screening on plant extracts for ovicidal activity … · nº de proyecto: fair5-pl97-3355. ......
TRANSCRIPT
SCREENING ON PLANT
EXTRACTS FOR OVICIDAL
ACTIVITY AGAINST
Deroceras reticulatum EGGS
University of Santiago de Compostela
Faculty of Biology
Spain
Autores:, José CASTILLEJO, Javier IGLESIAS,
* Investigación financiada por:
• la Unión Europea. Nº de Proyecto: FAIR5-PL97-3355.
Título: “Novel technologies for integrated control of slug damage in key horticultural crops”
• la Xunta de Galicia. Nº de Proyecto: PGIDT00AGR20001PR
Título: “Alternativas a los molusquicidas comerciales actuales”
Objectives
PLANTS
PLANT EXTRACTS
ACTIVE PRODUCT
SYNTHETIC
PRODUCT
® PATENT
COMERCIALIZATION
HOW and
WHEN to
apply it ?
AGAINST EGGS
AGAINST ADULTS
Preparing the extracts for testing:
Plants were collected and identified
Different parts of the plants were separated:
Rhizomes Leaves Fruits
After dry, were powdered with a domestic coffee grinder
They were stored in bottles
Seeds Flowers Roots
Preliminary tests
Water extracts
Acetone/water extracts (7:3)
Water extracts
The powder of the plant was allowed to macerate
during 24 hours
The extract was filtered and applied to the dishes
Control dishes receives only water
Then were dried at environment temperature
Acetone / Water extracts (7:3)
After macerate, the extract was filtered
Acetone was removed in the rotavapour
Control dishes received the mixture acetone/water
after remove the acetone
Testing the plants:
5 doses of the extract and one control. The doses were arranged in a ten-fold geometric series
3 replicates per dose
3 to 5 eggs per replicate
1 ml of extract was applied to each dish
After dry were remoistened with 1 ml of water
Digitalis purpurea L.
Common name: Foxglove (España:
dedalera)
Constituents:
1. glucosides : digitoxin, digitalin,
digitalein, digiton
2. volatile oil
3. fatty matter
4. gum
5. sugar
FOXGLOVE: any Eurasian scrophulariaceous plant of the
genus Digitalis, esp. D. purpurea, having spikes of purple or
white thimble-like flowers. The soft wrinkled leaves are a
source of digitalis
Digitalis’ extract tested
LEAVES
FRESH
Fp
WATER
(ppm)
80,000
40,000
8,000
ACETONE
/ WATER
(ppm)
100,000
Fp = Filter paper
Ss = Standard soil
Eucalyptus globulus Labill.
Common name: Tasmanian blue gum.
Constituents: Monoterpenes: a-pinene, limonene, b-pinene, p-cymene, terpinene, aromadendrene....
Sesquiterpenes, Monoterpene alchohols, sesquiterpenols, aldehydes.
Oxides: cineole
BLUE GUM: a tall fast-growing widely cultivated
Australian myrtaceous tree, Eucalyptus globulus,
having aromatic leaves containing a medicinal oil, bark
that peels off in shreds, and hard timber. The juvenile
leaves are bluish in colour
Eucalyptus’ extracts tested
LEAVES STEAMS
FRESH DRY FRESH DRY
Fp Fp Fp Fp
WATER
(ppm)
80,000
40,000
8,000
ACETONE/
WATER
(ppm)
100,000
Fp = Filter paper
Ss = Standard soil
Euphorbia helioscopia L.
Common name: Sun spurge (Euforbia)
Constituents: euphorbone; 12-deoxyphorbol; wax; resin; lignin; basorrin; volatile oils; euphorbo-resene; euphorbic acid; calcium malate
SPURGE: Any of various euphorbiaceous plants of the
genus Euphorbia that have milky sap and small flowers
typically surrounded by conspicuous bracts. Some species
have purgative properties
Euphorbia’s extracts tested LEAVES STEAMS ROOTS FRUIT
FRESH DRY FRESH DRY FRESH DRY FRESH DRY
Fp Fp Fp Fp Fp Fp Fp Fp
W
A
T
E
R (ppm)
100,000
80,000
50,000
40,000
8,000
ACENTO
NE /
WATER
(ppm)
100,000
Foeniculum vulgare Miller.
