Poster Presentation 2014

8/12/2019 Poster Presentation 2014

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Oral Presentationand Defense

Group 6 Antoine Becquerel

Conference RoomMSHS

January 28,2014 Tuesday







Karen T. Fields

Angelika Joy R. Paragas

Beatrice Maile G. Ramin

Danielle Eunice G. Tolentino

Anabelle Rose T. Vivo





In 1961, a tragedy occurred in West Germany whichphysically deformed an estimated number of ten thousandinfants and the main cause of this was the teratogenic propertyin thalidomide, a sedative drug. In the Philippines, anomalies andmalformations rank third place in the leading causes of death forinfants. This research proposal intends to help prevent the

increasing mortality rate of infants because of malformationscaused by the teratogenic property in Bataw (Lablab purpureus )seed extract. Zebrafish (Danio rerio ) embryos were used toconduct the experiment on testing the level of concentration forthe teratogenicity of Bataw seed extract. Preparations for theembryos were done as the Bataw seed from fresh Bataw plantswas extracted by the researchers using the aqueous extracting

method. The fertilized eggs were tested using varyingconcentrations of the Bataw seed extract on petri dishes. Anobservation and data collection will be conducted for theembryos every 1 dpf, 3dpf and 5dpf for any changes to occur.Normally, the Zebrafish embryos must develop most of theirorgans in a day, but at 3 dpf no embryos hatched in 0.1concentration. Thus, their heartbeats were abnormal. The

observations were tallied and encoded in SPSS version 17.0.According to the analysis of the data, at both 0.01 level ofsignificance the null hypothesis is rejected. This means, we canconclude that Bataw seed is teratogenic and the study issignificant and relevant to medicine, in pregnancy; agriculture, inbreeding animals; economy, environment and for the generationsto come.



The goal is to know the level of concentration that give

malformation to the embryos. By knowing this, the goal is reachedAnother goal is to know the effects of the teratogenic propertof the Bataw seed extract to the embryos. The effect would varysome mild and small effects, and some will give extremmalformations. It depends on the concentration levels.



In 1961, a tragedy occurred in West Germany which deformemost ten thousand babies. Babies came out with abnormal physicaharacteristics. This tragedy was caused by the teratogenic propertf Thalidomide. Thalidomide is a sedative drug introduced in the lat950s by German physicians and was used to relieved morning sicknesnd insomnia (Markus Müller, 2010). People were not aware that ould show teratogenicity. Through this study, the researchers ca

nd out the possible teratogenic property of Bataw (Lablab purpureu) seed extract and save the risk of having another tragedy like thisOne of the harvested and known crops in the country is the Suga

ane. “Sugarcane is a known teratogen”; and is known to stimulatomatic mutations (aneuploidy and polyploidy) in plants (Lewis anlvin-Lewis, 2003) . The plant also contains hydrocyanic acidccording to a study conducted by Watt and Breyer-Brandwij

962), the Molasses of Sugar cane in excess amounts, alone or mixeith feeds, may cause diarrhea, colic, urticaria, kidney irritationweating and paralysis in domestic stock; horses seem morusceptible, and toxicity could prove fatal (Francis William Fox esmond Hallowes, 1982). Since the Philippines is rich in natural resources, the researcher

hought that there are some harvested crops which are not ye

tudied and might show teratogenicity. By proving the teratogeniroperty of a particular crop, many feeders/animal owners coulinimize the intake of these plants to animals and prevent problemhen breeding. Knowledge about these things is important and woulreatly save the risk of having another tragedy brought by theratogenic property of certain food or products. And one of thesarvested crops is the Bataw seed.



In particular, Hyacinth beans or Bataw contains high levels oyanogenic glycoside in their seeds - a plant compound that containugar and produces cyanide (Lindsay Allen & Andrew Prentice, 2005hus, cyanogenic glycoside is proven toxic in large amounts becaus

ating parts of a plant that contains the chemical can cause symptomf cyanide poisoning as the cyanogenic glycoside is converted tyanide by the digestive process. The concentration of the chemicaaries amongst species of plant and often, significant quantities areeded to produce symptoms such as headache, chest tightnesshroat tightness, Muscle weakness, and Neuropathy. Neverthelessevere poisoning can result in death (Rightdiagnosis.com, 2011) .

