Brine Shrimp (Artemia salina) Bioassay of the medicinal plant Pseudelephantopus spicatus from Iligan City, Philippines

Abstract

Medicinal plants are commonly distributed in different regions of the Philippines. Based on ethnomedicinal significance, P. spicatus used in traditional medicine was collected and evaluated for biological activity using the Brine Shrimp Bioassay. Plant extracts were obtained through decoction, ethanolic extraction and extraction with ethanol-water. Four concentrations (10, 100, 500, 1000 ppm) of the P. spicatus extract were used. Mortality of the brine shrimp was observed after 6 hours and 24 hours. The results showed that the decoction and ethanol-water extract were inactive against brine shrimp. However, the ethanol extract showed a toxicity effect after 6h and 24h exposures with LC 50 values at 944.07 and 266.07 ppm, respectively. Results indicate that the ethanol extract may have substances that are cytotoxic and that active components of the plant are better extracted with absolute ethanol than with hot water or mixture of ethanol and water. The active components present may have medicinal importance with no no adverse effects, and may support the therapeutic use of P. spicatus.

Full text

International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202
Vol. 3(9), 47-50, September (2014) Int. Res. J. Biological Sci.
International Science Congress Association 47
Brine Shrimp (Artemia salina) Bioassay of the medicinal plant
Pseudelephantopus spicatus from Iligan City, Philippines
Lalisan Jeda A.
1
, Nuñeza Olga M.
1*
and Uy Mylene M.
2
1
Dept. of Bio. Sci., College of Science and Mathematics, Mindanao State University-Iligan Institute of Technology, Iligan City, PHILIPPINES
2
Department of Chemistry, College of Science and Mathematics, Mindanao State University-Iligan Institute of Technology, PHILIPPINES
Available online at: www.isca.in, www.isca.me
Received 28
th
March 2014, revised 29
th
May 2014, accepted 11
th
June 2014
Abstract
Medicinal plants are commonly distributed in different regions of the Philippines. Based on ethnomedicinal significance, P.
spicatus used in traditional medicine was collected and evaluated for biological activity using the Brine Shrimp Bioassay.
Plant extracts were obtained through decoction, ethanolic extraction and extraction with ethanol-water. Four concentrations
(10, 100, 500, 1000 ppm) of the P. spicatus extract were used. Mortality of the brine shrimp was observed after 6 hours and
24 hours. The results showed that the decoction and ethanol-water extract were inactive against brine shrimp. However, the
ethanol extract showed a toxicity effect after 6h and 24h exposures with LC
50
values at 944.07 and 266.07 ppm, respectively.
Results indicate that the ethanol extract may have substances that are cytotoxic and that active components of the plant are
better extracted with absolute ethanol than with hot water or mixture of ethanol and water. The active components present
may have medicinal importance with no no adverse effects, and may support the therapeutic use of P. spicatus.
Keywords: Cytotoxicity, decoction, ethanol extract, LC
50,
traditional medicine.
Introduction
In many developing countries, many people depend on
traditional and alternative medicine
1
. About four billion people
(80%) of the world’s population are estimated to use botanical
medicine
2
. Even though modern medicine may be accessible in
these countries, herbal medicines represent a considerable
proportion of the global drug market and have often maintained
popularity given that these countries, including the Philippines
possess rich floristic wealth. Herbal drugs are safe than the
synthetic drugs and are biofriendly and eco-friendly
3
. Many
plants used in traditional medicine contain chemical substances
called phytochemicals
4
that produce a physiological action on
the human body
5
. Pseudelephantopus spicatus is one of the
most important plants found in arid regions of the world. P.
spicatus
is used for treating poisonous snake bites in India
6
. In
Jamaica the plant is used for fever, sprains, and eye problems
7
.
In Taiwan, the plant which was originally useful to treat
dampness, nephritis, edema, scabies, and pneumonia, was
shown to induce acute hepatic damages in rats and exhibits
antifungal activity against Candida albicans, A. niger
8
and
performs antileishmanial activity
9
. In the Philippines, leaves are
considered specific for eczema and are used as a topical agent
and as vulnerary-a medicine used in the healing of wounds
However, data on the pharmacological properties and toxicity
of this plant are lacking and no study has been conducted that
shows the extensive diversity of its metabolites.
