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

• Determine the shelf-life of plant drugs

• Find out the quality of binders of tablet forms of the plant drugs for infectious coryza

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

Identification of local plants used by farmers

Preparation & dosage level of a particular herb

Diagnosis of diseases based on clinical signs

• Isolation of Haemonchus contortus

Blood agar/nutrient broth culture

• Isolation of Haemophilus paragallinarum in pure culture

Gram staining & Biochemical tests

Culture of eggs & infection of goats

Infection and isolation of H. paragallinarum

Culture of Infective Stage for In Vitro Assay

Blood agar

• Stock of pure culture of H. paragallinarum

• 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

Fecalysis (Floatation Technique)

Infected GoatsIdentified

Culture Feces for L3sof H. contortus

Retrieval & Identification of L3s

L3s of H. contortus

3 Goats InfectedWith L3s*

*Source of eggs for culture of L3s

Collect feces

isolation of eggs of H. contortus

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 andincubated 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

Ananas squamosa 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

Coleus blumei - Mayana (red) Leaves

Helianthus annuus* Sunflower Sunflower Seeds

Heliotropium indicum Elephant Grass

Trompa Elefante

Leaves

Spondias pinnata - Libas Leaves

Solanum spp. - Terramycin Plant

Fruits

Zingiber officinale Ginger Luya Root

*Fruits given raw to sick birds

In vitro assay of plant extracts* vs H. paragallinarum

**Sensi discs, BBL Microbiological systems

Treatment No.

Plant ExtractsZone of Inhibition (cm)

After 1 h

After 24 h

T0(-) Distilled Water NZ NZ

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

T10 Solanum spp. NZ NZ

T11 Zingiber officinale 1 NZ*1:2 (w/v) 1 part plant part: 2 parts distilled water by decoction, amount in each wellwas 0.05ml

In vivo assay of plant extracts* against H. paragallinarum infection in chicken (Lohman)

*1:2 (w/v) 1 part plant part: 2 parts distilled water by decoction, amount given to each bird was 5ml, given per os

Treatment No.

Plants No. of Birds

Examined

Water Eye & Nasal

Discharge

Facial Swelling

Mucus in Upper Respiratory

Tract

Congested 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

Chicken Showing Clinical Signs of Haemophilus paragallinarum Infection

Percent efficacy* of plant water extract by decoction according to animalspecies against Haemonchus contortus (Fernandez, 1991)

PlantsPlantsChicken Chicken

(% (% Efficacy)Efficacy)

Swine (% Swine (% Efficacy)Efficacy)

Goat (% Goat (% Efficacy)Efficacy)

Ananas comosus (fruit)Ananas comosus (fruit) 63.363.3 59.159.1 52.352.3

Ananas comosus Ananas comosus (leaves)(leaves)

NINI 54..754..7 56.756.7

Mangifera indicaMangifera indica 60.060.0 58.258.2 56.756.7

Manihot esculentaManihot esculenta 53.453.4 52.052.0 50.850.8

Tamarindus indicaTamarindus indica 59.459.4 58.458.4 55.655.6

Moringa oleiferaMoringa oleifera 69.069.0 64.764.7 61.561.5

Artemisia vulgarisArtemisia vulgaris 57.657.6 56.356.3 53.353.3

Mimosa pudicaMimosa pudica NINI NINI 79.7**79.7**

Chrysophyllum cainitoChrysophyllum cainito NINI NINI 70.3**70.3**

Tinospora rumphiiTinospora rumphii NINI NINI 85.6***85.6***

NI = not used as anthelmintic by local farmers in a particular animal species

*Based on formula of Reik and Keitz (1954)

Group

No.*

Scientific Names (LocalNames)

Plant Parts Preparati

ons

Dosages Administration

Chicken

Swine Goat

TO Control** - - - - - -

T1 Ananas comosus (pina)

