Transcript Slide 1

RESEARCH ON HERBAL DRUGS FOR
POULTRY & LIVESTOCK
BACKGROUND INFORMATION
• Two researches on local herbal plants conducted
 Herbal drugs for Haemophilus paragallinarum infection
 Herbal drugs for Haemonchus contortus infection
OBJECTIVES OF TWO RESEARCHES
• Screen the inhibitory activity of local plants used by local farmers
against infectious coryza/haemonchusis
• Find out the local practices adopted by farmers in using plants
for infectious coryza/haemonchusis
• Determine the LD50 and ED50 of plant drugs
• Find out the quality of binders of tablet forms
of the plant drugs for infectious coryza
• Determine the shelf-life of plant drugs
MATERIALS AND METHODS
Steps:
• Consultation with farmers
• Identification of local plants
• Diagnosis of diseases
• Culture of causative agents of diseases
• In vitro assay of local plants for their bactericidal/larvicidal actions
• Pharmacologic studies of herbal drugs
• Quality control study of herbal drugs
Farmer Consultations
• Interview with local farmers on:
 Clinical signs of a particular disease in poultry/livestock
 Local plants for treatment of a particular disease
in poultry/livestock
 Preparation & dosage level of a particular herb
Identification of local plants used by farmers
Diagnosis of diseases based on clinical signs
• Isolation of Haemophilus paragallinarum in pure culture
 Blood agar/nutrient broth culture
 Gram staining & Biochemical tests
 Infection and isolation of H. paragallinarum
• Isolation of Haemonchus contortus
 Culture of eggs & infection of goats
Culture of Infective Stage for In Vitro Assay
• Stock of pure culture of H. paragallinarum
 Blood agar
• Supply of eggs for culture of H. contortus
 Infection of goats with L3s for supply of eggs
Identification of
Infected Goats
by fecalysis
Collection of Fecal
Sample
isolation of eggs of H. contortus
Fecalysis
(Floatation Technique)
Infected Goats
Identified
Collect feces
Culture Feces for L3s
of H. contortus
Retrieval & Identification of L3s
L3s of H. contortus
3 Goats Infected
With L3s*
*Source of eggs for culture of L3s
Culture of Haemonchus larvae
Moistened cotton wads placed at the
bottom of shot glass
Feces from infected goats macerated
and placed on top of cotton wads
Sides of shot glass filled with tap water
Culture of Haemonchus larvae
Shot glass in Culture glass, filled with
water up to the brim of shot glass
Culture glass covered with wrappings and
incubated at room temperature for 6 days
Recovery of Infected Larvae (L3) of H. Contortus
Removal of wrappings of shot glass
Water in the culture glass transferred
into petri plate
Infective Larvae (L3s) recovered in Petri
plate identified
Preparation of Plant Extracts:
• Decoction using 1:2 (w/v)
• Dried plant parts were boiled for 15 minutes
• Crude extracts were filtered
• Filtrate was concentrated in a rotavapor (40oC) until
volume was reduced to 1/3 of its original volume
• Concentrated plant extract was placed in the oven (60oC) overnight
• Plant residues were collected & kept in the fridge for 1 wk.
IN VITRO ASSAYS OF LOCAL PLANT EXTRACTS
Techniques Used in In Vitro Assay
• Sensitivity test of Carter for Haemophilus gallinarum
• Larvicidal test for Haemophilus gallinarum
RESULTS
Plants and Parts used by local farmers for the treatment against H.
paragallinarum infection in chicken (Fernandez, 1990)
Scientific Names
Common
Names
Local Names
Plant Parts
Sweet Sop
Atis
Leaves
Capsicum frutescens
Wild Pepper
Siling labuyo
Fruits
Chrysanthemum indicum
Mansaninya
Hilbas
Leaves
Citrus grandis
Pomelo
Buongon
Rinds
Coleus aromaticus
Oregano
Karabo
Leaves
-
Mayana (red)
Leaves
Sunflower
Sunflower
Seeds
Elephant Grass
Trompa Elefante
Leaves
Spondias pinnata
-
Libas
Leaves
Solanum spp.
