Brine shrimp (Artemia salina) lethality assay of Indian Heliotrope (Heliotropium

indicum) stem decoction

A Research Proposal
Presented to the Faculty of the
Department of Research
Gullas college of medicine
University of the Visayas, Banilad, Cebu City
Philippines

IN PARTIAL FULFILLMENT OF THE REQUIREMENT IN

DOCTOR OF MEDICINE

By:-
GROUP 35-2B
CHAUHAN KAMIL
JAGTAP SHARDUL
KODIMELA HIMAJA
MISHRA UTTAM KUMAR
SINGHVI HARSHWARDHAN
THOTA HARITHA SAI

SHELBAY G.BLANCO, MD, MPH

ADVISER

FEB 2020
 TABLE OF CONTENTS

TITLE PAGE i

APPROVAL SHEET ii

ACKNOWLEDGEMENT iii

TABLE OF CONTENT iv

LIST OF FIGURES v

LIST OF TABLES vi

CHAPTER

I INTRODUCTION 1

Rationale 1

Objectives of the study 2

Statement of the hypothesis 2

Significance of the study 2

Scope and limitations 3

Definition of terms 3

II REVIEW OF RELATED LITERATURE 4

Heliotropium indicum 4

Botanical morphology 6

Total plant 8

Artemia salina 9
 Species ecology 9

Life cycle 9

Artemia salina lethality assay 10

Lethality test on other plants 11

Toxicity and other tests involving H.indicum 12

Conceptual framework 13

III METHODOLOGY 14

Research design 14

Research environment 14

Research subject 14

Research instrument 14

Research procedure 14

Preparation of plant decoction 14

Data processing and analysis 15

Ethical considerations 16

References 17

APPENDIXS

APPENDIX A LETTER TO DEAN

APPENDIX B LETTER TO CHAIRPERSON

APPENDIX C GANTT CHART

APPENDIX D BUDGET

CURRICULUM VITAE
 LIST OF FIGURES

Figure 1. Conceptual frame work

Figure 2. Map of research environment
 LIST OF TABLE

Table1. The modalities of Artemia salina reproduction

 APPROVAL SHEET

The research study entitled Brine shrimp (Artemia salina) lethality assay of Indian He-
liotrope (Heliotropium indicum) stem decoction prepared and submitted by CHAUHAN
KAMIL, JAGTAP SHARDUL, KODIMELA HIMAJA, MISHRA UTTAM KUMAR,
SINGHVI HARSHWARDHAN, THOTA HARITHA SAI in partial fulfilment of the require-
ments in the Degree of Doctor of Medicine are hereby recommended for acceptance and ap-
proval.

RECOMMENDED FOR APPROVAL:

_________________________

Chairman

__________________ __________________

__________________ __________________

Panellists

Approved by the Committee on Oral Examination with a grade of __________ on Febru-

ary____, 2020.

ACCEPTED and APPROVED in partial fulfilment of the requirements in the Degree of Doctor
of Medicine.

Dr. Nino Ismael S. Pastor, MD, PHSAE, DRDM, FPSMID Dean, Gullas College of Medicine
 ACKNOWLEDGEMENT

The researchers wish to extend their sincere gratitude and appreciation for giving precious contri-

butions towards the completion of this research proposal To our loving parents, for their moral and

financial support and providing the space to work with.

To the college librarian, for allowing us to make use of the available references to understand and

interpret the topic which made us finish this research work.

To our Research instructor, Dr.SHELBAY G.BLANCO for guiding us throughout our research

work. Also, for checking our problems computation And most especially to God, for giving us the

guidance and knowledge as well as good health in constructing this research work.

To God be the Glory.

