Upload
priyanka-gupta
View
673
Download
10
Tags:
Embed Size (px)
Citation preview
NEUROTOXICITY OF CYCASIN
IN
CHICK EMBRYO
ROBERT BROWN FOR THE FIRST TIME POINTED OUT THAT…..
WHAT HAD TILL BEEN REGARDED AS THE FEMALE FLOWER OF THE CYCADS WAS ACTUALLY
A NAKED OVULE
Robert Brown (1773–1858)
AS A RESULT OF THIS REMARKABLE DISCOVERY…………
CYCAS WAS EXCLUDED FROM DICOTYLEDONS &………
CAME TO RECOGNISED AS ..
GYMNOSPERM
……….However the first use of the Greek word gymnosperm have been already made as early as 300 b.c by the Greek philospher ……
THEOPHRASTUS in his “ENQUIRY INTO PLANTS”
GYMNOSPERM…..
An ancient group of plants dating back to the upper Devonian , roughly about 375 million year ago……..
A group of vascular plants whose seeds are not enclosed by a ripened ovary (fruit).
A group of plant which lack double fertilisation in the formation of endosperm
They are true land plants………..
Because they show……….
•Protection of gametophyte and young developing sporophyte from dessication and injury .
•Provision of nourishment for their proper development.
•Freedom from external water for the transference of the gametes
Gymnosperm achieved all these conditions by mustering 3 succesive generation in a single organ…….
SEED……..
•The parent sporophyte as …integuments & nucellus
•The gametophyte as ….endosperm
•The daughter sporophyte as……. embryo
integuments
nucellus
endosperm
embryo
gymnosperm
Cycadophyta (cycads)
Ginkophyta
(ginko)
Coniferophyta (conifers)
Gnetophyta (gnetae)
CYCAS
• SCIENTIFIC CLASSIFICATION
• Kingdom: PLANTAE Division: CYCADOPHYTAClass: CYCADOPSIDAOrder: CYCADALESFamily: CycadaceaePersoonGenus: Cycas
– They are evergreen, gymnospermous and dioceious plants having large pinnately compound leaves.
CYCAS…….
About 95 species are currently accepted. The best-known species is Cycas revoluta widely cultivated under the name "Sago Palm" or "King Sago Palm" due to its palm-like appearance although it is not a true palm
Approximate world distribution of Cycadales
EXPERIMENT……..
COMMENCMENT : 10 FEB 2008
FINISHED : 11 APRIL 2008
DURATION : ALMOST 2 MONTH
AIM…
TO STUDY THE EFFECT OF NEUROTOXIN PRESENT IN CYCAS SEEDS IN GROWING CHICK EMBRYO
MATERIAL REQUIRED….
1.PHYICAL APPARATUS
.CHINA DISH
.PISTAL & MORTAR
.BEAKER
.CUTTER
.PETRIDISH
.EGG STAND
.WATCH GLASS
2.CHEMICAL
.ETHANOL
.DISTILLED WATER
.FORMALDEHYDE
.SODIUM CHLORIDE(NaCl): 8g
.CALCIUM CHLORIDE(CaCl2):0.24g
.POTTASIUM CHLORIDE(KCl) :0.42g
.SODIUM BICARBONATE(NaHCO3):0.20g
3.BIOLOGICAL MATERIAL
.Cycas pectinata
.fertilised egg of hen
2 days old
4 days old
9 days old
15 days old
INSTRUMENTS……
.AN OVEN (set at 52 degree celcius )
this temperature depends on environmental condition.
.AN INCUBATOR (set at 37 degree celsius)
we used orbital shaker
.A DIGITAL CAMERA(for the pics)
Theory ……
Cycads produce several secondry compounds that arecarcinogenic and neurotoxic. Because of their uniqueplacement in terrestrial plant evolution, molecularstudies should help to define the origins of structuresthat led to the rise of seed plants and the role of neurotoxiccompounds that are found in cycads.
The subsequent search for a neurotoxiccompound in cycads lead to the identification of a
•Novel non-protein amino acid called BMAA (beta-methylamino L-alanine)
•methylazoxymethanol(MAM)-glycocides( cycasin macrozamin and neocycasin )
•sterol beta-D glucoside
STRUCTURE……
CYCASIN N MACROZAMIN
The toxic azoxyglycosides from the seeds of several species of Cycas.
Carcinogenic in mice, rats, hamsters, guinea-pigs, rabbits and fish.
Azoxyglycosides may have played an important ecological role as antiherbivore defenses.
ELECTRICAL
WEIGHING MACHINEWEIGHING LIMIT:
FOR MICROGRAM AND GRAM
Cycasin
1.3. Molecular formula, weight and structure:
2.C8H16N2O7; 253.233.4. Density: No data.4.5. Absorption spectroscopy: UV
absorption spectra have been reported (Riggs, 1956); λ (log ε)= 2187.