-
Terramycin Plant
Fruits
Ginger
Luya
Root
Ananas squamosa
Coleus blumei
Helianthus annuus*
Heliotropium indicum
Zingiber officinale
*Fruits given raw to sick birds
In vitro assay of plant extracts* vs H. paragallinarum
Zone of Inhibition (cm)
Treatment
No.
Plant Extracts
After 1 h
After 24 h
NZ
NZ
T0(-)
Distilled Water
TO(+)
Streptomycin disc**
3
5
T1
Anona squamosa
1
NZ
T2
Capsicum frutescens
NZ
NZ
T3
Chrysanthemum indicum
NZ
NZ
T4
Citrus grandis
1.5
NZ
T5
Coleus aromaticus
NZ
NZ
T6
Coleus blumei
NZ
NZ
T7
Helianthus annuus
NZ
NZ
T8
Heliotropium indicum
3
6
T9
Spondias pinnata
2
3
NZ
NZ
1
NZ
T10
Solanum spp.
T11
Zingiber officinale
*1:2 (w/v) 1 part plant part: 2 parts distilled water by decoction,
amount in each wellwas 0.05ml
**Sensi discs, BBL Microbiological systems
In vivo assay of plant extracts* against H. paragallinarum infection in chicken (Lohman)
Treatment
No.
Plants
No. of
Birds
Examined
Water Eye &
Nasal
Discharge
Facial
Swelling
Mucus in Upper
Respiratory Tract
Congeste
d Lungs
TO
Untreated Control
4
4
4
4
4
T1
Anona squamosa
6
4
3
3
0
T2
Capsicum frutescens
6
3
6
6
3
T3
Chrysanthemum indicum
6
4
5
5
2
T4
Citrus grandis
6
3
3
3
0
T5
Coleus aromaticus
5
4
4
4
0
T6
Coleus blumei
6
2
2
2
1
T7
Helianthus annuus
5
2
2
2
1
T8
Heliotropium indicum
5
0
0
0
0
T9
Spondias pinnata
6
3
3
3
0
T10
Solanum spp.
6
3
4
4
0
T11
Zingiber officinale
5
1
2
2
0
*1:2 (w/v) 1 part plant part: 2 parts distilled water by decoction,
amount given to each bird was 5ml, given per os
Chicken Showing Clinical Signs of Haemophilus paragallinarum Infection
Chicken Showing Clinical Signs of Haemophilus paragallinarum Infection
Percent efficacy* of plant water extract by decoction according to animal
species against Haemonchus contortus (Fernandez, 1991)
Chicken (%
Efficacy)
Swine (%
Efficacy)
Goat (% Efficacy)
63.3
59.1
52.3
NI
54..7
56.7
Mangifera indica
60.0
58.2
56.7
Manihot esculenta
53.4
52.0
50.8
Tamarindus indica
59.4
58.4
55.6
Moringa oleifera
69.0
64.7
61.5
Artemisia vulgaris
57.6
56.3
53.3
Mimosa pudica
NI
NI
79.7**
Chrysophyllum cainito
NI
NI
70.3**
Tinospora rumphii
NI
NI
85.6***
Plants
Ananas comosus (fruit)
Ananas comosus (leaves)
*Based on formula of Reik and Keitz (1954)
NI = not used as anthelmintic by local farmers in a particular
animal species
Preparations, dosage levels and mode of administration of plant parts used
by local farmers against parasitic infections in various animals
Group
No.*
Scientific Names
(Local
Names)
Plant
Parts
Dosages
Preparatio
ns
Administration
Chicke
n
Swine
Goat
TO
Control**
-
-
-
-
-
-
T1
Ananas comosus
(pina)
Unripe
fruit
pounding
15 g
30 g
40g
Mixed w/ feed
T2
Artemisia vulgaris
leaves
decoction
15 ml
30 ml
40 ml
Per os
T3
Bixa orellana
seeds
decoction
5 ml
10 ml
20 ml
Per os
roots
decoction
15 ml
30 ml
40 ml
Per os
seeds
pounding
15 g
30 g
40 g
Per os
(hilbas)
(Asuite)
T4
Cajanus cajan
(Kadios)
T5
Cassia alata
(sunting)
T6
Chrysophylum
cainito (caimito)
leaves
decoction
-
-
40 ml
Per os
T7
Clitorea ternatea
seeds
heating &
pounding
15 g
30 g
40 g
Mixed w/ feed
(balog-balog)
*No. of Animals/treatment = 4
**Untreated control
- not used by farmers
Preparations, dosage levels and mode of administration of plant parts used