 CHAPTER I 1

INTRODUCTION

Rationale In many developing/developed countries, a huge proportion of the population depends

vastly on traditional doctors (vaidyas) and medicinal plants to meet the basic health care needs. Al-

though allopathic medicines may be available in these countries, herbal medicines (phytomedi-

cines) have often been in popularity for historical, cultural, religious, and spiritual reasons. Today,

many citizens of developed countries have started trusting on alternative or complementary thera-

pies. Heliotropium indicum (H.indica) commonly known as the Indian heliotrope belongs to the

family Boraginaceae and is a native to India and Africa and has a long history of traditional medi-

cinal uses. Traditional folklore states that it has previously been used for curing various skin di-

seases, poison bites, stomachache, and nervous disorders. Whereas, advanced researches suggest

that it may also be helpful for the treatment of malaria, abdominal pain, and diseases like dermati-

tis. In different countries, different parts of H. indica are utilised by the locals to cure different

conditions. In the Philippines, decoction of dried roots is used to stimulate menstrual flow. In

Mexico, decoction of roots or any plant part used for asthma. In India, juice of leaves is used for

treating sores and insect bites. In Costa Rica, leaves are crushed and used as resolvent for abscesses

and furuncles. In other countries, H. indica are used for vomiting, amenorrhea, high blood pres-

sure. Stem decoction is also used for thrush; poultices used for herpes and rheumatism. With the

wide use of H. indica, the negative effects of this plant are still being studied. The researchers will

be focusing on its toxicity by evaluation of the plant stem decoction.

The researchers will use Artemia salina for determining the toxicity of H.indica as a simple

bioassay for natural product research which determines values of LC50 (lethal concentration) of

plant stem decoction in presence of A.salina . The researchers will use this method because it is

reliable, rapid and inexpensive.

 2
Objectives of the study

General This study generally aims to determine the toxicity of H.indica stem decoction us-

ing the lethality bioassay.

Specific aims :

1. Determine mortality after 6 and 24 hours in different concentrations and the control solu-

tions

2. Determine the concentration which rendered 50% mortality (LC50) using statistical meth-

ods

3. Perform one-way analysis of variance (ANOVA) to determine the statistical significance of

the mean mortality at each concentration

Statement of the hypothesis

The concentration which rendered 50% mortality (LC50) is expected to be observed in the

test tube with the highest concentration of stem decoction.

Significance of the study

Department of health.

1. The research will prove to be a preliminary tool for assessment of toxicity

2. screening for pharmacological activities in plants and natural marine products.

3. Based on the property of cytotoxicity, various plant extracts can be used for medicinal

purposes, thus advancing the pharmacological aspects of the society.

 3

Household/domestic purposes.

1. The cytotoxic properties of the plant are known, the extracts can be used in safer amounts

in several household or domestic products like soaps and disinfectants due to the antibacterial

properties possessed by it.

Scope and limitations

In this research, stem decoction from H. indica will be subjected to a toxicity test. The

method utilised in this research is A.salina lethality assay. Ten shrimps will be exposed to different

concentrations of the plant decoction. Exposure will only be conducted for 24 hrs. Observation will

happen after 6 and 24 hrs. The concentration that rendered 50% mortality (LC50) will be deter-

mined.

Definition of terms

Toxicity – ability of a chemical or solution to damage an tissue or organ system (kidneys, liver), or

disrupt a biochemical process, that may further lead to death

Mortality- number of deaths in a particular condition (time, place, or concentration)

 CHAPTER II 4

REVIEW OF RELATED LITERATURE

Heliotropium indicum

Heliotropium indicum (H.indica) is an annual, erect, branched, hirsute plant belonging to the fami-

ly Boraginaceae. It is a popular garden plant, but may also be found in the form of weeds. The

genus Heliotropium comprises about 250 species and is distributed in tropical, subtropical, and

warm temperate zones of all continents. Of these species, the H.indica is very common in India,

some parts of Africa and Bangladesh.