5.Solubility: Very soluble in water and dilute ethanol; less soluble in absolute ethanol; insoluble in
6.organic solvents.7.8. Description, appearance: Colorless, long,
needle-like crystals.
METABOLISM OF CYCASIN AND MACROZAMIN
The active component of all theses compounds is MAM (methylazoxyglycoside),
which is metabolised to a toxic intermediate by the p450 enzyme present in the liver and in the olfactory epithelium in the nose and inner membrane of mitochondria
TOXIC PRINCIPLEMETABOLISED
BY P450 ENZYME
NON TOXIC FORM
P450 ENZYME
Cytochrome P450 (abbreviated CYP, P450, infrequently CYP450) is a very large and diverse superfamily of hemoproteins found in all domains of life…
ncluding mammals,birds, fish, insects, worms, sea squirts, sea urchins, plants, fungi slime molds, bacteria and archaea
the most common reaction catalysed by cytochrome P450 is a monooxygenase reaction :RH + O2 + 2H+ + 2e– → ROH + H2O
PROCEDURE….
our first aim to isolate cycasin from cycas seeds..!
steps :
1 we were provided with 7 seeds of cycas pectinata
2 seed coat of cycas seed was quite hard so we first isolated seed form seed coat with the help of cutter.
3 we placed the naked cycas seeds in petridish
CYCAS SEED COVERED WITH HARD SEED COAT
MAIN CYCAS SEEDNAKED SEED
4 since before extraction of cycasin we need to quantitise the cycasin in cycas seeds, therefore we measured them in electrical weighing machine one by one and recorded them.RECORD : SEEDS WET WEIGHT(grams)
1 12.41
2 12.08
3 10.16
4 9.84
5 10.42
6 8.32
7 7.31
WET CYCAS SEEDS
PARTION BETWEEN THE SEED
5 since seeds were wet so we kept them in oven at 54 degree celcious to dry them.
6 our next step will be the crushing of dried seeds and soaking in water for extraction of cycasin.........
RECORD EARLIER WEIGHT : DRY WEIGHT1 12.41 : 6.50
2 12.08 : 4.71
3 10.16 : 4.922
4 9.84 : 5.884
5 10.42 : 4.69
6 8.32 : 4.87
7 7.31 : 3.10
After drying cycas seeds for 4 days in oven at 54degree celcius,
it was crushed in mortar and pistil untill it become fined powedered.
the powdered form of seeds were again weighed....,and it was found that their dry weight were almost 50% less than there originall weight..!
CYCAS FLOUR
CYCASIN SOLUTION
SOL A
CONC: 0.2 g/ml
Wash : 1st
Wash : 2nd
SOL A
CONC: 0.2 g/mlWash : 1stWash : 2nd
SOL B
CONC: 0.1 g/mlWash : 1stWash : 2nd
SOL C
CONC: 0.05 g/mlWash : 1stWash : 2nd
CYCAS SOLUTION
Solute: cycas flour
Solvent: distill water
Concentration :0.1 g /ml
DEALING WITH FERTILISED EGG
steps:
1 firstly we fixed the embryo in appropriate size beaker in such a way that narrow portion of head facing downward and broder portion facing ourself.
2 then with the help of needle a hole was made in the broder portion of egg.
3 then widened this hole with the help of scissor , this should be done very carefully without disturbing the vitelline membrane.
NORMAL EGG (CONTROL): WITHOUT CYCAS SOLUTION
EXPERIMENTAL EGG WITH CYCAS SOLUTION
INVITRO FERTILISED EGG
HOLES ARE MADE WITH NEEDLES WITHOUT DISTURBING THE VITELLINE MEMBRANE
HOLES ARE ALSO MADE IN NORMAL EGG FOR EQUIVALENT PARAMETERES
EGGS WERE EXAMINED AFTER 6 HOURS AND 24 HOURS
FERTILISED EGG YOLK
ALBUMIN TURNED GREENISH
YOLK
2 DAYS OLD EGG TREATED WITH CYCAS SOLUTION OBSERVED AFTER 24 HOURS
MORE TRANSPARENT
CONTROL EGG 2 DAYS OLD WITHOUT CYCAS SOLUTION
THIK YOLK
ALBUMEN
AFTER 24 HOURS (EXPERIMENTAL EGG 4 DAYS OLD)
A NORMAL (CONTROL) 4 DAY OLD EGG
RED SPOT INDICATE THE NERVE NETWORK
RED PORTION DENOTES SOMATOCYTES
NORMAL EGG 4 DAYS OLD
CYCAS SOLUTION TREATED 4 DAYS OLD EGG
NORMAL EGG 4 DAYS OLD
NERVE STRANDEXPERIMENTAL EGG
NERVE STRAND IN CONTROL EGG
A 9 DAY OLD CHICK EMBRYO
TREATED WITH CYCAS SOLUTION ..