Common name: Fennel (Hinojo)
Constituents: Monoterpenes: limonene; Monoterpenols: fenchol; Phenols: methyl chavicol; Aldehydes; Cetones: camphre; Oxides: cineole; Coumarines; Furocumarines. FENNEL: a strong-smelling yellow-flowered umbelliferous
plant, Foeniculum vulgare, whose seeds and feathery leaves
are used to season and flavour food
Foeniculum’s
extracts tested
LEAVES STEAMS
FRESH DRY
Fp Fp
WATER
(ppm)
80,000
40,000
8,000
ACETONE /
WATER
(ppm)
100,000
Laurus nobilis L.
Common name: Bay,
laurel.
Constituents: volatile
oils (cineole, pinene,
sabinene, eugenol acetate,
terpenoids...), tannic acid,
bitter principles.
LAUREL: a small Mediterranean lauraceous
evergreen tree, Laurus nobilis, with glossy
aromatic leaves, used for flavouring in cooking,
and small blackish berries
LEAVES
FRESH DRY
Fp Fp
W
A
T
E
R
(ppm)
80,000
40,000
8,000
Laurus’ extracts tested
Rosmarinus officinalis L.
Common name:
Rosemary (romero)
Constituents: 1. volatile oils: borneol, linalol,
esters, cineol, d-pinene,
camphene
2. Acids: phenolic, triterpenic,
rosmaricine, isorosmaricine
3. Flavonoids: diosmin
ROSEMARY: an aromatic European shrub,
Rosmarinus officinalis, widely cultivated for its
grey-green evergreen leaves, which are used in
cookery for flavouring and yield a fragrant oil
used in the manufacture of perfumes: family
Labiatae (labiates). It is the traditional flower of
remembrance
LEAVES
FRESH DRY
Fp Fp
WATER
(ppm)
80,000
40,000
8,000
ACETONE
/ WATER
(ppm)
100,000
Rosmarinus’ extracts tested
Rubia peregrina L.
Common name: Wild
madder.
Constituents: rubian,
rubiadin, ruberythric acid,
purpurin, tannin, sugar, alizarin,
pseudopurpurin,
xanthopurpurin.
MADDER: any of several rubiaceous plants of
the genus Rubia, esp. the Eurasian R. tinctoria,
which has small yellow flowers and a red fleshy
root.
Rubia’s extracts tested
LEAVES
FRESH
Fp
WATER
(ppm)
80,000
40,000
8,000
Ruta graveolens L.
Common name:
Rue (Ruda)
Constituents:
furocouamarines, rutin, volatile
oils, alkaloids, coumarines.
RUE: any rutaceous plant of the genus Ruta,
esp. R. graveolens, an aromatic Eurasian shrub
with small yellow flowers and evergreen leaves
which yield an acrid volatile oil, formerly used
medicinally as a narcotic and stimulant. Archaic
name: herb of grace
Ruta’s extracts tested LEAVES STEAMS FLOWERS
FRESH DRY FRESH DRY FRESH DRY
Fp Ss Fp Ss Fp Ss Fp Ss Fp Ss Fp Ss
W
A
T
E
R (ppm)
100,000
80,000
50,000
40,000
10,000
8,000
1,000
100
A
C
E
T
O
N
E
/
W
A
T
E
R
(ppm)
100,000
80,000
50,000
40,000
10,000
8,000
1,000
100
Fp = Filter paper
Ss = Standard soil
Cistus ladanifer L.
Common name: Rock
rose (Heliantemo)
Constituents:
Monoterpenes: pinene,
camphene; Monoterpene
alcohols; Esters: linalyle
acetates; Phenols: eugenol,
thymol; Aldehydes; Cetones.
ROCKROSE: any of various cistaceous shrubs or
herbaceous plants of the Mediterranean genera
Helianthemum, Tuberaria, and Cistus, cultivated
for their yellow-white or reddish roselike flowers.
Cistus’ extracts tested
LEAVES
DRY
Fp
W
A
T
E
R
(ppm)
100,000
50,000
25,000
12,500
6,250
DEFINITIVE Ruta’S EXPERIMENTS ON
FILTER PAPER
LEAVES
DRY
Fp
W
A
T
E
R
(ppm)
80,000
40,000
20,000
10,000
5,000
ACETONE /
WATER
(ppm)
80,000
40,000
20,000
10,000
5,000
Ruta’s dry leaves
water extracts
Mortality over time
0
20
40
60
80
100
0 5 10 15 20
Time in days
% m
ort
ali
ty
80000 ppm 40000 ppm 20000 ppm
Dose-response curve/ LD50
22939
18455
32707
0
20
40
60
80
100
1000 10000 100000
Dose in ppm (log scale)
% m
ort
ali
ty
5 days 7 days 10 days LD50
Hatches
0
20
40
60
80
100
0 5 10 15 20 25
Time in days
% h
atc
h
Control 10000 ppm 5000 ppm
Ruta’s dry leaves
acetone / water (7:3) extracts
Mortality over time
0
20
40
60
80
100
0 5 10 15 20 25
Time in days
% m
ort
ali
ty
80000 ppm 40000 ppm 20000 ppm
10000 ppm 5000 ppm
Dose-response curve
27841786216907
0
20
40
60
80
100
1000 10000 100000
Dose in ppm (log scale)
% m
ort
ali
ty
2 days 3 days 5 days LD50
Hatches
0
20
40
60
80
100
0 5 10 15 20 25
Time in days
% h
atc
h
Control 5000 ppm 2500 ppm 1250 ppm
Asphodelus album Mill.