The whole plant is claimed to possess medicinal properties. Locals used H.indica for treat-

ing various conditions. The stem paste, when applied externally, can cure rheumatism and skin in-

fections. It may also be used for ringworms, eye diseases, fever, and urticaria. The roots are aphro-

disiac; used for curing night blindness. A decoction of the leaves mixed with the roots is believed

to cure whooping cough in small children. The flowers are considered emmenagogue (stimulate

blood flow in the pelvic area and the uterus) in small doses and abortifacient (causing miscarriage)

in large doses. The dried inflorescence of the plant is also believed to produce permanent sterilisa-

tion. Ancient folklore and traditional methods also suggest that it has previously been used to treat

skin diseases, poison bites, nervous disorders, stomachache, fever, diarrhoea, and skin rashes. Dif-

ferent extracts of H.indicum have been studied for possible biological activities in various animal

models which show the significant antimicrobial, anti fertility, anti tumour, antituberculosis, anti-

inflammatory, histogastroprotective, anti-cataract, analgesic and wound healing properties of the

plant(anupam roy et al., 2014). Further studies suggest that these properties are exhibited due to the

various alkaloids, steroids and essential oils possessed by the plant. Thus, this plant provides a

wide area for research in pharmacological actions due to its potential source of many chemical

constituents, and the ability to cure a wide range of severe health problems.
 5

The inflorescence of Heliotropium indicum turned their rows of flowers towards the sun. ‘Helios’

meaning in Greek languages is ‘sun,’ and the ‘tropium’ word comes from Greek word ‘tropein.’

The meaning of this word is ‘to turn’ Heliotropium indicum Linn. is comes under the family Borag-

inaceae. Maximum plants under in the family Boraginaceae are herbs. Many of the plants of this

family Boraginaceaeare used as garden plants, and few others are treated as weeds. The Bengali

name of this plant species is Hatishur. The genus Heliotropium comprised approximately 250 plant

species and distributed in tropical and temperate zones of different parts of the world Heliotropium

indicum is termed as ‘Indian heliotrope’ is highly endemic in India and Bangladesh. It is also found

in different parts of the world, especially in many African countries.

The plant is an annual, erect; ascending hirsute branched about 20 to 60 cm tall and coarse

foetid herb. The leaves are opposite or sub-opposite, alternate or straight forward or sub- alternate,

ovate to obovate, hairy, and acute, 5 to 10 cm long. Leaf margins undulate, nerves on either side,

serrulate or cordate, or veins conspicuous beneath the leaves. The petiole is about 4 to 10 cm long.

The flowers are green and approximately 5 mm in diameter. Flowers develop apically within the

cymose, at maturity nutlets are present at the base of the inflorescence (cyme). Flowers are white

or whitish violet, regular, sessile, axillary. Sepals-5, 3 mm long, diffused with hairs in outside, deep

green, linear to lanceolate and uneven or unequal. Distributed branched, hirsute with hairs in the

stem and the root is tap root and branch. The fruits are dry 2 to 4 lobed, with or without united nut-

lets, 3 to 6 mm long. It is an endemic weed in disturbed areas, garden or lawns, roadside, anthro-

pogenic habitats or waste places and settled areas. It is found in sunny locations with around 800 m

altitude. Throughout the year flowers are observed in this plant species.

All the plant parts of these medicinal weed possess different pharmacological activities. The

plant leaves highly used in an ethnomedicinal systems such as eye diseases, conjunctivitis, cataract,
 6

and pharyngodynia. The roots of Heliotropium indicum are used as astringent, expectorant, infu-

sion and febrifuge. The water extract of leaves was showed activity against Schwart’s leukemia.

Taxonomic classification

Domain: Eukaryota

Kingdom: Plantae

Phylum: Spermatophyta

Subphylum: Angiospermae

Class: Dicotyledonae

Order: Boraginales

Family: Boraginaceae

Genus: Heliotropium

Species: Heliotropium indicum

Botanical morphology

Habitat: It is an endemic hirsute medicinal weed in disturbed areas, garden or lawns, roadsides,

anthropogenic habitats and waste places It is found in approximately 1000 m altitude.

