of 24 h, so a concentration below these concentrations of the respective plant extracts was considered safe to be consumed by Zebrafish embryos. LC₅₀ values of three whole plant ethanolic extract A1, A2, A3 with six different concentrations each on Zebrafish embryos will aid in determining the therapeutic dose of the respective plant extracts.

ACKNOWLEDGEMENT: The authors like to express gratitude to West Bengal University of Health Sciences, Kolkata, Guru Nanak Institute of Dental Sciences and Researches, Kolkata, Guru Nanak Institute of Pharmaceutical Sciences and Research, Kolkata.

CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

REFERENCES:

1. Ugwah-Oguejiofor CJ, Okoli CO and Ugwah MO: Acute and sub-acute toxicity of aqueous extract of aerial parts of Caralluma dalzielii E. Brown in mice and rats. Heliyon. 2019; 5(1): e01179.
2. Ismail HF, Hashim Z, Soon WT, Rahman NSA, Zainudin AN and Majid FA: A comparative study of herbal plants on the phenolic and flavonoid content, antioxidant activities and toxicity on cells and zebra fish embryo. Journal of Traditional and Complementary Medicine 2017; 7: 452-65.
3. Lieschke GJ and Currie PD: Animal models of human disease: zebrafish swim into view. Nat Rev Genet 2007; 8: 353-67.
4. Westerfield M: The Zebrafish Book: A Guide for the Laboratory use of Zebrafish (Danio Rerio); University of Oregon Press 1995.
5. Nusslein-Volhard C and Dahm R: Zebrafish: Practical Approach (the Practical Approach Series); Oxford University Press 2002.
6. Median lethal dose. Wikipedia, the free encyclopedia. 21 July 2020, at 08:31 (UTC). Available at: https://en. wikipedia.org/wiki/Median_lethal_dose.Accesssed August 13,2020
7. OECD Guidelines for The Testing of Chemicals: Fish Embryo Acute Toxicity (FET) Test. 236 Adopted: 26 July 2013
8. Al-Nuaimi AAHD: Extracts of plants used as traditional medicines have toxic effect on the liver and kidney. MOJ Anat Physiol 2018; 5(1): 32–41.
9. Neuman MG, Cohen L, Opris M, Nanau RM and Hyunjin J: Hepatotoxicity of pyrrolizidine alkaloids. J Pharm Pharm Sci 2015; 18: 825-43.
10. Seremet OC: Toxicity of plant extracts containing pyrrolizidine alkaloids using alternative invertebrate models.Molecular Medicinal Report 2018; 17(60): 7757-63.
11. Phua DH, Zosel A and Heard K: Dietary supplements and herbal medicine toxicities when to anticipate them and how to manage them. Int J Emerg Med 2009; 2(2): 69e76.
12. Asif M: A brief study of toxic effects of some medicinal herbs on kidney. Adv Biomed Res 2012; 1: 44.
13. Saleh-e-In, Sultana N, Hossain MN, Hasan S and Islam MR: Pharmacological effects of the phytochemicals of Anethum sowa Root extracts. BMC Complementary and Alternative Medicine 2016; 16: 464.
14. Arsirapoj S, Sudthonghong C and Bullangpoti V: Acute toxicity of Amanranthus viridus extract on guppies, Poecilia reticulata. Commun Agric Appl Biol Sci 2010; 75(2): 199-202.
15. Umar KJ, Hassan LJ, Dangoggo SM, Maigandi SA, Muhammad S and Sani NA: Toxicity studies of Amaranth viridis Linn (Spiny Amaranth) using albino rats. IJSBAR 2015; 20(1): 368-77.
16. Emmanuel AM, Nadia BA, Amadou CF and KoffiK: Evaluation of acute and subacute toxicity induced by methanol extract of Amaranthus viridis (Amaranthaceae) leaves in wistar rats. The Pharma Innovation Journal 2018; 7(7): 625-30
17. Zahir S, Pal TK, Sengupta A, Biswas S and Bar S: Phytochemical screening and antibacterial activities of Amaranthus viridis, Cynodon dactylon & Aervasan guinolenta. J Indian Med Assoc 2018; 116: 27-30.
18. Matsuura H and Fett-Neto AG: Plant Alkaloids: Main Features, Toxicity, and Mechanisms of Action. Plant Toxins 2015. DOI 10.1007/978-94-007-6728-7_2-1
19. Shirwaikar A, Issac D and Malini S: Effect of Aervalanata on cisplatin and gentamicin models of acute renal failure. Journal of Ethnopharmacology 2004; 90(1): 81-86.
20. Gunatilake M, Lokuhetty MD, Bartholameuz NA, Edirisuriye DT, Kularatne MU and Date A: Aervalanata (Polpala): Its effects on the structure and function of the urinary tract. Pharmacognosy Res 2012; 4(4): 181-88.
21. Omotoso KS, Aigbe FR, Salako OA, Chijioke MC and Adeyemi OO: Toxicological evaluation of the aqueous whole plant extract of Aerva lanata (l.) Juss. ex Schult (Amaranthaceae). J Ethnopharmacol 2017; 208: 174-84.
22. Khalid M, Rahman S, Bilal M and Dan-feng H: Role of flavonoids in plant interactions with the environment and against human pathogens - A review. Journal of Integrative Agriculture 2019; 18(1): 211-30.
23. Panche AN, Diwan AD and Chandra SR: Flavonoids: an overview. J Nutr Sci 2016; 5: e47.
24. Devi HC, Moirangth RS, Vikram SS, Devi KS, Devi KP and Rita S: Hepatoprotective activity of aqueous extract of Cynodon dactylon on paracetamol induced hepatotoxic albino rats. Sch J App Med Sci 2017; 5(1C): 199-204.
25. Yadav AK and Nath P: Anthelmintic effects and toxicity of Cynodon dactylon (L.) Pers. in rodent models. J Intercult Ethnopharmacol 2017; 6(4): 407-12.
26. de Araújo Pinho FVS: Phytochemical Constituents and Toxicity of Duguetia furfuracea hydroalcoholic extract in Drosophila melanogaster. Evidence–based Complementary and Alternative Medicine 2014; (ID 838101): 11.
27. Hoo JY, Kumari Y, Shaikh MF, Hue SM and Goh BH: Zebrafish: A Versatile Animal Model for Fertility Research. Biomed Res Int 2016; 9732780.
28. Sarmah S and Marrs JA: Zebrafish as a Vertebrate Model System to Evaluate Effects of Environmental Toxicants on Cardiac Development and Function. Int J Mol Sci 2016; 17(12): 2123.
29. Reynolds M: A Toxicological Study using Zebrafish (Daniorerio) as a Model. The Journal of Toxicological Education 2013; 1: 10‐20.
    

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    How to cite this article:

    Zahir S, Pal TK, Sengupta A, Biswas S, Bar S and Bose S: Determination of lethal concentration fifty (LC₅₀) of whole plant ethanolic extract of Amaranthus viridis, Cynodon dactylon & Aerva sanguinolenta on Zebrafish (Danio rerio) embryos. Int J Pharm Sci & Res 2021; 12(4): 2394-04. doi: 10.13040/IJPSR.0975-8232.12(4).2394-04.

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