Toxicоlogical Evaluation of Psidium guajava Leaf Extracts using Brine Shrimp (Artemia salina L.) Model

 

Akshay R. Yadav*, Shrinivas K. Mohite

Department of Pharmaceutical Chemistry, Rajarambapu College of Pharmacy, Kasegaon, Maharashtra,

India - 415404.

 

ABSTRACT:

The search for new plant-derived drugs has gained renewed interest among researchers worldwide in the hunt for new drugs that have the potential to combat the threat of drug-resistant pathogenic microorganisms, antitumor and anticancer agents. Plants can be useful in either their crude or advanced forms, offering their pure state a source of drugs. Brine Shrimp Lethality Assay is the most convenient system to monitor the biological activity of different species of plants. This approach is very useful for the toxicity evaluation of plant extracts in advance. This assay has many advantages: rapidity, simplicity and low requirements. However, several requirements need to be fulfilled, especially with standardized experimental conditions (temperature, medium pH, salinity, aeration, and light). Psidiumguajava leaves have long been in use in the Indian Local Medicine System for their antioxidant and anti-inflammatory properties. The toxicity of Psidiumguajava leaf extracts using this assay was determined within a concentration range of 1mg/ml, 100μg/ml, 10μg/ml and 1μg/ml of the herbal extract being tested. After 24 hours of exposure to the studied sample, most toxicity studies using the Brine Shrimp Lethality Assay assess toxicity. The Brine Shrimp Lethality Assay is an excellent predictive tool for the toxic potential of plant extracts in humans.

 

 

 

INTRODUCTION:

Medicinal plants are an important component of flora and are widely distributed in India. Pharmacological assessment of plant substances is an established method for the identification of lead compounds which may lead to the development of novel and safe medicinal products¹. Psidiumguajava is a small evergreen tree (Myrtaceae), commonly known as guava in English, an important food crop and medicinal plant native to South America, grown in tropical and subtropical lands and also found in India². Now-a-days brine shrimp (Artemiasalina, fairy shrimp or sea monkeys) lethality assay is commonly used to check the bioactive chemicals cytotoxic effect³. This is a preliminary screening of plant extracts for toxicity. Subsequently animal model for establishment is recommended⁴. Other top assays at the bench are inhibition of crown gall tumors on potato tuber disks, frond proliferation inhibition in duckweed and yellow fever larvae lethality test⁵. Between them, the lethality test for brine shrimps is the shortest, low cost and effective one. The larvae (nauplii; singular nauplius), about 22mm long, are large enough to observe in a laboratory without high magnification and small enough to hatch in vast amounts without extensive workspace⁶. This is a rapid and thorough test for bioactive compounds of either natural or synthetic origin. It is also a cheap and simple test, since no aseptic techniques are required⁷. It easily uses a large number of species for statistical testing and needs no special equipment, and needs fairly low sample amounts (2-20 mg or less)⁸. This in vivo test has been employed successively since its introduction for the bioassay-guide fractionation of active cytotoxic and antitumor agents. Furthermore, several studies have shown that there is a strong link between the results for the lethal concentration that kills 50 per cent of the exposed population (LC50) obtained using Artemiasalina with the Brine Shrimp Lethality Assay⁹. Toxicity is an expression of being poisonous, indicating the state of adverse effects led by the toxicant/cell interaction. This interaction can differ depending on the toxic chemical properties and the cell membrane, as it can occur on the cell surface, in the cell body, or in the tissues below, as well as in the extracellular matrix. The toxic effects may take place prior to the binding of the toxicants to the vital organs such as liver and kidneys^10-11. Evaluation of a substance's toxic properties is also important when thinking for the safety of public health, since exposure to chemicals can be dangerous and can result in adverse human effects. In practice, there are typically acute, subchronic, chronic and carcinogenic effects in the evaluation. In the present analysis of Psidiumguajavaleaf extracts for the brine shrimp lethality test, to determine its toxic properties¹².

 

MATERIALS AND METHODS:

Plant material:

Psidiumguajavahas been collected from Karad, Maharashtra, India. Department of Botony, YashwantraoChavan College of Science, Karad has identified the plant and authenticated it.