Growth: An annual coarse foetid herbaceous plant with erect, branched growth form upto 60 cm

long. Slightly woody at the base of the plant

Foliage: Leaves are 4-10 cm long and 2-5 cm wide, opposite or sub-opposite, alternate or sub-al-

ternate, ovate to obovate, and acute, with a wavy or undulate, serrulate or cordate leaf margin,

nerves on either side or veins conspicuous beneath the leaves. The leaf surface is covered in short

hairs which may be quite stiff. The leaf blade extends down the length of the petiole. Distinctly

petiolate, petioles 5 cm tall with a sub-truncate base or ovate.

 7

Flowers: Flowers (4-5 mm wide) are regular, sessile, axillary, slightly purple or white or whitish

violet (lilac) with a small yellow center and having a narrow tube with lobes formed a plate shape.

The thin cyme inflorescence is similar as a string or twisted of beads with a prominent curl at the

apex. The flowers are green and approximately 5 mm in diameter. Flowers develop apically within

the cymose inflorescence, tip coiled; at maturity of the flower, nutlets are there at the base of the

inflorescence. Flowers arranged at one side of cyme inflorescences. Sepals number is five, 3 mm

long, diffused with hairs in outside, deep green, linear to lanceolate and uneven or unequal. Calyx

lobes ciliate, 3 mm long; Stamens are five and borne in a corolla tube, terminal, corolla-tube 4-6

mm long; petals rounded. The ovary is 4- lobed.

Fruits: The fruits are known as nutlets. Fruits are dry, indehiscent 2 to 4 lobed, 3 to 6 mm long,

with or without united nutlets, ovate, ribbed separated into two nutlets, each nutletis two-celledand

beaked.

Stem and roots: Wide distributed, branched or unbranched, hirsute with hairs in the stem and the

root system is long tap root, and it is highly branched

Genetics: Chromosome number of Heliotropium indicum Linn. is 2n=22, 24.

Aerial Parts: Aerial parts of the plants contain pyrrolizidine alkaloids,indicine, echinitine, supi-

nine, heleurine, heliotrine, lasiocarpine, acetyl indicine, indicinine, and indicine-n-oxide. The plant

also possesses rapone and lupeol and an ester of retronecine

Root: Roots contain a high amount of estradiol. Joao Sammy N. Souza et al., 2005, isolated a new

pyrrolizidine alkaloid with unusual structural features helindicine, together with the known lycop-

samine, from the roots

Seeds: Presence of cynoglossine, europine-N-oxide, heleurine-N- Oxide, heliotridine-N-Oxide,

heleotrine-N-Oxide and heliotrine have been identified from the seeds. Seeds containheliotrine as

major chemical constituents. The seeds contain 12% oil and 1.8% N2
 8

Leaves: Aqueous leaf extracts of Heliotropium indicum from seedlings contains phenolic com-

pounds. The alkaloids trachelanthamidine and retronecine and the pyrrolizidine precursor amines

(in leaves and inflorescence) putrescine, spermidine and spermine were isolated and identified in

the leaves .

Total Plant

Apart from alkaloids, several triterpenes and steroids including β-amyrin, lupeol, chalinasterol, β-

sitosterol, stigmasterol and campesterol, hexacosane-1-ol, an ester of retronecine have been report-

ed from the whole plant. Other compounds reported from the entire plant parts

Pharmacological property

Among the other experimental herbaceous plants various solvent extracts of Heliotropium indicum

have been investigated, and it showed different bioactivities in animal models and reported to pos-

sess anti-oxidant, wound healing, anti-tussive, anti-bacterial, anti-fertility, anti-tumor, anti- hyper-

glycemic, anti-tuberculosis, anti-inflammatory, histo- gastro protective, anti-ulcer, anti-cataract,

analgesic and anti- pyretic properties. These properties are described as follows.

Wound Healing Property

Alcoholic extract of Heliotropium indicum showed wound healing activities in an animal model.

