RESEARCH ON HERBAL DRUGS FOR

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RESEARCH ON HERBAL DRUGS FOR POULTRY
LIVESTOCK
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BACKGROUND INFORMATION
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• Two researches on local herbal plants conducted

• Herbal drugs for Haemophilus paragallinarum
  infection

• Herbal drugs for Haemonchus contortus infection

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OBJECTIVES OF TWO RESEARCHES
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• 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

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

• Determine the shelf-life of plant drugs

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MATERIALS AND METHODS
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Steps

• Consultation with farmers

• Identification of local plants

• Diagnosis of diseases

• Culture of causative agents of diseases

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• In vitro assay of local plants for their
  bactericidal/larvicidal actions

• Pharmacologic studies of herbal drugs

• Quality control study of herbal drugs

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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

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• Preparation dosage level of a particular
  herb

Identification of local plants used by farmers
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Diagnosis of diseases based on clinical signs

• Isolation of Haemophilus paragallinarum in
  pure culture

• Blood agar/nutrient broth culture

• Gram staining Biochemical tests

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• Infection and isolation of H. paragallinarum

• Isolation of Haemonchus contortus

• Culture of eggs infection of goats

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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

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• Infection of goats with L3s for supply of eggs

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isolation of eggs of H. contortus
Identification of Infected Goats by fecalysis
Collection of Fecal Sample
Fecalysis (Floatation Technique)
Infected Goats Identified
Collect feces
Culture Feces for L3s of H. contortus
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Retrieval Identification of L3s
L3s of H. contortus
3 Goats Infected With L3s
Source of eggs for culture of L3s
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Culture of Haemonchus larvae

Moistened cotton wads placed at the
bottom of shot glass
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Feces from infected goats macerated and
placed on top of cotton wads
Sides of shot glass filled with tap water
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Culture of Haemonchus larvae

Shot glass in Culture glass, filled with water
up to the brim of shot glass
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Culture glass covered with wrappings
and incubated at room temperature for 6 days
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Recovery of Infected Larvae (L3) of H. Contortus
Removal of wrappings of shot glass
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Water in the culture glass transferred
into petri plate
Infective Larvae (L3s) recovered in Petri
plate identified
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Preparation of Plant Extracts

• Decoction using 12 (w/v)

• Dried plant parts were boiled for 15 minutes

• Crude extracts were filtered

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• 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.

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IN VITRO ASSAYS OF LOCAL PLANT EXTRACTS
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Techniques Used in In Vitro Assay

• Sensitivity test of Carter for Haemophilus
  gallinarum

• Larvicidal test for Haemophilus gallinarum

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RESULTS
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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
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Fruits given raw to sick birds
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In vitro assay of plant extracts vs H.
paragallinarum
Treatment No. Plant Extracts Zone of Inhibition (cm) Zone of Inhibition (cm)
Treatment No. Plant Extracts 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
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12 (w/v) 1 part plant part 2 parts distilled
water by decoction, amount in each wellwas
0.05ml
Sensi discs, BBL Microbiological systems
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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 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
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12 (w/v) 1 part plant part 2 parts distilled
water by decoction, amount given to each bird
was 5ml, given per os
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Chicken Showing Clinical Signs of Haemophilus
paragallinarum Infection
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Chicken Showing Clinical Signs of Haemophilus
paragallinarum Infection
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Percent efficacy of plant water extract by
decoction according to animal species against
Haemonchus contortus (Fernandez, 1991)
Plants Chicken ( Efficacy) Swine ( Efficacy) Goat ( Efficacy)
Ananas comosus (fruit) 63.3 59.1 52.3
Ananas comosus (leaves) 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
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Based on formula of Reik and Keitz (1954)
NI not used as anthelmintic by local farmers in
a particular animal species
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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 Preparations Dosages Dosages Dosages Administration
Group No. Scientific Names (Local Names) Plant Parts Preparations Chicken Swine Goat Administration
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 5 ml 10 ml 20 ml Per os
T4 Cajanus cajan (Kadios) roots decoction 15 ml 30 ml 40 ml Per os
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
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No. of Animals/treatment 4
Untreated control
- not used by farmers
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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 Preparations Dosages Dosages Dosages Administration
Group No. Scientific Names (Local Names) Plant Parts Preparations Chicken Swine Goat Administration
T8 Karicq papaya (kapayas) seeds pounding 15 g - 40 g Per os
T9 Lansium domesticum (lansones) seeds pounding 15 g 30 g 40 g Per os
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/ expressing 3 ml 6 ml 12 ml Per os
T15 Moringa oleifera (malunggay) seeds pounding 15 g 30 g 40 g Per os
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No. of Animals/treatment 4
- not used by farmers
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Preparations, dosage levels and mode of
administration of plant parts used by local
farmers against parasitic infections in various
animals
Group No. Scientific Name (Local Name) Plant Parts Preparation Dosages Dosages Dosages Administration
Group No. Scientific Name (Local Name) Plant Parts Preparation chicken Swine Goat Administration
T16 Quisqualis indica (niyog-niyogan) seeds pounding 15 g 30 g 40 g Per os
T17 Tamarindus indica (Sambag) leaves decoction 15 ml 30 ml 40 ml Per os
T18 Tinospora rumphii (panyawan) stem decoction - - 40 ml Per os
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No. of Animals/treatment 4
- not used by farmers
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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
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NI not used by local farmers
highly effective
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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
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
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NI not used by local farmers
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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
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NI not used by local farmers
highly effective
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On-Going Study on Haemonchus Contortus
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Rationale
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Goat Population
As of 2002, 3.29 million heads
25 are concentrated in the Visayas
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99.9 of 3.29 millions are raised by small
holders ( in rural areas)
Source Philippines Recommends for Goat
Production, PCARRD (2004)
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Constraints in Goats Production
Endoparasitism hampers productivity full
development of goat
subsector
Ways endoparasites hampered productivity of goats
1. High rate mortality at weaning
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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 US3.55 M
annually
Source Philippines Recommends for Goat
Production, PCARRD (2004)
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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
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Two classes of anthelmintic that could be
resorted to
1. Commercial or synthetic anthelmintic
2. Herbal anthelmintic
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Advantage of synthetic anthelmintic
1. Being pure, synthetic anthelmintic is very
effective
Advantage is outweighed by the following
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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
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Use of alternative dewormer Herbal anthelmintic
Advantages of Herbal anthelmintic