by local farmers against parasitic infections in various animals
Group
No.*
T8
Scientific Names
(Local
Names)
Karicq papaya
Plant
Parts
Dosages
Administration
Preparatio
ns
Chicken
Swine
Goat
seeds
pounding
15 g
-
40 g
Per os
(kapayas)
T9
Lansium
domesticum
(lansones)
seeds
pounding
15 g
30 g
40 g
Per os
T10
Leucaena
leucocephala (ipil-
seeds
Pounding
15 g
30 g
40 g
Per os
Mangifera indica
seeds
Pounding
15 g
10 g
30 g
Per os
ipil)
T11
(paho)
T12
Manihot esculenta
(balanghoy)
bark/
roots
Decoction
15 ml
30 ml
40 ml
Per os
T13
Mimosa pudica
leaves
decoction
-
-
40 ml
Per os
(makahiya)
T14
Momordica
charantia (paliya)
leaves
Heating/
expressing
3 ml
6 ml
12 ml
Per os
T15
Moringa oleifera
seeds
pounding
15 g
30 g
40 g
Per os
(malunggay)
*No. of Animals/treatment = 4
- not used by farmers
Preparations, dosage levels and mode of administration of plant parts used
by local farmers against parasitic infections in various animals
Dosages
Group
No.*
Scientific Name
(Local Name)
T16
Quisqualis
indica
Plant
Parts
Preparation
Administration
chicken
Swine
Goat
seeds
pounding
15 g
30 g
40 g
Per os
(niyog-niyogan)
T17
Tamarindus
indica (Sambag)
leaves
decoction
15 ml
30 ml
40 ml
Per os
T18
Tinospora
rumphii
stem
decoction
-
-
40 ml
Per os
(panyawan)
*No. of Animals/treatment = 4
- not used by farmers
Percent efficacy of plant parts according to animal species
Plants
Chicken
(% Efficacy)
Swine
(% Efficacy)
Goat
(% Efficacy)
Control
-7.1
-13.7
-7.8
Ananas comosus
63.3
59.1
52.3
Artememisia vulgaris
57.6
56.3
53.3
Bixa orellana
66.0
61.4
58.3
Cajanus cajan
57.7
53.3
53.3
Cassia alata
62.8
59.1
57.0
NI
NI
70.3
Clitorea ternatea
62.8
58.7
55.6
Karica papaya
59.4
NI
58.8
Chrysophyllum cainito
NI = not used by local farmers
*highly effective
Percent efficacy of plant parts according to animal species
Plants
Chicken
(% Efficacy)
Swine
(% Efficacy)
Goat
(% Efficacy)
Lansium domesticuml
65.6
61.0
60.7
Leucaena leucocephala
68.0
62.1
62.1
Mangifera indica
60.0
58.2
56.7
Manihot esculenta
53.4
52.0
50.8
NI
NI
79.7
Momordica charantia
74.7
70.0
64.5
Moringa oleifera
69.0
64.7
61.5
Clitorea ternatea
62.8
58.7
55.6
Quisqualis indica
70.7
70.0
62.4
Mimosa pudica
NI = not used by local farmers
Percent efficacy of plant parts according to animal species
Plants
Chicken
(% Efficacy)
Swine
(% Efficacy)
Goat
(% Efficacy)
Tamarindus
indica
59.4
58.4
55.6
Tinospora
rumphii
NI
NI
85.6*
NI = not used by local farmers
*highly effective
On-Going Study on Haemonchus Contortus
Rationale
Goat Population:
As of 2002, 3.29 million heads
25% are concentrated in the Visayas
99.9% of 3.29 millions are raised by small holders
( in rural areas)
Source: Philippines Recommends for Goat Production, PCARRD (2004)
Constraints in Goats Production:
Endoparasitism hampers productivity & full development
of goat subsector
Ways endoparasites hampered productivity of goats:
1. High rate mortality at weaning
2. Drop in milk production
3. Reduced feed conversion efficiency
4. Mortality of productive goats
Economic Impact due to roundworm infection in goats
is valued at US$3.55 M annually
Source: Philippines Recommends for Goat Production, PCARRD (2004)
Haemonchus contortus is one of these endoparasites
considered most pathogenic (Soulsby, 1982)