As researchers, they want to study on the chemical, cyanogeniycoside in Bataw seed to see the plant’s possible occurrence o

eratogenicity that would lead abnormalities on a growing embryo oetus. Through this study, it can generate new information about thossible teratogenic property of Bataw seeds and could save the risf having another tragedy regarding teratogenicity.



At least two to three percent of newborn babies worldwide havecongenital abnormalities. According to Dr. Beda Espineda,president of the Philippine Society of Pediatric Surgeons, about

30 are born with major anomalies, 80 percent of which are life-hreatening or need immediate surgery in the Philippines. By

proving the teratogenic property of Bataw seeds, it can helppeople to be aware and can also minimize the mortality andmalformation rate of babies in the Philippines. The research studycan benefit the following people:

Pregnant Women – The research intends to help people, especiallyhose who undergo pregnancy, to be aware of the possibleeratogenic property of Bataw (Lablab purpureus L.) seed extract

and can prevent malformations in the fetus.

Animal breeders - Since most jobs, especially in provinces, areabout taking care of animals, this research would be of great help

by minimizing the Bataw seed intake to animals to preventproblems when breeding.

Future Researchers– The study can also help future researcherso gain information presented in this study and can also serve as a

guide for them.

Organisms in the environment- Many organisms that feed on Bataweed may also profit from this research. The mortality and

abnormality rate of these organisms can be minimized.

Environment – The study could also reduce the mortality rate ofome animals that feed on Bataw seed which can affect the food

chain and the food web of other animals. Through this study, the

environment can maintain homeostasis.





Research Design

The researchers chose the Completely Randomized Designhis design involves a group of test plants or animals whicre studied only once but subsequent treatment is applied tetermine the cause of change. Thus, the experimental unitre homogenous.This study utilized 72 Zebrafish embryos and 12 replicate

or each treatment. These embryos were grouped in to twelvest animals each micro titer plate. Different groups underg

arying treatments with each group having individuareatments each. The experimental group consisted of fouoncentrations: A. 10 g/ml of 100% (22.5g/500moncentration of Bataw seed aqueous extract diluted to 10l distilled water, B. 1 g/ml of 100% (22.5g/500moncentration of Bataw seed aqueous extract diluted to 10

l distilled water, C. 0.1 g/ml of 100% (22.5g/500moncentration of Bataw seed aqueous extract diluted to 10l distilled water, and D. 0.01 g/ml of 100% (22.5g/500moncentration of Bataw seed aqueous extract diluted to 10l distilled water. For the control group, the negative controthe M. test medium/ fish water and the positive contro

ill be 2% Ethanol. In addition, the sets of Zebrafish embryoere numbered 1 to 72 and randomization will be utilized bssigning a corresponding letter of the concentration of thataw seed aqueous extract. Letters were drawn from a boxherefore, assigning each embryo randomly to the differenet-ups.



Legend:A. 10 g/ml of 100% (22.5g/500ml) concentration of Bataw see

queous extract diluted to 100 ml distilled waterB. 1 g/ml of 100% (22.5g/500ml) concentration of Bataw see

queous extract diluted to 100 ml distilled waterC. 0.1 g/ml of 100% (22.5g/500ml) concentration of Bataw see

queous extract diluted to 100 ml distilled waterD. 0.01 g/ml of 100% (22.5g/500ml) concentration of Bataw seequeous extract diluted to 100 ml distilled water

E. Positive Control (2% Ethanol extract)F. Negative Control (Test medium/ fish water)

E1 A E13 D E25 B E37 F E49 E E61 C

E2 D E14 A E26 C E38 B E50 F E62 E

E3 C E15 A E27 E E39 B E51 D E63 F

E4 B E16 D E28 F E40 C E52 E E64 A

E5 E E17 F E29 A E41 D E53 C E65 B

E6

BE18

DE30

FE42

EE54

AE66

C

E7 E E19 C E31 B E43 F E55 D E67 A

E8 A E20 E E32 C E44 B E56 D E68 F

E9 D E21 F E33 A E45 E E57 B E69 C

E10 C E22 A E34 E E46 B E58 F E70 D

E11 B E23 D E35 C E47 F E59 A E71 E

E12 F E24 D E36 A E48 E E60 C E72 B



Sampling Design and Technique

The researchers used the pure random sampling technique sinche population has no differentiated levels. There are 6 setups. Th

