toxicological assessment using brine shrimp lethality ......in brine shrimp lethality bioassay,...

Toxicological Assessment using Brine Shrimp Lethality Assay and

Antimicrobial activity of Capparis Grandis

Vaibhav S. Suryawanshi*, Akshay R. Yadav, Dr. Shrinivas K. Mohite,

Dr. Chandrakant S. Magdum

Department of Pharmaceutical Chemistry, Rajarambapu College of Pharmacy, Kasegaon, Dist.

Sangli, Maharashtra, India-415404

ABSTRACT

Medicinal plants constitutes an important component of flora and are widely distributed in India.

The pharmacological evaluation of substances from plants is an established method for the

identification of lead compounds which can leads to the development of novel and safe medicinal

agents. Based on the ethnopharmacological literature, several species of medicinal plants used

in traditional medicine in india were collected. In the present study, Capparis grandis extracts

were screened for cytotoxicity using brine shrimp lethality test. The present studies widen the

scope of the brine shrimp model that may prove quite helpful as a preliminary screen to

determine toxic properties. In Brine shrimp lethality bioassay, compounds produced dose

dependent cytotoxicity effect to brine shrimp nauplii and Capparis grandis extract were screened

for antimicrobial activity, ethanolic and methanolic extracts shows significant activity.

Keywords: Capparis grandis, Brine shrimp lethality test, cytotoxicity, Antimicrobial activity.

1. INTRODUCTION

Capparis grandis Russ.ex Wall (Capparidaceae) locally known as pachunda. It is small tree up to

10 m tall, distributed from Rajasthan southwards to Karnataka. Infusions of leaves are used

intemally for swelling and eruptions1. Capparis plant species are used since long time ago in

ayurveda and unani for their ethnomedicinal potential. Traditionally used of Cappris speices for

various ailments like arthritis anemia, dropsy, and gout. And also the fruit of Capparis Species

have good nutritional values due the fruits are used in making pickle and curry2. Capparis species

Journal of University of Shanghai for Science and Technology ISSN: 1007-6735

Volume 22, Issue 11, November - 2020 Page-746

contain an enormous amount of the antimicrobial and anti-oxidant activity against bacterial

pathogens and new research suggest a possible use of Capparis plant species as a source of

natural antioxidant and antimicrobial agents. Plants and their products are being used as a source

of medicine since long3. According to World Health Organization (WHO) more than 80% of the

world’s population, mostly in poor and less developed countries depend on traditional plant

based medicines for their primary healthcare needs. India is richly endowed with a wide variety

of plants having medicinal value. These plants are widely used by all sections of the society

either directly as folk remedies or indirectly as pharmaceutical preparation of modern medicine4-

5. Microwave extraction has proved to be more effective and efficient than its conventional

counterpart i.e, Soxhlet extraction method. Extraction systems are used to conduct routine

solvent extractions of soils, sediments, sludge, polymers and plastics, pulp and paper, biological

tissues, textiles and food samples6-10

. Experiments have proved that microwaves, in comparison

with the Soxhlet extraction, use a lesser volume of solvent and sample and perform extraction at

a much faster rate. Growth of green chemistry holds necessary potential for the reduction of by

product, a reduction in the waste production and a lowering of energy costs11-15

. Due to its ability

to couple directly with reaction molecule and passing thermal conductivity leading to fast rise in

the temperature microwave irradiation had used to improve many extraction techiques. The

Principle behind the heating in microwave oven is because interaction of charged particle of

reaction material with electromagnetic wavelength of particular frequency. The phenomena of

the producing heat by electromagnetic irradiation are either by conduction or collision16-18

. In

order to study the toxicity of these medicinal plants we performed brine shrimp lethality bioassay

which based on the ability to kill laboratory cultured brine shrimp (Artemia nauplii). The brine

shrimp assay was proposed by Michael et al., and latter developed by Vanhaecke et al., Sleet and

