RESEARCH PLAN
Rationale
Exponential growth of various diseases is now being studied to
mitigate and prevent the further increase of diseases in the world. Some
of these diseases are being inclined to the production of free radicals
inside human body and to the helminth infection which is among the
most common infections in man affecting large proportion of population
all over the world.
These free radicals can cause large chain chemical reactions in the
body as it react so easily with other molecules through the process called
oxidation and it can be either beneficial or harmful. Oxidation is a
normal and necessary process that takes place in your body however if
could be damaging when there’s an imbalance between free radical
activity and antioxidant activity. When there are more free radicals
present than can be kept in balance by antioxidants, the free radicals can
start doing damage to fatty tissue, DNA, and proteins in your body
(Mosquera et al., 2007).
On the other hand, helminthic infections or helminthiasis is a
major cause of morbidity and mortality worldwide and is a prominent
among the so-called neglected tropical diseases (NTDs) such as
schistosomiasis and soil transmitted helminths’ (STH) (O’Dempsey,
2010). In developing countries they pose a large threat to public health
and contribute to the prevalence of malnutrition, eosinophilia, anemia as
1
well as pneumonia. Helminthiasis have been of concern to the medical
field for centuries and the helminthes still cause considerable problems
for human beings as well as animals.
Hence, the search for effective, nontoxic natural compounds with
anthelminthic and antioxidative activity has been intensified in recent
years to mediate various diseases caused by helminths and oxidation.
Therefore, there is a big need to investigate the properties of the plants
and their ability to be a potential source of natural anthelminthic and
antioxidant since the time immemorial, traditional system of medicine
and folklore claimed that medicinal plants as a whole or their parts are
being used in all types of diseases successfully including antibacterial
and anthelmintic, anti-inflammatory and many other pharmacological
property.
Thus in this study, Sesbania grandiflora L. locally known as
Katuday will be tested to determine its potency as an anthelminthic and
antioxidative agent. Respectively, it will also be tested to identify its
secondary metabolites and find out its bioactivity.
Statement of the problem:
This study aims to determine the potential of the Katuday
(Sesbania grandiflora L.) leaf extract in producing antioxidants.
Specifically it sought to answer the following questions:
2
1. Does Katuday ethanolic leaf extract contain the phytochemicals
alkaloids, steroids, flavonoids, saponins, tannins and polyphenols?
2. At what level of concentration does the Katuday (Sesbania
grandiflora L.) ethanolic leaf extract exhibit bioactivity based on
Brine Shrimp Lethality Test (BLST)?
3. Does the Katuday leaf extract exhibit antioxidant activity based on
DPPH Free Radical scavenging assay?
4. Is there a significant difference between the antioxidant activities of
the Katuday leaf extract and ascorbic acid?
5. Does the Katuday leaf extract exhibit anthelmintic activity against
Eudrilus eugeniae?
6. Is there a significant difference between the anthelmintic activity of
Sesbania grandiflora L. leaf extract and the reference drug?
Methodology
In this study the ethanolic leaf extract of the Katuday plant was
subjected to four different tests: phytochemical screening, cytotoxicity
test, antioxidant test and in vitro anthelmintic assay. The phytochemical
screening was conducted to detect the secondary metabolites present in
the Katuday (Sesbania Grandiflora L.) leaf extract particularly tannins
and polyphenols, saponins, alkaloids, flavonoids and steroids. It
employed a Qualitative Experimental Design (QED) where the presence or
absence of each secondary plant metabolite are determined using to
independent tests. A Cytotoxicity test was also performed to determine
the bioactivity of the Katuday plant. The Brine Shrimp Lethality Assay
3
was performed for this purpose where the % mortality was measured at
different concentrations (1000, 100 and 10 ppm) in order to determine
the lethal concentration required to kill 50% of the test organisms, (LC50)
through estimation following Finney’s linear regression analysis. The test
used four treatments (T1= saline solution as control, T2= 10 ppm, T3=
100 ppm and T4= 1000 ppm) each having three (3) replications. DPPH
Free Radical scavenging assay was done to determine the antioxidant
activity of the Katuday leaf extract using ascorbic acid as reference.
Different concentrations were allowed to react with a fixed amount of
DPPH Free radical solution and the absorbance was measured at 517 nm
with the inhibitory percentage of DPPH decoloration was then calculated.
Finally, In vitro anthelmintic assay was conducted to determine the
anthelmintic activity of Katuday (Sesbania Grandiflora L.) with the use of
albendazole as reference drug against African Nightcrawler earthworm
(Eudrilus eugeniae) as test organism. This study was carried out in a
Complete Randomized Design (CRD) having four treatments. And each
treatment had three replications (R1, R2 and R3).And each treatment will
have three replications (R1, R2, and R3) to obtain a valid and more
reliable estimate than which is possible with one observation only.