Research investigation concluded that alcoholic extract possessed wound healing activity in the n-

butanol crude extract and from this extract, two alkaloids Pestalamide B and Glycinamide, N-(1-

oxooctadecyl) glycyl-Lalanylglycyl-L-histidyl were isolated and identified. These two bioactive

compounds are responsible for wound healing. In research study petroleum ether, chloroform,

methanol, and aqueous extracts were used to evaluate for their wound healing activity in an animal

model. The results showed that a significant increase in wound healing with both methanolic and

water extracts in comparison with other extracts in this study. Promising increase in the granulation
 9

tissue weight, hydroxyl proline content, and increased activity of SOD and catalase percentage in

the methanolic extract in dead space wound model. The study validates its ethnomedicinal uses.

Artemia salina

Artemia salina (L.) is a primitive aquatic arthropod (salt lakes) of the Artemiidae family (figure 1)

with an age of about 100 million years. Linný (1758) described it as Cýncer salinus but 61 years

later, Leach (1819) transferred it to Artemia salina. It was reported for the first time in Urmia Lake

in 982 by a Iranian geographer (Asem, 2008).

Species ecology

Artemia salina lives only in lakes and ponds with high salinity, which varies between 60-300 ppt. It

was also discovered in Elkhorn Slough (California), which communicates directly with the sea. It

is a species endemic to the Mediterranean, but is found on all continents (figure 2). In our country

is reported in salt lakes (Bear Lake, Ocna Sibiu, Techirghiol, Braila Salt Lake, etc.) contributing to

the formation of sapropelic mud used in peloidotherapy. A. salina is associated with current or past

commercial exploitation of salt (Abatzopoulos et al., 2002, quoted by Abatzopoulos et al., 2010).

Can tolerate large amounts of salt (up to 300 grams of salt per liter of water) and can live in quite

different solutions of seawater such as potassium permanganate and silver nitrate.Iodine, which is

found frequently in salt for human use, is harmful to this species.

These animals have the ability to reduce the osmotic pressure of haemolymph by NaCl excre-

tion against the concentration gradient. Has been shown to developed a mechanism to maintain

haemolymph extreme hypotonic in saline extreme media (Croghan, 1957).Also, this species can

survive in water with high oxygen deficiency. Minimum concentration of oxygen for an adult is

very low, 0.5 milligrams per liter, and for naupliia even less than 0.3 milligrams per liter. Typical
 10

salt lakes have seasonal or cyclical droughts, periods when completely dry. During this process salt

water concentrations ranging from ideal to barely tolerable

Life cycle

Mode of reproduction is controlled by environmental factors: the concentration of oxygen in water

and its fluctuation, the type of food, salinity, etc. (table 1). There was a correlation between water

salinity level and method of reproduction. Ovoviviparous to less than 150 ppt salinity and

oviparous predominantly between 150-200 ppt salinity

Table 1. The modalities of Artemia salina reproduction

REPRODUCTION

Oviparous Ovoviviparous

low O2-content high oxygen content

(such as in high (such as in low

salinity) salinity)

-- strong O2-fluctua- -minor O2 fluctua-

tions tions
- fe-rich food (green - fe-low food (organic
debris)
algae)

Artemia salina lethality assay

lethality bioassay is a simple, high throughput cytotoxicity test of bioactive chemicals. It is

based on the killing ability of test compounds on a simple zoological organism Artemia salina. It is

widely used in the evaluation of toxicity of heavy metals, pesticides, medicines especially natural

plant extracts. (Chao et al, 2014).

 11

One important aspect of this assay is that the solvent used in this assay may give false posi-

tive signals due to the toxicity of the solvent itself. It has been well-known that some organic sol-

vent and detergents have high cytotoxicity in vivo. A systematic study on how high concentration

of solvent affects the results from lethality bioassay and guidance for maximum working concen-

tration of solvents is needed (Chao et al, 2014).