Brendel19-20

. The assay is considered a useful tool for preliminary assessment of toxicity and it

has been used for the detection of fungal toxins, plant extract toxicity, heavy metals, pesticides

and cytotoxicity testing of dental materials. The brine shrimp assay is very useful tool for the

isolation of bioactive compounds from plant extracts21-22

. The method is attractive because it is

very simple, inexpensive and low toxin amounts are sufficient to perform the test in the

microwell scale23-25

. Infectious disease are the world’s leading cause of premature deaths, killing

almost 50,000 people every day. Morbidity and mortality due to diarrhea continues to be a major

problem in many developing countries, specially amongst children. Infections due to variety of



bacterial etiologic agents such as pathogenic Escherichia coli and Staphylococcus aureus are

most common. In recent years drug resistance to human pathogenic bacteria has been commonly

reported from all over the world. With the continuous use of antibiotics microorganism have

become resistant26-27

. Addition to this problem, antibiotics are sometimes associated with adverse

effects on host which include hypersensitivity, immunosuppressant and allergic reactions This

has created immense clinical problems in the treatment of infectious diseases. Therefore, there is

a need to develop alternative antimicrobial drugs for the treatment of infectious diseases; one

approach is to screen local medicinal plants for possible antimicrobial properties. Plant materials

remain an important recourse to combat serious diseases in the world. According to WHO

(1993), 80% of the world’s population is dependent on the traditional medicine and a major part

of the traditional therapies involves the use of plant extracts or their active constituents28-29

. Yet a

scientific study of plants to determine their antimicrobial active compounds is a comparatively

new field. The traditional medicinal methods, specially the use of medicinal plants, still play a

vital role to cover the basic health needs in the developing countries. Since ancient times, herbs

and their essential oils have been known for their varying degrees of antimicrobial activity. In

recent times, the search for potent antibacterial agents has been shifted to plants. Most plants are

medicinally useful in treating disease in the body and in most of cases the antimicrobial efficacy

value attributed to some plants is beyond belief. Conservative estimates suggest that about 10%

of all flowering plants on earth have at one time, been used by local communities throughout the

world but only 1% have gained recognition by modern scientists. There are about 120 plant-

based drugs prescribed worldwide and they come from just 95 plant species. Approximately

250,000 species of flowering plants and only 5000 have had their pharmaceutical potential

assessed. The treatment of infectious diseases with antimicrobial agents continues to present

problems in modern-day medicine with many studies showing a significant increase in the

incidence of bacterial resistance to several antibiotics. Due to increased resistance of many

microorganisms towards established antibiotics, investigation of the chemical compounds within

traditional plants has become desirable30-32

. Due to failure of ADME so it necessary to perform

docking studies before pharmacological activity. An outbreak of coronavirus disease (COVID-

19) caused by the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) raises

an unparalleled challenge in the discovery of appropriate drugs for prevention and treatment33-37

.

Given the rapid pace of scientific research and clinical data produced by the large number of



people quickly infected with SARS-CoV-2, clinicians need reliable proof of successful medical

care for this infection as in intial stage with help of molecular docking software it is easy to do

research. The chemical modification of drug delivery system for protein and peptide drugs is

important in improving both enzymatic stability and membrane permeations can help to have

good biological activity from any compound modification. Someday soon, you might be making

your own medicines at home. That’s because researchers have tailored a 3D printer to synthesize

pharmaceuticals and other chemicals from simple, widely available starting compounds from

phytoconstituents. Chemotherapy is unable to obtain clinical responses in patients with highly

invasive metastatic disease. Several limitations are reported with pharmacotherapy using, among

them, drug resistance and risk of toxicities are the most important ones. Therefore, there is an

essential need for more effective approaches to treatment of microbial infections38-47

.

2. MATERIALS AND METHODS

Collection of plant material

Stem of Capparis grandis were collected from the residential areas of Vita, Sangli, Maharashtra,

India and authenticated from the Department of Botany, Y.C. College of Science, Karad.

Preparation of plant extract

Shade drying was done for almost a month as to avoid chemical degradation due to sunlight.

Grinding of the dried material was done, with the aid of a grinder and converted into coarse

powder. Extraction of Capparis grandis was done by microwave extraction further filtered and

excess solvent present was evaporated and dried extract were collected and subjected for further

studies.

Biological Evaluation:

Brine Shrimp Lethality Assay:

Brine shrimp lethality test has been used as a bioassay for a variety of toxic substances. A

general bioassay that appears capable of detecting a broad spectrum of bioactivity, present in

extracts, rather than more tedious and expensive in-vitro and in-vivo antitumor assays.

Furthermore, it does not require animal serum as is needed for cytotoxicity.