etups are treated with varying concentrations of the Bataw seequeous extract and Control groups. Since there are 72 Zebrafismbryos, a corresponding number were assigned to each one. Letterere drawn from a box, therefore, assigning each subject randomly tfferent treatments of Bataw seed aqueous extract.This sampling technique helped the researchers mak

andomization of each subject to different treatments easier.



TESTING THE EFFECT DIFFERENTSET-UPS

TERATOGENIC TESTING

OBTAINING OF ZEBRAFISH

DATA GATHERING PROCEDURE

PREPARATION OF SET-UPS

FEEDING OF ZEBRAFISH

REARING OF ZEBRAFISH

COLLECTION OF FERTILIZED EGGS

AUTHENTICATION OF BATAWSEEDS

Set-upA

10 g/mlof 100%(22.5g/5

00ml)

Set-upB

1 g/ml of100%

(22.5g/500ml)

Set-upC

0.1 g/mlof 100%(22.5g/5

00ml)

Set-upD

0.01 g/mlof 100%(22.5g/5

00ml)

POSITIVECONTROL

2%Ethanolextract

NEGATIVECONTROL

Embryomedium





There are two hypotheses in the study, the null

hypothesis and the alternative hypothesis. The statistical testfor the study is Analysis of the Variance (ANOVA) becausethere is only one factor or independent variable being studied,which is the Bataw seed. And this factor has 6 different levelsand concentrations that were tested on the dependentvariable, the Zebrafish. Overall, there are more than twomeans involved in the data table of research design. Thus, ithelps on the comparison of effects of varying concentrations.There are 6 setups, 4 experimental groups and 2 control group.The level of significance to be used is 0.05. Through this test,the researchers discerned on what concentration of theextract exhibits most abnormalities in the embryo of theZebrafish. The post-hoc test named Duncan’s Multiple RangeTest, is able to compare means of each concentration and tellif they are significantly different or not from each other. Thiswas able to discern what concentration is most teratogenic.





• Phytochemical analysis concluded on the Bataw seed extractsrevealed the presence of cyanogenic glycosides.

• Cyanogenic glycosides are compounds that contain sugar andproduce hydrogen cyanide (HCN). (Linsay Allen, AndrewPrentice, 2005).

• Hydrogen cyanide performs the metal-containing enzymes.

• Cytochromoxidase is one example of metal-containing enzymewhich contains iron; this enzyme system is responsible for theenergy providing processes in the cell where oxygen isutilized. (Petersen Jk, 2007).

1. Experiments to determine the sublethal concentrations ofBataw (Lablab Purpureus L. ) seeds aqueous extract which isbetween the 0.1%-1% concentration.

2. Further studies to understand the underlying possiblemechanisms of the observed activities as well as need toisolate, purify, and to characterize active phytochemicalsof Bataw (Lablab Purpureus L. ) seeds, including cyanogenicglycoside that is said to be responsible for the bioactivity.



Conclusion

The results showed the effects of the varyingconcentrations of Bataw seed aqueous extract on the

Zebrafish embryos in terms of survival rate at 1dpf and5dpf,hatch rate at 3dpf,heart rate (beats per minute),number of melanocytes and larval length (in mm). Thetests that were conducted revealed that the Bataw seedaqueous extract only showed teratogenicity in the set-upwith 1% concentration. Although the embryos did not showphysical malformations, it showed hindrance in its

development. Other concentrations that were lower than1% showed normal development whereas the concentrationhigher than 1%, which is 10%, caused death to theembryos. The researchers concluded that Bataw seedaqueous extract was teratogenic within a certain range.Although they were not able to find the exact sublethalconcentration wherein the Bataw seed aqueous extract’s

teratogenicity would be most evident, they were able todetermine the range which is from 1% to 10%.



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http://www.maribehlla.com/vegetables/bataw/

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Poster Presentation 2014