A.salina lethality assay is a convenient system for monitoring biological activities of vari-

ous plant species. The rapidness monitoring of this test could be extended to maximum 60 hours,

but in most cases, relevant data for the LC50 calculations are obtain only after 24 hours of expo-

sure. This test does not require any aseptic techniques and special equipments (Меntor R. Hamidi,

Blagica Jovanova, Таtјаnа Kadifkova Panovska et al., 2014).

In previous studies, when the concentration of the test solution taken were 10 µg/ml, 100

µg/ml , 500 µg/ml , 1000 µg/ml, of all the four concentrations , 1000 µg/ml showed the highest

percent mortality at 92.23% , followed by 500 µg/ml at 76.67%, 100 µg/ml at 21.13%and 10 µg/ml

the least , at 15.57% (Caňa et al., 2014).

lethality test on other plant species

lethality assay was carried out to investigate the cytotoxicity of extracts of medicinal plants of In-

dia. Researchers conducted experiment along with control ,different concentrations (1-5000 µg ml-

1) of the test substances in a set of three tubes per dose. After doing this experiment they got the

result that the LC50 values of the plant extracts (24 h) were obtained by a plot of percentage of the

shrimps killed against the concentrations of the extracts and the best-fit line was obtained from the

data by means of regression analysis. LC50 was obtained from the best-fit line method and they

came on conclusion that The fact that eleven out of the 118 plants screened for toxicity had LC50
 12

values less than 60 µg ml-1 is interesting and lend support to the traditional use of these plants.

(Krishnaraju et al., 2006) The seeds of Swietenia mahagoni have been applied in folk medicine for

the treatment of hypertension, diabetes, malaria, amoebiasis, cough, chest pain, and intestinal para-

sitism. Using lethality assay, the LC50 for the extract is 0.68 mg/ml at 24 hours of exposure. These

results may indicate that S. mahagoni crude methanolic seed extract may contain bioactive com-

pounds of potential therapeutic significance which are relatively safe from toxic effects, and can

compromise the medicinal use of this plant in folk medicine (Sasidharan et al.,2010 ).

Toxicity and other tests involving H.indicum.

The toxic effect of H.indicum roots and stem were tested on rats. In this, research, H. in-

dicum roots and stem are reported to possess significant antifertility activity when studied in rats

(Anupam roy et al., 2014) .

In another experiment H. indicum were studied for anti-fertility activity in rats using anti-implanta-

tion and abortifacient model. The study revealed that H. indicum possesses promising abortifacient

activity (Anupam roy et al., 2014). Several pyrrolizidine alkaloids have been isolated from this

species. But the most alkaloids are hepatoxic. Therefore, internal use of Heliotropium species is not

recommended (Anupam roy et al., 2014).This provides a wide area for research into the detailed

pharmacological action of this drug which has not been explored much as compared to its utility.

One research also showed that the extracts of the roots and stem bark exhibited relatively

mild cytotoxic activity against larvae with LC50 values ranging from 113.75–4356.76 and 36.12–

1458.81 µg/ml, respectively. The stem wood extracts exhibited the highest toxicity against the

A.salina (LC50 values 2.58–14.88 µg/ml (Mbwambo et al., 2007).

 13

Conceptual Framework

Figure 1 illustrates the outline or flow of the study. In this study, H. indicum stem decoction

will be subjected to a toxicity test using A.salina lethality assay. In this toxicity assay, mortality at

each concentration will be determined. Using the data that will be obtained, LC50 will be calculat-

ed. The obtained data and results will be used to analyze possible implications especially on

health, furthermore, to formulate recommendations.

Heliotropium indicum stem decoction

A.Salina Lethality Assay

Mortality at each concentration

LC50

Implications/Recommendations

Figure 1. Conceptual Framework of the study

 CHAPTER 3 14

METHODOLOGY

Research design

This research is an experimental research involving the manipulation of an independent

variable (stem decoction concentration) and the measurement of a dependent variable (mortality).

Research environment

Mature H. indica leaves will be collected from Cebu, Philippines and will be brought to

the laboratory setup in Elizabeth place talamban Cebu city.