10
Documentation of antimicrobial properties and toxicity of
medicinal plants is essential to build a comprehensive database
from which it may be potential to search new leads in
development of drugs
11,1 2
.
The study of bioactive components from plant extracts in the
laboratory is frequently hindered by the lack of simple, suitable,
and fast screening procedure
13, 14
. Many bioassay methods are
applied using whole animals, biochemical system or isolated
tissues but these procedures are somewhat expensive and
complicated. Therefore, brine shrimp bioassay is a more
convenient procedure for general toxicity screening. The
lethality assay using Artemia salina was used in this study
because it has been proven to effectively biomonitor the
isolation of insecticidal, cytotoxic, antineoplastic, antimalarial,
and antifeedant compounds from plant extracts
15
. The method is
attractive because it is very simple, inexpensive and little toxin
amounts are enough to perform the test in the micro-well scale
16,
17
. Since its introduction, this in vivo lethality bioassay has been
successively used for providing a frontline screen that can be
backed up by more specific and more sophisticated bioassays
once the active compounds are isolated
18, 19
.With this, the P.
spicatus safety or toxicity is assessed, since the findings are
important considering the usage of the plant by human beings.
Thus, the findings of this present work may provide baseline
information on the promising plant species that could be used as
a basis for the development of new tools of great therapeutic
importance.
Material and Methods
Collection of Plant Material: For the purpose of botanical
identification, small branches or twigs with reproductive
structures, healthy leaves, stipules, bark and wood samples from
each plant were collected in duplicate following accurate
documentation. Mature P. spicatus leaves were collected from

International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202
Vol. 3(9), 47-50, September (2014) Int. Res. J. Biological Sci.
International Science Congress Association 48
Dalipuga, Iligan City, Philippines and were identified by
Professor Aranico from the Department of Biological Sciences,
Mindanao State University-Iligan Institute of Technology,
Philippines.
Preparation of Plant Extract: For the preparation of the crude
extract, about 2-3kg of plant or plant parts were cleaned by
washing with tap water followed by distilled water. The sample
was air-dried for about 2-3 weeks, and the dried samples were
pulverized using a sterile electric blender. The half portion of
the pulverized leaves was soaked in pure absolute ethanol, and
the other was soaked in 50:50 ethanol-water mixtures for three
days. Each solution was filtered with Whatman No. 1 filter
paper and concentrated at 40°-50°C under reduced pressure
using the rotary evaporator
21
.
For the preparation of the plant decoction, about 1 kg fresh and
clean samples of the plant were cut into pieces and boiled in
sufficient amount of distilled water (1:2 ratio) for 5 minutes.
The mixture was filtered, cooled and stored in glass containers
and freeze-dried until all the water was removed to give
concentrated decoction. It was then kept until required.
Stock solutions were prepared. Thirty milligrams of dried
samples from decoction, crude ethanol extract and crude
ethanol-water extract, were dissolved with 3000 ppm ethanol
and distilled water respectively and then sonicated to dissolve
the dried samples. From the stock solution, 10, 100, 500, and
1000 ppm concentrations were prepared by the addition of 5
ppm, 50 ppm, 250 ppm and 500 ppm of solution, respectively in
a 20 mL test tube. Addition of a minimal amount of dimethyl
sulfoxide (DMSO) was done to completely dissolve the solution
in each test tube.
Brine Shrimp Lethality Bioassay: Hatching of Brine
Shrimp: Brine shrimp (Artemia salina) lethality bioassay was
carried out to investigate the cytotoxicity of extracts of
medicinal plants. Artificial seawater was prepared by dissolving
40 grams of natural table salt in every liter of distilled water.
Sea water was kept in a small tank, and A. salina eggs were
added to the divided tank
21
. Constant temperature (around 37°
C) was maintained and constant supply of oxygen was carried
out. Brine shrimps were allowed to mature and hatch as nauplii
for two days. The newly hatched shrimp was collected using a
dropper.