Procedure

Preparation of seawater

38 g sea salt (without iodine) was weighed, dissolved in one liter of distilled water and filtered

off to get clear solution.

Hatching of brine shrimp

Artemia salina 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 side. 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 nauplii was taken carefully by micropipette.

Preparation of test samples

In each experiment, 0.5 mL of test compound of different concentration i.e (50, 100 and 150

μg/mL) was added to brine solution and maintained at room temperature for 24 hr under the light

and surviving larvae were counted after 24hr. Vehicle treated used as control for the test. Test

solutions were used in sets of three tubes per dose. Replicas should be maintained to get accurate

results. The effectiveness or the concentration-mortality relationship of test compounds is usually

expressed as a LC5049-50

.

Antimicrobial Activity:

Preparation of Inoculum

The gram positive (Bacillus subtilis and Staphylococcus aureus) and gram negative bacteria

(Escherichia coli) were pre-cultured in nutrient broth overnight in a rotary shaker at 37°C,

centrifuged at 10,000 rpm for 6 min, pellet was suspended in double distilled water and the cell

density was standardized spectrophotometrically (A nm). The fungal inoculums (Candida

albicans, Aspergillus niger, and Dreschlera turcica) were prepared from 6 to 10 day old culture

grown on Potato dextrose agar medium. The Petri dishes were flooded with 9 to 11 ml of



distilled water and the conidia were scraped using sterile spatula. The spore density of each

fungus was adjusted with spectrophotometer (A595 nm) to obtain a final concentration of

approximately 105 spores/ml.

Antibacterial testing

Antibacterial activity was measured using agar dilution technique. Briefly, the methanol extracts

were dissolved in dimethyl sulfoxide (DMSO, Merck) and serially diluted in molten Mueller

Hinton Agar (MHA, Sigma) in petri dishes (100 mm×15 mm) to obtain final concentrations:

100, 50, 25 and 12.5 μg/ml. The solvent did not exceed 1% concentration and did not affect the

growth of the organisms. All bacterial strains were grown in Mueller Hinton Broth (MHB,

Sigma) for 4 h at 37°C. Bacterial suspensions with 0.5 McFarland standard turbidity, which is

equivalent to 108 cfu/ml were prepared by dilution with Mueller Hinton broth. The diluted

inoculum was added to a Steer’s replicator calibrated and incubated for 24 hr at 37 °C. After

incubation, all dishes were observed for microbial inhibition by the disc diffusion method

Streptomycin sulphate (10 μg/mlG) used as positive control and methanol solvent (100 μg/mlG)

used as negative control. The antibacterial assay plates were incubated at 37°C for 24 hr. The

diameters of the inhibition zones were measured in mm.

Antifungal Activity

The antifungal activity was tested by disc diffusion method. The potato dextrose agar plates were

inoculated with each fungal culture (10 days old) by point inoculation. The filter paper discs (5

mm in diameter) impregnated with 100 μg mlG concentrations of the extracts were placed on test

organism-seeded plates. Methanol was used to dissolve the extract and was completely

evaporated before application on test organism-seeded plates. Blank disc impregnated with

solvent methanol followed by drying off was used as negative control and Nystatin (10 μg discG)

used as positive control. The activity was determined after 72 hr of incubation at 28°C. The

diameters of the inhibition zones were measured in mm.

Preparation of test samples

For the antimicrobial tests, ethanolic extracts were diluted in dimethylsulfoxide (DMSO):

methanol (1/1: v/v) solvent to a concentration of 20 mg/ml.



Antimicrobial bioassay

For bioassays, suspension of approximately 1.5 × 108 bacterial cells/ml in sterile normal saline

were prepared and about 1.5 ml of it was uniformly seeded on Mueller-Hinton-Agar medium

with 3–4 mm thickness in 12 cm × 1.2 cm glass petridishes, left aside for 15 min and excess of

suspension was then drained and discarded properly. Wells of 6 mm in diameter and about 2 cm

apart were punctured in the culture media using sterile cork borers. Wells were filled with 0.1 ml

of 20 mg/ml concentration of each sample (2 mg/well) and incubated at 37 °C for 48 hr.

Bioactivity was determined by measuring Diameter of Inhibition Zones (DIZ) in mm. Each

experiment was repeated three times and the mean of the diameter of the inhibition zones was

calculated. Pure solvent was used as negative control51-54

.