Unripe fruit

pounding 15 g 30 g 40g Mixed w/ feed

T2 Artemisia vulgaris (hilbas)

leaves decoction

15 ml 30 ml 40 ml Per os

T3 Bixa orellana (Asuite)

seeds decoction


T4 Cajanus cajan(Kadios)

roots decoction


T5 Cassia alata(sunting)

seeds pounding 15 g 30 g 40 g Per os

T6 Chrysophylum cainito (caimito)

leaves decoction

- - 40 ml Per os

T7 Clitorea ternatea (balog-balog)

seeds heating &

pounding

15 g 30 g 40 g Mixed w/ feed

Preparations, dosage levels and mode of administration of plant parts usedby local farmers against parasitic infections in various animals

*No. of Animals/treatment = 4

**Untreated control

- not used by farmers

Group

No.*

Scientific Names (LocalNames)

Plant Parts Preparati

ons

Dosages Administration

Chicken

Swine Goat

T8 Karicq papaya(kapayas)

seeds pounding 15 g - 40 g Per os

T9 Lansium domesticum (lansones)


T10 Leucaena leucocephala

(ipil-ipil)

seeds Pounding 15 g 30 g 40 g Per os

T11 Mangifera indica (paho)

seeds Pounding 15 g 10 g 30 g Per os

T12 Manihot esculenta

(balanghoy)

bark/ roots

Decoction 15 ml 30 ml 40 ml Per os

T13 Mimosa pudica(makahiya)

leaves decoction - - 40 ml Per os

T14 Momordica charantia (paliya)

leaves Heating/ expressin

g


T15 Moringa oleifera (malunggay)








Group No.*

Scientific Name (Local

Name)

Plant Parts

PreparationDosages

Administration

chicken Swine

Goat

T16 Quisqualis indica(niyog-

niyogan)


T17 Tamarindus indica

(Sambag)

leaves decoction 15 ml 30 ml 40 ml Per os

T18 Tinospora rumphii

(panyawan)

stem decoction - - 40 ml Per os

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

Chrysophyllum cainito NI NI 70.3

Clitorea ternatea 62.8 58.7 55.6

Karica papaya 59.4 NI 58.8

NI = not used by local farmers

*highly effective



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

Mimosa pudica 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




*highly effective


Swine (% Efficacy)

Goat (% Efficacy)

Tamarindus indica

59.4 58.4 55.6

Tinospora rumphii

NI NI 85.6*

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:

2. Drop in milk production

1. High rate mortality at weaning

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)

Two classes of anthelmintic that could be resorted to:

Haemonchus contortus infection in ruminants is addressed by use of athelmintic or dewormers

1. Commercial or synthetic anthelmintic

2. Herbal anthelmintic

Advantage of synthetic anthelmintic:

1. Being pure, synthetic anthelmintic is very effective

5. Not available in remote areas

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

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:

PlantsPlantsCompounds & ConcentrationCompounds & Concentration

FlavonoiFlavonoidsds

AnthraquinoAnthraquinonesnes

AlkaloiAlkaloidsds

TanniTanninsns

Chrysophyllum Chrysophyllum cainitocainito

++++ -- ++++++++ --

Tinospora Tinospora rhumpiirhumpii

++++++++ -- ++++ --

Mimosa Mimosa pudicapudica

-- ++++ -- ++++++++

C. caimitoM. pudicaT. rumphii

(Ratio of Extracts)

FlavonoidsTannins

AnthraquinonesAlkaloids

Model Animal

Monoclonal Antibodies

Isolate

Bioactive Compounds

Antigen

Compounds

Inject Hyperimmunize

Hybridoma Cell /

E. coli

Anthelmintic activity

Purified dewormer

Testing

Final product

Hatch

L1 and L2

Develop

L3 on Grass

Ingested

Goat Infected

Feces

Developmental Cycle of Haemonchus spp.