Assay Proper: Ten brine shrimp larvae were introduced into
each sample vials containing different concentrations of the
extracts. Seawater was added to make a total volume of 5 ml.
The vials were maintained under illumination. Survivors were
counted after 6, and 24 hours and the deaths at control and each
dose level were determined.
Lethal concentration Determination: After 6h and 24h, the
lethal concentrations of the P. spicatus extract resulting to 50%
mortality of the brine shrimp (LC
50
) were determined. Then, by
means of a trendline fit linear regression analysis (MS Excel
version 7) the dose-response data were transformed into a
straight line. From the best-fit line obtained the LC
50
was
derived.
Statistical Analysis: Reed-Muench statistical method was used
to determine the relative toxicity of the P. spicatus extracts to
living organisms. It was done by testing the response of A.
salina under various concentrations of the extract. LC
50
represents the dose lethal to the half members of the A. salina.
This was determined by plotting the mortality (y-axis) versus
log of concentration (x-axis). The concentration that rendered
50% mortality was the LC
50
.
Results and Discussion
Table 1 shows the toxicity of the P. spicatus extracts on the
brine shrimp after 6 and 24-hour exposure. The extracts
obtained from decoction and ethanol-water extract exhibited no
lethality on the brine shrimps at any of the concentrations at 6h
and 24h. The brine shrimps were still actively moving, and no
signs of behavioral changes were observed. Crude plant extract
with LC
50
value of less than 1000 ppm is toxic while non-toxic
(inactive) if it is higher than 1000 ppm
20
. Since the LC
50
in the
both of this extract taken from decoction and ethanol-water
mixture was higher than 1000 ppm, it was considered inactive.
It may be because the active components present in the P.
spicatus were not extracted through the two methods mentioned
above. Even though decoction process is economical due to its
low cost in terms of instrumentation and reagents
21
it may be an
inefficient process given that ingredients may be damaged
during the prolonged heating of substances, and other
ingredients may be oxidized and lose activity
22
. During the
decoction process, many aromatic herbs with high levels of
volatile oils are easily lost through evaporation
23
. Also, ethanol-
water mixture extraction process was still ineffective and it is in
accordance with the previous study in which the alcohol/water
mixture (typically 20–40% alcohol) is actually a poor medium
for extraction. It is because it causes the desired components to
condense out of the liquid therefore none is left in the finished
product
24
. The ethanol extract of P. spicatus showed a toxicity
effect at 6h and 24h, with LC
50
value at 944.07 and 266.07 ppm,
respectively. This suggests that the extract could have
compounds that are cytotoxic as the LC
50
value was lower than
1000 ppm (table 1). The brine shrimp mortality rate at different
concentrations in the ethanol extract was found to increase with
increasing concentration of the sample, and it clearly shows that
the extraction with ethanol was a better way of obtaining P.
spicatus extract bioactive components. The previous studies
show that ethanolic extract of P. spicatus demonstrated strong
biological activity toward Leishmania amazonensis
8
. Ursolic
acid and the two hirsutinolides (the 8-acetyl-13-O-ethyl-
piptocarphol and 8, 13-diacetyl-piptocarphol 8-acetyl-13-O-
ethyl-piptocarphol) isolated through phytochemical screening
might be responsible for its pharmacological activities thus
giving support to its use in Peru
9
.

International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202
Vol. 3(9), 47-50, September (2014) Int. Res. J. Biological Sci.
International Science Congress Association 49
Table- 1
Effects of the P. spicatus Extracts on the Brine Shrimp A. salina after 6- and 24-hr Exposure
Extracts Concentration of the Extract (ppm) Brine Shrimp Mortality (%) Acute LC
50
(after 6 hrs)
Chronic LC
50
(after 24 hrs)
After 6 hrs. After 24 hrs.