Fig.2

Research subjects

Mature H. indica leaves will be used. For the purpose of botanical identification, a whole

plant will be collected in duplicate following accurate documentation. A. salina eggs will be ob-

tained from a pet shop and will be hatched using artificial seawater.
 15

Research instruments

Researches will introduce eggs in tank fitted with aerator to provide medium for

hatching. Micropipettes will be used in taking accurate concentrations of plant decoction.

Study lamp will also be used for illumination.

Research procedure

Collection of Plant Material.

Mature H. indica leaves will be collected from Cebu, Philippines and will be brought to

the Elizabeth place talamban. For the purpose of botanical identification, a whole plant will be

collected in duplicate following accurate documentation

Preparation of the plant decoction.

One kilogram of fresh and clean samples of the plant stem will be cut into pieces and boiled in

sufficient amount of distilled water (1:2 ratio) for 5 minutes. The mixture will be filtered, cooled and

stored in glass containers and freeze-dried until all the water will be removed to give concentrated

decoction. It will then be kept until required. Stock solution will be prepared by dissolving 30 mg

with 3000 ppm ethanol and distilled water. From the stock solution, 10, 100, 500, and 1000 ppm con-

centrations will be prepared by the addition of 5 ppm, 50 ppm, 250 ppm and 500 ppm of stock solu-

tion, respectively in a 20 mL test tube. Addition of a minimal amount of dimethyl sulfoxide (DMSO)

will be done to completely dissolve the solution in each test tube .Artificial seawater will serve as

the positive control while Potassium dichromate will serve as the positive control.

Hatching of Eggs.

Artificial seawater will be bought from a pet shop in Cebu City, Philippines. This will then be

kept in a small tank, and A. salina eggs will be added to the divided tank. Constant temperature

(around 37° C) will be maintained and constant supply of oxygen will be carried out. A.salina will be

allowed to mature and hatch as nauplii for two days. The newly hatched A.salina will then be collect-

ed using a dropper.
 16

Lethality Assay.

Ten larvae will be introduced into each sample vials containing different concentrations of

the extracts. Artifical seawater will be added to make a total volume of 5 ml. A drop of yeast sus-

pension (3 mg in 5 ml seawater) will be added as food to each vial. The vials will be maintained

under illumination. Survivors will then be counted after 6, and 24 hours and the deaths at control

and each dose level will be determined.

Data processing and analysis

After 6h and 24h, the lethal concentrations of the H. indica decoction resulting to 50% mor-

tality (LC50) will be determined. Then, by means of a trendline fit linear regression analysis (MS Ex-

cel), the dose-response data will be transformed into a straight line. From the best-fit line obtained,

the LC50 will be derived. If LC50 >1000 ppm, it is nontoxic. If LC50 is 20 ppm1000 ppm, it is tox-

ic. If LC50 < 20 ppm, the solution is highly toxic. Statistical significance of the mean mortality at

each concentration will be analyzed using one-way analysis of variance (ANOVA).

Ethical Considerations.

The current study will not involve humans and higher animals as subjects. Hence, no ethi-

cal concerns will be considered in this study.

 17

REFERENCES

Caña, D. B., & Guinit, J. C., (2009) Toxic effect of Heliotropium indicum (trompa ng elepante) on

artemia salina (brine shrimp) nauplii. R07-USC-14052312360622.

Dash, G. K., & Abdullah, M. S., (2017) A Review On Heliotropium indicum L. (Boraginaceae).