Developmental Cycle of Haemonchus contortus

Eggs in Pasture area

L1 and L2

L1 and L2

Purposes of Phase I

• The effective concentration against L3s of H. contortus will be the basis for the determination of LD50 of plant cocktail

• Extraction by solvent will be the basis for isolation of bioactive compounds

• Effective plant cocktail dewormer will be made into drug forms and these will be studied for their ED50

L1 and L2

Objectives of Phase I

• 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

General:

• Determine the ratios of the plant cocktail that would kill at least 80% of the L3s of H. contortus

Specific:

• 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 StemChopped Stem of T. rumphii

Air-Drying of C. cainito Leaves Chopped leaves of C. cainito

Leaves of Mimosa pudica Leaves of Chrysophyllum cainito

Stem of Tinospora rumphii

Plant Sample

Petroleum Ether

Petroleum Ether Extract

Crude PetroleumEther Fraction

Assay*

Rotavap

Plant Residue

Ethanol

Ethanol Extract

Crude Ethanol Fraction

Assay*

Rotavap

Plant Residue

Water

Water Extract

Crude Aqueous Fraction

Assay*

Rotavap

Plant Residue

Discard

*Highly effective extract by solvent will be combined for herbal cocktail

Extraction of Individual Plant Parts

Combination of Plant Extracts*

Assay of Plant Cocktail forlarvicidal activity

Effective Combination of Plant Cocktail against L3

Dosage Level for LD50 and ED50

Studies (Phase II)

Isolation of Bioactive Compounds (Phase II)

*Highly Effective Extract by Solvent will Be combined to form a plant cocktail

Multiplication of Bioactive Compounds

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

Tinospora rumphiiTinospora rumphii Chrysophyllum cainitoChrysophyllum cainito Mimosa pudicaMimosa pudica

1 part1 part 1 part1 part 1 part1 part

1 part1 part 1 part1 part 2 parts2 parts

1 part1 part 1 part1 part 3 parts3 parts

1 part1 part 2 parts2 parts 1 part1 part

1 part1 part 2 parts2 parts 2 parts2 parts


1 part1 part 3 parts3 parts 1 part1 part



2 parts2 parts 1 part1 part 1 part1 part

2 parts2 parts 1 part1 part 2parts2parts

Table 1. Varying ratios used in combining the 3 plants (makabuhay, caimito, and makahiya) extracts

Table 1. Continued

2 parts2 parts 1 part1 part 3 parts3 parts

2 parts2 parts 2 parts2 parts 1 part1 part

2 parts2 parts 2 parts2 parts 3 parts3 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 with0.5 ml of the varying concentrations of plantcocktail

Movement of L3s monitored for 30 Minutes after exposure

Absence of movement upon touchingof L3s with end of inoculating needle

Exposure of L3s with Plant Extracts

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

% 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

Formula of Reik and Keitz (1954) on Percent Efficacy of Anthelmintic

Experimental Design:

Experimental Design:

Layout of Experiment 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

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

Statistical Analysis:

Experiment on Plant Cocktails:

Experiment of Individual Plant Extracts:

Analysis of Variance (ANOVA)

Significant among treatment means: DMRT

Analysis of Variance (ANOVA)

R E S U L T S

Mean Percent Efficacy of Individual Panyawan-, Caimito-, & Makahiya- Pet Ether Extracts

0102030405060708090

1000.

5%Iv

erm

ectin

1% P

etEt

her

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Concentrations

% E

ffica

cy

PANYAWAN

CAIMITO

MAKAHIYA

Mean Percent Efficacy of Individual Panyawan-, Caimito-, & Makahiya- Ethanol- Extracts

0

10

20

30

40

50

60

70

80

90

100

0.5%

Iver

mec

tin1%

Pet

Ethe

r

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Concentrations

% E

ffica

cy

PANYAWAN

CAIMITO

MAKAHIYA

Mean Percent Efficacy of Individual Panyawan-, Caimito-, & Makahiya- Water- Extracts

0102030405060708090

1000.

5%Iv

erm

ectin

1% P

etEt

her

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Concentrations

% E

ffica

cy

PANYAWAN

CAIMITO

MAKAHIYA

Petroleum Ether-Ethanol Extraction

Treatments Treatments (P:C:M)(P:C:M)

MeanMean

TT0(+) 0(+) (0.5% (0.5%

Ivermectin)Ivermectin)