Haemonchus contortus infection in ruminants is addressed
by use of athelmintic or dewormers
Two classes of anthelmintic that could be resorted to:
1. Commercial or synthetic anthelmintic
2. Herbal anthelmintic
Advantage of synthetic anthelmintic:
1. Being pure, synthetic anthelmintic is very effective
Advantage is outweighed by the following:
1. Imparts residue to meat which is hazardous to consuming public
2. Parasite develops resistance against synthetic anthelmintic
3. Synthetic anthelmintic pollutes environment
4. Expensive for small holders, being imported
5. Not available in remote areas
Use of alternative dewormer: Herbal anthelmintic
Advantages of Herbal anthelmintic:
1. Does not impart drug residue in meat
2. Not very expensive
3. Available all year round
4. Parasite does not develop resistance
5. No chemical that would pollute environment
Requisites for an Effective Dewormer:
1. Kills all worm burden (Cytotoxic action)
2. Expels dead worm (Cathartic or purgative)
3. Heals injury brought about by inflammatory
reaction by worms (Astringent)
Compounds in 3 Plants with Their Corresponding Compounds and actions:
Compounds & Concentration
Plants
Chrysophyllum
cainito
Tinospora rhumpii
Mimosa pudica
Flavonoids
Anthraquinones
Alkaloids
Tannins
++
-
++++
-
++++
-
++
-
-
++
-
++++
C. caimito
M. pudica
T. rumphii
(Ratio of Extracts)
Flavonoids
Tannins
Anthraquinones
Inject
Alkaloids
Model
Animal
Hyperimmunize
Monoclonal
Antibodies
Compounds
Isolate
Bioactive
Compounds
Antigen
Hybridoma Cell /
E. coli
Testing
Anthelmintic activity
Final product
Purified dewormer
Feces
Goat Infected
Eggs in
Pasture area
Hatch
Ingested
L3 on
Grass
L1
L2
L1 and
and L2
Develop
Developmental
Haemonchus
spp.
DevelopmentalCycle
Cycle ofof
Haemonchus
contortus
Purposes of Phase I
• The effective concentration against L3s of H. contortus will
be the basis for the determination of LD50 of plant cocktail
• Effective plant cocktail dewormer will be made into drug forms and
these will be studied for their ED50
• Extraction by solvent will be the basis for isolation of bioactive
compounds
L1 and L2
Objectives of Phase I
General:
• Evaluate the effect of different solvents on extraction of active
compounds of the plant extract that would kill at least 80% of
L3s of H. contortus
• Determine the ratios of the plant cocktail that would kill at least
80% of the L3s of H. contortus
Specific:
L1 and L2
• Find out the concentration of the individual plant according to
solvent that would kill at least 80% of L3s of H. contortus
• Determine the concentration of the plant cocktail that would kill
at least 80% of L3s of H. contortus
MATERIALS AND METHODS
Collection of Plant Parts
Air-Drying of T. rumphii Stem
Air-Drying of C. cainito Leaves
Chopped Stem of T. rumphii
Chopped leaves of C. cainito
Leaves of Mimosa pudica
Leaves of Chrysophyllum cainito
Stem of Tinospora rumphii
Extraction of Individual Plant Parts
Plant Sample
Petroleum Ether
Petroleum Ether
Extract
Plant Residue
Rotavap
Ethanol
Crude Petroleum
Ether Fraction
Ethanol Extract
Assay*
Plant Residue
Water
Rotavap
Crude Ethanol
Fraction
Water Extract
Plant Residue
Rotavap
Assay*
*Highly effective extract by solvent
will be combined for herbal cocktail