Decoction
10 0 0
>1,000ppm >1,000ppm
100 0 0
500 0 0
1000 0 0
Ethanol
10 0 0
944.07ppm 266.07ppm
100 6.67 3.33
500 26.67 16.67
1000 36.67 76.67
Ethanol-water
10 0 0
>1,000ppm >1,000ppm
100 0 0
500 0 0
1000 0 0
Herbal medicines have received high interest as a substitute to
clinical treatment, and the demand for herbal remedies has
currently increased rapidly. The increase in the number of
herbal users as opposed to the insufficiency of scientific
evidences on its safety has raised concerns regarding its
detrimental effects
25
and related concerns apply to the P.
spicatus in this study. Although there was no mortality in the
control group, in the extract obtained by decoction and mixture
of ethanol and water, it is evident that the ethanol extract
possessed a mild toxicity effect with acute and chronic values of
LC
50
944.07 ppm and 266.07 ppm, respectively with the percent
mortality increasing with concentration. This present study
shows that P. spicatus plant extract has some evident toxicity
and may be used as herbal medicine in known dosages,
especially in rural areas, where conventional medicine is too
expensive. In addition, the P. spicatus extract can be further
tested for acute toxicity on the animal model to compare the two
methods of toxicity evaluation.
Conclusion
This study presents the toxicity of the P. spicatus ethanol
extract, which should be very useful for any future in vivo or
clinical study of this plant extract. Results indicated that
bioactive compounds present might account for the plant’s
pharmacological or toxicological effects. The results somehow
support the use of this plant species in traditional medicine. In
addition, the present study confirms the utilization of the brine
shrimp (Artemia salina) bioassay as a reliable, simple, and
convenient method in monitoring bioactivity of medicinal
plants.
References
1. Elamin A.E., Abdmageed M.A.M., Shyoub M.E. and
Mohammed R. R., Evaluation of Toxicity of some plants
having traditional uses in Sudan on Brine Shrimp, Global
Journal of Traditional Medicinal Systems, 2(1), 19-23
(2013)
2. World Health Organization, Who launches the first global
strategy on traditional medicine. Geneva: Press release
WHO 2002/38, http://www.who. int/mediacentre/
news/releases/release38/en. (2002)
3. D’ Britto V., Dev P.P., Prasad B.L.V., Dwahan A., Mantri
V.G. and Prabhune A., Medicinal plant extracts used for
blood sugar and obesity therapy shows excellent inhibition
of invertase activity: synthesis of nanoparticles using this
extract and its cytotoxic and genotoxic effects,
International Journal of Life Science and Pharma
Research, 2(3), 61-74 (2012)
4. Olowa L.F. and Nuñeza O.M., Brine Shrimp Lethality
Assay of the Ethanolic Extracts of Three Selected Species
of Medicinal Plants from Iligan City, Philippines,
International Research Journal of Biological Sciences,
2(11), 74-77 (2013)
5. Vanitha K and Thangarasu S., Phytochemical Profiling And
GC-MS Study of Antigonum leptopus Hook & ARN,
International Journal of ChemTech Research. 5(4), 1630-
1640(2013)
6. Gomes A., Das R. and Sarkhel S., Herbs and herbal
constituents active against snake bite, Indian Journal of
Experimental Biology, 48, 865-878 (2010)
7. Picking D., Youngerb N., Mitchellc S. and Delgoda R., The
prevalence of herbal medicine home use and concomitant
use with pharmaceutical medicines in Jamaica, Journal of
Ethnopharmacology, 137(1), 305-311 (2011)
8. Lin C.C., Yen M.H., and Chiu H.F., The pharmacological
and pathological studies on Taiwan folk medicine (VI): The
effects of Elephantopus scaber subsp. oblanceolata, E.
mollis and Pseudelephantopus spicatus, American Journal
of Chinese Medcine, 19(1), 41-50 (1991)
9. Oddone G., Herbette G., Eparvier V., Bourdy G., Rojas
R., Sauvain, M. and Stien D., Antileishmanial sesquiterpene
lactones from Pseudelephantopus spicatus, a traditional

International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202
Vol. 3(9), 47-50, September (2014) Int. Res. J. Biological Sci.