 

Preparation of Psidiumguajavaleaf powder:

Fresh guava leaves were collected and air dried for 10 days. The dried leaves were then crushed into a blender and churned to form a coarse powder. The powder was collected in an air-tight jar, and stored away from sunlight in a cool, dry place.

 

Preparation of plant extract:

Extraction of malvastrumcoromandelianum was done by microwave extraction further filtered andexcess solvent present was evaporated and dried extract were collected and subjected for activity studies.

 

Preparation of seawater:

38gm sea salt (without iodine) was weighed, dissolved in one liter of distilled water and filtered off to get clear solution.

 

Hatching of Brine Shrimp:

Artemiasalina leach (brine shrimp eggs) collected from pet shops was used as the test organism. Seawater was taken in the small tank, and shrimp eggs were moved to one side of the tank, and sealed on this side¹³. The shrimp was allowed to hatch for two days and be matured like nauplii. Constant supply of oxygen was rendered during the process of hatching. The hatched shrimps are drawn to the light (phototaxis), and so egg shell-free nauplii from the illuminated portion of the tank was collected. The nauplii was taken by a pipette from the fish tank and filtered to improve visibility in fresh clear sea water, and 10 naupliiwas taken carefully by micropipette¹⁴.

 

Preparation of Reagents:

Serial dilution of extract:

Clean test tubes have been taken and labelled. An analytical balance was measured against plant extract of 10mg. Dissolving 10mg of plant extract (soluble in water) in 1ml of water then prepared stock solution. Concentrations of 1 mg/ml, 100μg/ml, 10μg/ml, and 1μg/ml were prepared from stock solution using serial dilution. Then 1 ml of prepared solution was taken into the test tubes which contain 10 nauplii and 1 ml of seawater. After 24 hours the number of dead nauplii was counted^15-16.

 

CALCULATIONS:

The mortality endpoint of this bioassay is defined as the absence of controlled forward motion during 30 seconds of observation. The percentage of nauplii lethality for each concentration was calculated. For each tube, count the number of dead and the number of live nauplii, and determine the % death^17-18.

 

 

RESULTS AND DISCUSSION:

Different extracts of Psidiumguajava used for the cytotoxicity test with help of brine shrimp. It is very useful for BSLA to screen a wide range of extract for their different bioactivities. From results it wasfound that the ethyl acetate extract shows less % percent death of nauplii in different concentrations compared to the other extracts.

Table 1: Results of Brine shrimp lethality assay of Psidiumguajava extracts

Sr. No	Extract	% death nauplii
		1mg/ml	100 µg/ml	10µg/ml	1µg/ml
1	Methanolic extract	100	50	40	20
2	Ethyl acetate extract	80	30	20	10
3	Ethanolic extract	100	70	60	50
4	Aqueous extract	100	60	50	30

 

CONCLUSION:

Many plants which have been described as curative may also have harmful effects. The concentration of a substance is the most important determinant of the outcome: it could lead to toxic effects if it reaches a sufficiently high concentration in the susceptible biological system. Although the saline shrimp lethality assay is somewhat inadequate in terms of the elucidation of the mechanism of action, it is very useful to assess the bioactivity of plant extracts. In fact, in the course of our studies, the saline shrimp lethality assay proved to be a convenient system for monitoring the biological activities of a number of plant species used in traditional medicine. It was of great need to involve in vivo testing using animal model in order to gain relevant data which can be extrapolated to human population. Researchers have used rats and other animal models for years that exhibit a high correlation with the human population. This type of testing has recently been restricted due to ethical and economic considerations. Alternative toxicity assays are therefore widely used to test the potential for toxicity of plant products. Brine Shrimp Lethality Assay seemed a good approach, particularly since it could still be classified as in vivo testing. Artemiasalinanauplii is one of the alternatives for herbal extracts biological toxicity assays and this test has proved to be significantly correlated with several other animal models. The preliminary toxicity data obtained through the Brine shrimp lethality assay provides a convenient platform for further study of toxicity.

 

ACKNOWLEDGEMENT:

I express my sincere thanks to Vice-principal Prof. Dr. S. K. Mohite for providing me all necessary facilities and valuable guidance extended to me.

 

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Received on 02.06.2020         Modified on 07.07.2020

Accepted on 21.07.2020       ©A&V Publications All right reserved