Crude Aqueous
Fraction
Assay*
Discard
Combination of Plant
Extracts*
Assay of Plant Cocktail for
larvicidal activity
Effective Combination of
Plant Cocktail against L3
Isolation of Bioactive
Compounds (Phase II)
Multiplication of Bioactive Compounds
*Highly Effective Extract by Solvent will
Be combined to form a plant cocktail
Dosage Level for
LD50 and ED50
Studies (Phase II)
Weighing of Plant Parts
Transfer of Plant Parts in Amber
Bottles
Extraction by Infusion
Straining of Plant Part Extracts
Setting up of Rotavapor
Concentration of Plant Part Extracts in Rotavapor
Plant Extracts Ready for Concentration in a Rotary evaporator
Plant Extracts by Solvents & 0.5% Ivermectin
Table 1. Varying ratios used in combining the 3 plants (makabuhay, caimito, and makahiya) extracts
Tinospora rumphii
Chrysophyllum cainito
Mimosa pudica
1 part
1 part
1 part
1 part
1 part
2 parts
1 part
1 part
3 parts
1 part
2 parts
1 part
1 part
2 parts
2 parts
1 part
2 parts
3 parts
1 part
3 parts
1 part
1 part
3 parts
2 parts
1 part
3 parts
3 parts
2 parts
1 part
1 part
2 parts
1 part
2parts
Table 1. Continued
2 parts
1 part
3 parts
2 parts
2 parts
1 part
2 parts
2 parts
3 parts
2 parts
3 parts
1 part
2 parts
3 parts
2 parts
2 part
3 parts
3 parts
3 parts
1 part
1 part
3 parts
1 part
2 parts
3 parts
1 part
3 parts
3 parts
2 part
1 part
3 parts
2 parts
2 parts
3 parts
2 parts
3 parts
3 parts
3 parts
1 part
3 parts
3 parts
2 parts
A S S A Y OF PLANT EXTRACTS
L3s Transferred into Wells of Hollow Glass Slide
Exposure of L3s with Plant Extracts
L3s in wells of hollow glass slide counted
L3s in wells of hollow glass slide exposed with
0.5 ml of the varying concentrations of plant
cocktail
Exposure of L3s with Plant Extracts
Movement of L3s monitored for 30
Minutes after exposure
Absence of movement upon touching
of L3s with end of inoculating needle
Computation for the Dosage Level of the Combined Plant Extracts:
A x 100
B
Where:
A = Weight (mg/g) of Combined Plant Extract
B = Volume (ml) of Distilled water
Formula of Reik and Keitz (1954) on Percent Efficacy of Anthelmintic
% Efficacy = No. of Dead L3s
x 100
No. of L3s Exposed
Where:
Below 70 % efficacy, the plant extract is said to be ineffective;
71%-80 % efficacy, the plant extract is said to be effective
81-100% efficacy, the plant extract is said to be highly effective
Experimental Design:
Layout of experiment of Individual Plant Extracts: Completely Randomized Design (CRD)
T0(-) = 1% of appropriate solvent
T0(+) = 0.5% Ivermectin
Treated Groups: nth Concentrations of appropriate extract
Replicates: 2 with at least 30 L3s per replicate
Layout of Experiment
Experimental
Design:on Plant Cocktails: Completely Randomized Design (CRD)
TO(-) = 1% Ethanol
TO(+) = 0.5% ivermectin
Treated Groups =nth concentrations of plant cocktail
Replicates = 2 with at least 30 L3s per replicate
Statistical Analysis:
Experiment of Individual Plant Extracts:
Analysis of Variance (ANOVA)
Experiment on Plant Cocktails:
Analysis of Variance (ANOVA)
Significant among treatment means: DMRT
RESULTS
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
1% Pet
Ether
100
90
80
70
60
50
40
30
20
10
0
0.5%
Ivermectin
% Efficacy
Mean Percent Efficacy of Individual Panyawan-, Caimito-,