International Science Congress Association 50
remedy from the Chayahuita Amerindians (Peru), Journal
of Ethnopharmacology, 137(1), 875-879 (2011)
10. Ragasa C. and Rideout J., An Antifungal Cadinanolide
from Pseudelephantopus spicatus, Chemical and
Pharmaceutical Bulletin, 49(10), 1359-1361 (2001)
11. Nguta J.M., Mbaria J.M. and Mvula M.D., Brine shrimp
toxicity and in vitro antimicrobial activity of Piliostigma
thonningii (Schum.) Milne-Redh, [Leguminosae-
Caesalpinioideae] from Kenya and Malawi against some
pathogens of human and veterinary importance, Journal of
Veterinary Medicine and Animal Health, 5(9), 251-256
(2013)
12. Babajide O.J., Mabusela W.T., Green I. R., Ameer F.,
Weitz F., and Iwuoha E.I., Phytochemical screening and
biological activity studies of five South African indigenous
medicinal plants, Journal of Medicinal Plants Research,
2(18), 1924-1932(2010)
13. Tawaha K.A., Cytotoxicity Evaluation of Jordanian Wild
Plants using Brine Shrimp Lethality Test, Journal of
Applied. Sciences, 8(1), 12-17 (2006)
14. Montanher A. P., Pizzolatti M. G and Costa I.M, An
Application of the Brine Shrimp Bioassay for General
Screening of Brazilian Medicinal Plants, Acta Farm.
Bonaerense, 21(3), 175-8 (2002)
15. Ahmed T. and Hashim A., Brine Shrimp Bioassay of
Ethanol Extracts of Sesuvium verrucosum, Salsola
baryosma and Zygophyllum quatarense, Medicinal Plants
from Bahrain. Phytotherapy Research, 14, 48–50 (2000)
16. Krishnaraju A.V., Rao T.V.N., Sundararajua D., Vanisreeb
M., Tsay H. and Subbarajua G.V., Assessment of
Bioactivity of Indian Medicinal Plants Using Brine Shrimp
(Artemia salina) Lethality Assay, International Journal of
Applied Science and Engineering, 3(2), 125-134 (2005)
17. Omale J and Omajali J.B., Evaluation of bio-safety and
antioxidant activity of the fruit and leaf of Saba florida
(Benth.) from Ibaji forest, International Journal of
Medicine and Medical Sciences, 2(3), 100-105 (2010)
18. Apu A.S., Bhuyan S.H., Khatun F., Liza M.S., Matin M.,
and Hossain F., Assessment of cytotoxic activity of two
medicinal plants using brine shrimp (Artemia salina) as an
experimental tool, International Journal of Pharmaceutical
Science and Research, 4(3), 1125-1130 (2013)\
19. Pillai L.S., and Nair B. R., Evaluation of cytotoxic activity
of Cleome viscose L. and Cleome burmanni W. &A.
(Cleomaceae), International Journal of Pharmacy and
Pharmaceutical Sciences, 5(4), (2013)
20. Meyer B.N., Ferrigni N.R., Putnam J.E., Jacobsen L.B.,
Nichols D.E. and McLaughlin J.L., Brine shrimp: A
convenient general bioassay for active plant constituents,
Planta Medica, 45, 31-34 (1982)
21. Kiranmai M., MahendraK. and Ibrahim M., Comparison of
total flavanoid content of Azadirachta indica root bark
extracts prepared by different methods of extraction,
Research Journal of Pharmaceutical, Biological and
Chemical Sciences, 2(3), 254-265 (2011)
22. Bandaranayake W. M., Quality Control, Screening,
Toxicity, and Regulation of Herbal Drugs. In I. Ahmad, F.
Aqil, & M. Owais (Eds.), Modern Phytomedicine: Turning
Medicinal Plants into Drugs, Wiley-VCH Verlag GmbH &
Co. KGaA, Weinheim, Germany (2006)
23. Plant S., The Use of Herb Powders (San Ji) and the Draught
(Zhu San) Method in Chinese Herbal Medicine, Journal of
Chinese Medicine, 99, 37-40 (2012)
24. Dharmananda S., Dosage and form of herbs: decoctions,
dried decoctions, powders, pills, etc.
http://www.itmonline.org/arts/dosage.htm (2005)
25. Saad B., Azaizehm H., Abu-Hijleh G., Said O., Safety of
Traditional Arab Herbal Medicine, Evid Based
Complementary Alternative Medicine, 3(4), 433–439
(2006)