All the data that will be gathered and obtained in cytotoxicity test,
antioxidant test and anthelminthic assay will be subjected for statistical
analysis using Statistical Software: Statistical Package for Social Sciences
(SPSS version 16.0) ANOVA and the means were compared using the
Least Significant Different (LSD) to determine the significant coefficient of
4
the differences between samples, wherein p values < 0.05 were
considered to be significant.
Lastly, all instruments and materials that will be used in the whole
duration of the experiment will be washed with soap and running water
to free it from any residues. Pressure cooker will be utilized to heat for
the materials such as petri dishes, test tubes in order to eliminate the
traces of microorganisms that will be used.
After drying, it will be
returned to the laboratory. The experiment will be conducted and
performed from October 2018 to September 2019.
Research Procedure
The diagram below illustrates the step by step procedure in
conducting the study from preparation of materials up to the statistical
analysis of the results obtained.
Preparation of
Materials
Collection of Plants
Plant Extraction
Antioxidant Test
Cytotoxicity Test
Phytochemical
Screening
In vitro Anthelmintic
Assay
Analysis of Data
Proper Disposal
5
Bibliography
Fojas, F.R., Barrientos, C.M., Capal, T.V., Cruzada, S.F., Sison, F.M., Co,
Y.C., Chua, N.G. and Gavina T.L. Premilinary Phytochemical and
Pharmacological Studies of Sesbania grandiflora (L.) Pers. National
Institute of Science and Technology. Manila, Philippines:157.
Guevara, Beatrice. A Guidebook to Plant Screening: Phytochemical and
Biological. Manila: UST Publishing House, 2005.
Gowri, S. Shyamala, and K. Vasantha. "Antioxidant activity of Sesbania."
International Journal of Engineering Science and Technology 2
(2010): 4350-4356.
Gowri, S. Shyamala, and K. Vasantha. "Free Radical Scavenging and
Antioxidant Activity of Leaves from Agathi (Sesbania grandiflora
L.)." American-Eurasian Journal of Scientific Research 5, 2010: 114119.
Kachroo,
Vipin,
Arun
Kumar
Gupta,
and
Rajesh
Gupta.
"Pharmacognostical investigations on Sesbania grandiflora (L.)
Pers."
International
Journal
of
Pharmaceutical
Sciences
and
Research, 2011: 1069-1072.
Maurya, R., and P.P. Singh G. & Yadav. "Antiosteoporotic agents from
Natural sources. In: Atta-ur-Rahman (Ed.) Studies in Natural
Products Chemistry." Elsevier, 2008: Pp 517-545.
Mavi, A, Z Terzi, U Ozgen, A Yildirim, and M Coskun. Antioxidant
properties of some medicinal plants: Prangos ferulacea (apiaceae),
Sedum sempervivoides (crassulaceae) Malva neglecta (malvaceae),
6
Cruciata taurica (rubiaceae), Rosa pimpinellifolia (rosaceae), Galium
verum subspecies verum (rubiaceae), Urtica. Biol Pharm Bull, 2007.
Mosquera, OM, YM Correa, DC Buitrago, and J. Nifno. Antioxidant
activity of twenty five plants from Colombian biodiversity. Mem Inst
Oswaldo Cruz, 2007.
Neethu
S.
Kumar,
Dhanyaraj
F.S.
"Phytochemical
Analysis
and
Antimicrobial Activities of Sesbania Grandiflora (L) Leaf Extracts ."
International Journal of Pharmaceutical Sciences Review and
Research, 2015: Pp. 144-148 .
O'Dempsey, Tim. "Helminthic Infection." In Antibiotic and Chemotherapy,
by David Greenwood, S. Ragnar Norrby and Richard Whitley Roger
Finch, 842-859. 2011.
Rajagopal, P.L., Premaletha, K. and Sreejith, K.R. Anthelmintic activity of
the
flowers
of
Sesbania
grandiflora
Pers.
Department
of
Pharmacognosy and Phytochemistry, Academy of Pharmaceutical
Sciences, Pariyaram Medical College, Kannur, Kerala, India.
Journal of Innovations in Applied Pharmaceutical Sciences.
www.jiapsonline.com.
Sable, S., Dhawale, S., Dawalbage, A. Phytochemical analysis and invitro anthelmintic activity of Musa paradisiacal linn. And Sesbaina
grandiflora.
School
of
Pharmacy,
Swami
Ramanand
Teerth
Marathwada University, Nanded, Maharashtra, India. International
Journal of Chemical and Pharmaceutical Sciences: Vol. 4(2)-69-73.
2013.
7
Sarker, S.D. & Nahar, L. Chemistry for Pharmacy Students General,
Organic and. England: Wiley and Sons, 2007.
Sequin, Margarita. The Chemistry of Plants: Perfumes, Pigments and
Poisons. Royal Society of Chemistry, 2012.
Sumayya,
AR.,
"Preliminary
Sivagami
Srinivasan,
screening
of
and
Nabeelah
phytochemicals
in
Amatullah.
Agatikeerai."
International Journal of Pharmaceutical and Chemical Sciences Vol.
2 (2013): 594-596.
8