& Makahiya- Pet Ether Extracts
PANYAWAN
Concentrations
CAIMITO
MAKAHIYA
Mean Percent Efficacy of Individual Panyawan-, Caimito-,
& Makahiya- Ethanol- Extracts
100
90
70
60
50
40
30
20
10
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
1% Pet
Ether
0
0.5%
Ivermectin
% Efficacy
80
PANYAWAN
Concentrations
CAIMITO
MAKAHIYA
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
1% Pet
Ether
100
90
80
70
60
50
40
30
20
10
0
0.5%
Ivermectin
% Efficacy
Mean Percent Efficacy of Individual Panyawan-, Caimito-,
& Makahiya- Water- Extracts
PANYAWAN
Concentrations
CAIMITO
MAKAHIYA
Petroleum Ether-Ethanol Extraction
Treatments
(P:C:M)
Treatments
Mean
Mean
T12 (2:1:3)
100.00a
T13
(2:2:1)
98.04a
T14 (2:2:3)
98.60a
T15
(2:3:1)
100.00a
T16
(2:3:2)
98.96a
T17
(2:3:3)
98.15a
T18
(3:1:1)
100.00a
T0(+) (0.5%
37.90b
T0(-)
(1% Ethanol)
14.83c
T1
(1:1:1)
97.88a
T2
(1:1:2)
100.00a
T3
(1:1:3)
100.00a
T4
(1:2:1)
100.00a
T5
(1:2:2)
100.00a
T6
(1:2:3)
100.00a
T7
(1:3:1)
100.00a
T8
(1:3:2)
100.00a
T22 (3:2:2)
99.12a
T9
(1:3:3)
100.00a
T23 (3:2:3)
100.00a
T10 (2:1:1)
100.00a
T24 (3:3:1)
100.00a
T11 (2:1:2)
100.00a
T25 (3:3:2)
100.00a
Ivermectin)
T19 (3:1:2)
99.02a
T20
(3:1:3)
98.99a
T21
(3:2:1)
100.00a
Means with different letters are statistically significant (p<0.01) by DMRT
Plant Cocktail by Petroleum Ether-Ethanol Extraction
Treatments
T25
T24
T23
T22
T21
T20
T19
T18
T17
T16
T15
T14
T13
T12
T11
T10
T9
T8
T7
T6
T5
T4
T3
T2
T1
1% Ethanol
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
0.5% Ivermectin
% Efficacy
Mean Percent Efficacy of Pet Ether to Ethanol- Plant- Cocktail Extracts at 100% Concentration
Plant Cocktail by Ethanol Extraction
Treatments
T25
T24
T23
T22
T21
T20
T19
T18
T17
T16
T15
T14
T13
T12
T11
T10
T9
T8
T7
T6
T5
T4
T3
T2
T1
1% Ethanol
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
0.5% Ivermectin
% Efficacy
Mean Percent Efficacy of Ethanol- Plant Cocktail Extracts at 100% Concentration
PHASE II ACTIVITIES
Pharmacologic Studies of Plant Cocktail
• LD50 of plant cocktail
• ED50 of plant cocktail
Studies on Drug Forms
• Studies on the appropriate binders for tablet forms of plant cocktail
• Studies on the appropriate capsules for plant cocktail
Pharmacologic Studies of Plant Cocktail
• LD50 of plant cocktail
• ED50 of plant cocktail
Studies on Drug Forms
• Studies on the appropriate binders for tablet forms of plant cocktail
• Studies on the appropriate capsules for plant cocktail
Bioactive Compounds Elucidation
Plant Extracts
Isolate
Flavonoids
Tannins
Anthraquinone
Alkaloids
Inject
Animal
Model
Monoclonal
Antibodies
Isolate
Bioactive Compounds.
Bioactive Compounds Elucidation
Solid Phase
Ligand
Antibody
Antigen
Bioactive Compounds Elucidation
Antigen
(bioactive compounds)
Inoculate
Hybridoma/E. coli
Testing
Purified Anthelmintic
Testing
Final Product
Herbal Anthelmintic Product Path
Product
Development
Discovery
Bioactive
cmpds
Monoclonal
antibodies
Hybridomal
E. coli
Plant
drug
dev’t.
Plant/
Raw
Materials
Screening
Yr 1
Pure Form
Lab
evl’n.
Drug
Forms
Product Enhancement
Field
evl’n.
Quality
control
Drug
Forms
Pilot
comm’l
prod’n
Mktg
sales
Crude Form
Yr 1 - 3
Yr 2
Yr 3
Yr 4
IP/Biosafety Audit
Biosafety Approval
Regulatory approvals
(BFAD)
Post
market