3. RESULTS AND DISCUSSION

Brine Shrimp Lethality Assay

The lethality of a test sample in a simple zoological organism such as the shrimp (Artemia

salina) has been utilized in the Brine shrimp cytotoxicity test (BSCT). It is a very useful tool to

screen a wide range of chemical compounds for their various bioactivities. It has been

demonstrated that BSCT correlates reasonably well with cytotoxic and other biological

properties. The brine shrimp bioassay has been established as a safe, practical and economic

method for determination of bioactivities of synthetic compound as well as plant products. The

brine shrimp lethality bioassay also indicates antifungal effects, pesticidal effects, teratogenic

effects, toxicity to environment and many more. Table 1 shows the lethality of different test

sample to the brine shrimp nauplii. All the extracts of Capparis grandis were tested for

cytotoxic activity by the brine shrimp lethality assay. Among them compounds methanolic and

ethanolic extract showed a dose dependent cytotoxic activity at concentrations of 1.72 µg/ml and

1.28 µg/ml. The remaining extracts exhibited less activity when compared to the other

compounds at various concentration levels.



Table 1. Brine shrimp lethality assay data of Capparis grandis extracts

Sr. no Extracts LC50 (µg/ml)

1 Methanol 1.72

2 Petroleum ether 5.79

3 Ethanol 1.28

4 Chloroform 8.45

Antimicrobial Activity

The antimicrobial activity of plant parts from four medicinal plant species has been evaluated in

vitro against pathogens including three bacterial species (Escherichia coli, Bacillus subtilis and

Staphylococcus aureus) and three fungus species (Candida albicans, Aspergillus niger, and

Dreschlera turcica). Table 2 illustrated that ethanolic and methanolic extract showed

antimicrobial activity against all test microorganisms. It was observed that ethanolic extracts

exhibited the highest significant antibacterial activity against E. coli with mean inhibition zone

equal to 23±0.04 and methanolic extract exhibited the highest significant activity against C.

albicans with mean inhibition zone equal to 21±1.67 mm, respectively.



Table 2. Antibacterial activity of Caparis grandis extracts (100 μg mlG1) and antibiotic (10 μg

mlG1) against bacterial species tested by disc diffusion assay Zone of inhibition (mm)

Sr.

no

Extract Diameter of zone of inhibition (mm)

E. coli B. subtilis S.aureus

1 Methanol 19±1.58 16±0.02 18±0.22

2 Petroleum

ether

17±0.31 13±1.08 11±0.42

3 Ethanol 23±0.04 18 ±0.91 22±0.10

4 Chloroform 20±0.82 15±0.02 16±1.33

Table 3. Antifungal activity of Caparis grandis extracts (100 μg mlG1) and fungicide (10 μg

mlG1) against fungal species tested by disc diffusion Zone of inhibition (mm)

Sr.

no

Extract Diameter of zone of inhibition (mm)

C. albicans A. niger D. turcica

1 Methanol 21±1.67 19±0.28 20±2.44

2 Petroleum

ether

12±0.41 10±3.51 13±0.27

3 Ethanol 18±2.56 16 ±1.72 15±3.22

4 Chloroform 10±2.64 9±3.12 11±0.29

4. CONCLUSION

Brine shrimp lethality bioassay is considered as a useful tool for the preliminary assessment of

toxicity. Toxicity of the Caparis grandis extracts was evaluated using artemia salina nauplii and

cytotoxicity was inferred based on the same brine shrimp lethality assay. Although the brine

shrimp lethality bioassay is an excellent choice to elementary toxicity investigations it was



concluded that extract shows dose dependent concentrations. The current study was initiated

because of the increasing resistance to antibiotics including bacteria and fungi. Plant extracts and

compounds are of new interest as antiseptics and antimicrobial agents. As a result, the

antimicrobial activity of different Caparis grandis extracts was screened against the most

common pathogens. In general, ethanolic and methanolic extracts of Caparis grandis extracts to

be effective source of active antimicrobial agents.

5. ACKNOWLEDGEMENT

We are very thankful to the vice principal Dr. S. K. Mohite and principal Dr. C. S. Magdum for

their guidance, motivation and providing all necessary requirements to carry out this study.

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mouthwash against oral pathogens. Asian J. Res. Pharm. Sci. 2020; 10(3): 149-152.



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