37.90b37.90b

TT0(-)0(-) (1% Ethanol)(1% Ethanol) 14.83c14.83c

TT1 1 (1:1:1)(1:1:1) 97.88a97.88a

TT2 2 (1:1:2)(1:1:2) 100.00a100.00a

TT3 3 (1:1:3)(1:1:3) 100.00a100.00a

TT4 4 (1:2:1)(1:2:1) 100.00a100.00a

TT5 5 (1:2:2)(1:2:2) 100.00a100.00a

TT6 6 (1:2:3)(1:2:3) 100.00a100.00a

TT7 7 (1:3:1)(1:3:1) 100.00a100.00a

TT8 8 (1:3:2)(1:3:2) 100.00a100.00a

TT9 9 (1:3:3)(1:3:3) 100.00a100.00a

TT10 10 (2:1:1)(2:1:1) 100.00a100.00a

TT11 11 (2:1:2)(2:1:2) 100.00a100.00a

TreatmentsTreatments

MeanMean

TT12 12 (2:1:3)(2:1:3) 100.00a100.00a

TT13 13 (2:2:1)(2:2:1) 98.04a98.04a

TT1414 (2:2:3)(2:2:3) 98.60a98.60a

TT15 15 (2:3:1)(2:3:1) 100.00a100.00a

TT16 16 (2:3:2)(2:3:2) 98.96a98.96a

TT17 17 (2:3:3)(2:3:3) 98.15a98.15a

TT18 18 (3:1:1)(3:1:1) 100.00a100.00a

TT1919 (3:1:2)(3:1:2) 99.02a99.02a

TT20 20 (3:1:3)(3:1:3) 98.99a98.99a

TT21 21 (3:2:1)(3:2:1) 100.00a100.00a

TT22 22 (3:2:2)(3:2:2) 99.12a99.12a

TT23 23 (3:2:3)(3:2:3) 100.00a100.00a

TT24 24 (3:3:1)(3:3:1) 100.00a100.00a

TT25 25 (3:3:2)(3:3:2) 100.00a100.00a

Means with different letters are statistically significant (p<0.01) by DMRT

Plant Cocktail by Petroleum Ether-Ethanol Extraction

Mean Percent Efficacy of Pet Ether to Ethanol- Plant- Cocktail Extracts at 100% Concentration

05

101520253035404550556065707580859095

100

0.5%

Iver

mec

tin

1% E

than

ol

T1 T2 T3 T4 T5 T6 T7 T8 T9

T10

T11

T12

T13

T14

T15

T16

T17

T18

T19

T20

T21

T22

T23

T24

T25

Treatments

% E

ffica

cy

Mean Percent Efficacy of Ethanol- Plant Cocktail Extracts at 100% Concentration

05

101520253035404550556065707580859095

100

0.5%

Iver

mec

tin

1% E

than

ol T1 T2 T3 T4 T5 T6 T7 T8 T9

T10

T11

T12

T13

T14

T15

T16

T17

T18

T19

T20

T21

T22

T23

T24

T25

Treatments

% E

ffica

cyPlant Cocktail by Ethanol Extraction

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

Bioactive Compounds Elucidation

Plant Extracts

Isolate

FlavonoidsTannins

AnthraquinoneAlkaloids

Inject

Animal Model

MonoclonalAntibodies

Bioactive Compounds.

Isolate


Solid Phase

Ligand

Antibody

Antigen


Antigen (bioactive compounds)

Hybridoma/E. coli

Purified Anthelmintic

Final Product

Inoculate

Testing

Testing

Plantdrug

dev’t.

HybridomalE. coli

Monoclonalantibodies

Bioactivecmpds

Postmarket

Mktgsales

Pilotcomm’lprod’n

Qualitycontrol

Field evl’n.

Herbal Anthelmintic Product Path

IP/Biosafety Audit

Biosafety Approval

Regulatory approvals(BFAD)

DiscoveryProduct

Development Product Enhancement

Yr 1 Yr 1 - 3 Yr 2 Yr 3 Yr 4

Pure Form

Plant/Raw

MaterialsScreening

Labevl’n.

Crude Form

Drug Forms

Drug Forms

RESEARCH ON HERBAL DRUGS FOR POULTRY & LIVESTOCK

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Transcript of RESEARCH ON HERBAL DRUGS FOR POULTRY & LIVESTOCK