REMAINED ALIVE AFTER 6 HOURS WITH ALL THE CONCENTRATIONS HOWEVER DIED AFTER 24 HOURS WHEN TREATED WITH SOLUTION A
A 9 DAYS OLD CHICK EMBRYO
DIED AFTER 24 HOURS WHEN TREATED WITH SOL A(HIGHLY CONCENTRATED ,1ST WASH)
A 9 DAYS OLD CHICK EMBRYO( ALIVE) NORMAL I.E NOT TREATED WITH CYCAS SOLUTION
A 18 DAYS OLD CHICK EMBRYO
SURVIVE WITH ALL THE CONCENTRATION AND WASHS
EGG EXPERIMENT OBSERVATION INFERENCE
2 DAYS
OLD
SOL A
WASH 1
WASH 2
ALBUMEN TURNED GREENISH IN BOTH CASE
MAY BE BECAUSE OH HIGH CONCENTRATION TOXIC IN CYCAS SOLUTION
SOL B
WASH 1
WASH 2
ALBUMEN TURNED YELLOWNO CHANGE
FIRST WASH CONTAIN RELATIVELY HIGH AMOUNT OF TOXIN THAN 2ND WASH
SOL C
WASH 1
WASH 2
NO CHANGENO CHANGE
EXTREMLY SMALL QUANTITY OF TOXIN IN BOTH WASH
OBSERVATION &DESCUSSION
EGG EXPERIMENT OBSERVATION INFERENCE
4 DAYS OLD
SOL A
WASH 1 –6HOURS
24 HOURS
WASH 2-6 HOURS
24 HOURS
NERVE NETWORK DEVELOPMENT RETARTED IN E.E IN COMPARISON TO N.EEGG SPOILED AND A VERY FOUL SMELL CAME & NERVE DEVELOPMENT WAS VERY LITTLE IN COMPARISON 2 N.ENERVE NETWORK REMAINS ALMOST SAME IN BOTHNERVE RETARTED BUT FOUL SMELL DIDN’T COME
HIGH CONCENTRATION OF TOXIN MIGHT KILLED SOME OF THE SOMATOCYTES HIGH CONCENTRATION MIGHT KILLED ALL THE SOMATOCYTES IN E.E & DESTROYED THE EGG IN SUCH A LONG TIME
COMPARETIVELY LOW CON. AND VERY LESS TIMELOW CON. AND LONG ACTION TIME
EGG EXPERIMENT OBSERVATION INFERENCE
4 DAYS
OLD
SOL B
WASH 1 –6HOURS
24 HOURS
WASH 2-6 HOURS
24 HOURS
NOT DISHTINGUISHABLE OBSERVATION
NERVE NETWORK SHOW RETARDATION IN DEVELOPMENT
NO CHANGE ALMOST SAME
NO CHANGE ,E.E DIDN’T SPOIL
LOW CONCENTRATION &LESS ACTION TIME
LOW CONCENTRATION &BUT RELATIVELY LONG ACTION TIME
EXTREMLY LOW CONCENTRATION
EGG EXPERIMENT OBSERVATION INFERENCE
4 DAYS
OLD
SOL C
WASH 1 –6HOURS
24 HOURS
WASH 2-6 HOURS
24 HOURS
NO CHANGE
NO CHANGE
NO CHANGE
NO CHANGE
MIGHT BE DUE TO EXTREMLY LOW OR ALMOST NEGLIGIBLE CONCENTRATION OF TOXIN
EGG EXPERIMENT OBSERVATION INFERENCE
18 DAYS
OLD
SOL A
WASH 1 –6HOURS
24 HOURS
WASH 2-6 HOURS
24 HOURS
NO EXTERNAL OBSERVATION
CHICK REMAINED ALIVE IN ALL THE CONDITIONS
HIGH RESISTANCE TO TOXIN DUE TO WELL DEVELOP IMMUNE SYSTEM
MIGHT BE THERE WERE CHANGES IN NERVOUS SYSTEM ACCORDING TO STUDIES BUT WE WERE UNABLE TO FIND THAT
THANX……..
DR.RITA MAM
2. JIBAN SIR
3. B.T DEPARTMENT
4.SAKSHI MAM
5.SWATI MAM
6.INTERNET SOURCES,
7. INTRODUCTION TO GYMNOSPERM BY DIVYA DARSHAN PANT.
8 PLANT ENCYCLOPEADIA
9CHICK EMBRYOLOGY..
ROMEIO N JULLIET