Common name: Asphodel
Constituents: alcohol, inuline, volatile oil
ASPHODEL: any of various S European liliaceous
plants of the genera Asphodelus and Asphodeline,
having clusters of white or yellow flowers. Compare
bog asphodel.
Asphodelus’ extracts tested
FRUITS
DRY
Fp
W
A
T
E
R
(ppm)
100,000
50,000
25,000
12,500
6,250
Asphodelus’ dry fruits
water extracts
Mortality over time
0
20
40
60
80
100
0 5 10 15 20
Time in days
% m
ort
ali
ty
100000 ppm 50000 ppm
Dose-response curve
53938
0
20
40
60
80
100
10000 100000
Dose in ppm (log scale)
% m
ort
ality
3 days LD50
Hatches
0
20
40
60
80
100
0 5 10 15 20 25
Time in days
% h
atc
h
Control 50000 ppm 25000 ppm
12500 ppm 6250 ppm
CONCLUSIONS
Up to now, Ruta and Euphorbia extracts showed the highest ovicidal activity on paper at the lowest doses and showed some ovicidal activity on soil.
From now on, although we shall continue the tests with new plants, we shall concentrate the research on these two plants.
We have already started to do a number of
new extracts of both Ruta and Euphorbia.
Using a variety of solvents in order to locate
and if possible, to identify the active
constituents responsible of the ovicidal
activity.
Also, for this year we plan to test some of
the extracts on natural soil under semi-
natural conditions.
FRACTIONAL
EXTRACTION OF
SOLVENTS
PLANT EXTRACTS
The process has 4 steps:
Maceration 3 times with MeOH
Concentration and test
Division of solvents
Concentration and test
First and second step: maceration
with MeOH, concentration and test.
Dry powder plant was put in an Erlenmeyer flask.
The solvent was put on it. After 10 hours the solution was passed through a filter paper.
The solvent was concentrated in a rotavapour. Over the plant was placed more MeOH.
This process was repeated 3 times.
The solvent was accumulated in the same glass ball. Then it was concentrated to vacuum in the rotavapour.
Part of the extract obtained was used in the tests, the rest of them was used in the extraction process.
Powder of the plant with MeOH
ROTAVAPOUR
Third and fourth step: division of
solvents, concentration and test.
The solvents used were:
o Cl4C
o CH2Cl2
o Hexane
o BuOH
SUMMARY
METHANOLIC EXTRACT
Hexanic extract
Cl4C extract
CH2Cl2 extract
n-BuOH extract Water extract
MeoH:H2O 8:2
Cl4C
MeOH:H2O 9:1
Hexane (x2)
MeOH:H2O 6:4
CH2Cl2
1º) remove MeOH
2º) n-BuOH (x4)
CONTROL DE LAS PLAGAS DE BABOSAS EN CULTIVOS
HORTÍCOLAS. EFECTOS COLATERALES DE LOS
MOLUSQUICIDAS SOBRE LA FAUNA EDÁFICA*.
1.- Indica los tipos de disolventes empleados para extraer los principios activos
empleados como ovicidas
2.- Indica 2 plantas cuyos extractos tienen efecto ovicida
3.- Dibuja un huevo de la babosa Deroceras reticulatum y señala sus partes
4.- Indica las partes de las plantas donde se encuentran los principios activos
con acción ovicida y señala los procesos previos que se realizan antes de
proceder a la extracción de estos
5.- Indica las causas por las que los extractos de Ruda tiene efecto ovidica sobre
papel de filtro, pero no sobre suelo estándar (artificial)
6.- Factores exógenos que afectan al ciclo biológico de las babosas
* En el respeto al equilibrio está el éxito de nuestra lucha particular
Fin de la Comunicación
*