International Journal Of Pharmaceutical Science And Research, SJR : 0.137 , Projected Impact

Factor : 0.59

Hamidi, M. R., Blagica, J., Tatjana, K. P., (2014) Toxicological evaluation of the plant products

using Brine Shrimp (Artemia salina L.) model. Macedonian pharmaceutical bulletin, 60 (1) 9 – 18,

ISSN 1409 –8695, UDC: 615.322.099

Krishnarajun, A. V., Tayi, V. R., Dodda, S., Mulabagal, V., Hsin-Sheng, T., Gottumukkala, V. S,

(2006) Biological Screening of Medicinal Plants Collected from Eastern Ghats of India Using

Artemia salina (Brine Shrimp Test). International Journal of Applied Science and

Engineering, 4, 2: 115-125

Lee, D., (2001) INDIA HELIOTROPE Heljotropium indicum Michx, Retrieved from https://

plants.usda.gov/plantguide/pdf/pg_hein.pdf

Mbwambo, Z. H., (2007) Antimicrobiał activity and brine shrimp toxicity of extract of Terminalia

brownie roots and stem. (BMC Complement Altern Med. 2007), Retrieved from
 18

Meyer, B. N., Ferrigni, N. R., Putnam, J. E., Jacobsen, L. B., Nichols, D.E., McLaughlin, J. L.,

(1982) Brine shrimp: A convenient general bioassay for active plant constituents. Planta Medica.

1982; 45: 31-34.

Quazi, S. S., Chowdhury, F. A., Mir, M., (2017) visual experiment, Brine shrimp lethality assay.

12: 186-189, DOI: 10.3329/bjp.v12i2.32796

 CURRICULUM VITAE

PERSONAL INFORMATION

Name: CHAUHAN KAMIL

Date of Birth: 16th MAR 2000

Place of Birth: JUNAGADH

Permanent Address: 17, NATWAR NAGAR, DHARI WALA DELO, BA-

GASARA, AMRELI, GUJRAT, INDIA.

Contact number: 09495834631

E-mail address: chauhankamil00@gmail.com

EDUCATIONAL BACKGROUND

PRIMARY AND SECONDARY

SAHAJANAND SCHOOL OF ACHIEVER

2005-2017

BACHELOR OF SCIENCE IN BIOLOGY

UNIVERSITY OF VISAYAS, GULLAS COLLEGE OF MEDICINE, BANILAD, CEBU CITY

,2017

Previous study- Effects of Internet on Medical Students of Gullas College of Medicine Banilad
Cebu City.

Motto- To inspire and be inspired

PERSONAL INFORMATION

Name: Jagtap Shardul Rangnath

Date of Birth:07-23-1999

Place of Birth: Pune

Permanent Address:Ap-shikrapurtale-shirurdist-puneShantainagar1st banglow

Contact number: 09235257547

E-mail address:sharduljagtap7000@gmail.com

EDUCATIONAL BACKGROUND

SECONDARY

Abasaheb garware clg of science-pune

Year-2013-17

BACHELOR OF SCIENCE IN BIOLOGY

University of the Visayas

2017

Previous study- Effects of Internet on Medical Students of Gullas College of Medicine Banilad
Cebu City.

Motto- to never give up

PERSONAL INFORMATION

Name: Harshwardhan Singhvi

Date of Birth: 04 October 1999

Place of Birth: Jodhpur , Rajasthan, INDIA

Permanent Address: B-37 ,UIT colony, Pratap nagar

Contact number: +639254699134 m

E-mail address: harshsinghvi99@gmail.com

EDUCATIONAL BACKGROUND

PRIMARY

Delhi public school 2016

SECONDARY

Adarsh central senior secondary school

2017

BACHELOR OF SCIENCE IN BIOLOGY

University of the Visayas

Previous study- Effects of Internet on Medical Students of Gullas College of Medicine Banilad
Cebu City.

Motto- To serve humanity

MISHRA UTTAM KUMAR

Home address :MunderwaThakurain , Bisheshwarganj , Payagpur ,

Dis – Bahraich , U.P , India
Residential address : Elizabeth Place , 0363 Tudtud Street ,
Nasipit Road , Talamban , Cebu City , 6000 , Philippines
E-mail address :uttammishra989@gmail.com
Mobile No : +63 9254699136

Previous study- Microbial Quality of Cooked Vegetables in University of the Visayas – Gullas
Campus Cafeterias,Cebu City, Philippines

Motto- to sever selflessly