1. Does not impart drug residue in meat

2. Not very expensive
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3. Available all year round
4. Parasite does not develop resistance
5. No chemical that would pollute environment
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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)
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Compounds in 3 Plants with Their Corresponding
Compounds and actions
Plants Compounds Concentration Compounds Concentration Compounds Concentration Compounds Concentration
Plants Flavonoids Anthraquinones Alkaloids Tannins
Chrysophyllum cainito - -
Tinospora rhumpii - -
Mimosa pudica - -
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C. caimito M. pudica T. rumphii (Ratio of
Extracts)
Flavonoids Tannins Anthraquinones Alkaloids
Model Animal
Monoclonal Antibodies
Inject
Hyperimmunize
Isolate
Compounds
Bioactive Compounds
Antigen
Hybridoma Cell / E. coli
Testing
Anthelmintic activity
Final product
Purified dewormer
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Feces
Eggs in Pasture area
Goat Infected
Hatch
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Ingested
L1 and L2
L1 and L2
L3 on Grass
Develop
Developmental Cycle of Haemonchus spp.
Developmental Cycle of Haemonchus contortus
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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

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

L1 and L2
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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

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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

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MATERIALS AND METHODS
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Collection of Plant Parts
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Air-Drying of T. rumphii Stem
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Chopped Stem of T. rumphii
Chopped leaves of C. cainito
Air-Drying of C. cainito Leaves
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Leaves of Chrysophyllum cainito
Leaves of Mimosa pudica
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Stem of Tinospora rumphii
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Plant Sample
Extraction of Individual Plant Parts
Petroleum Ether
Plant Residue
Petroleum Ether Extract
Rotavap
Ethanol
Crude Petroleum Ether Fraction
Ethanol Extract
Plant Residue
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Water
Rotavap
Assay
Crude Ethanol Fraction
Water Extract
Plant Residue
Rotavap
Assay
Crude Aqueous Fraction
Discard
Highly effective extract by solvent will be
combined for herbal cocktail
Assay
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Combination of Plant Extracts
Assay of Plant Cocktail for larvicidal activity
Dosage Level for LD50 and ED50 Studies (Phase
II)
Effective Combination of Plant Cocktail against
L3
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Isolation of Bioactive Compounds (Phase II)
Multiplication of Bioactive Compounds
Highly Effective Extract by Solvent will Be
combined to form a plant cocktail
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Weighing of Plant Parts
Transfer of Plant Parts in Amber Bottles
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Extraction by Infusion
Straining of Plant Part Extracts
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Setting up of Rotavapor
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Concentration of Plant Part Extracts in Rotavapor
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Plant Extracts Ready for Concentration in a
Rotary evaporator
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Plant Extracts by Solvents 0.5 Ivermectin
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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
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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
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A S S A Y OF PLANT EXTRACTS
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L3s Transferred into Wells of Hollow Glass Slide
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Exposure of L3s with Plant Extracts
L3s in wells of hollow glass slide counted
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L3s in wells of hollow glass slide exposed
with 0.5 ml of the varying concentrations of
plant cocktail
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Exposure of L3s with Plant Extracts
Movement of L3s monitored for 30 Minutes after
exposure
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Absence of movement upon touching of L3s with end
of inoculating needle
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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
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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
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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
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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
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Statistical Analysis
Experiment of Individual Plant Extracts
Analysis of Variance (ANOVA)
Experiment on Plant Cocktails
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Analysis of Variance (ANOVA)
Significant among treatment means DMRT
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R E S U L T S
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Petroleum Ether-Ethanol Extraction
Treatments (PCM) Mean
T0() (0.5 Ivermectin) 37.90b
T0(-) (1 Ethanol) 14.83c
T1 (111) 97.88a
T2 (112) 100.00a
T3 (113) 100.00a
T4 (121) 100.00a
T5 (122) 100.00a
T6 (123) 100.00a
T7 (131) 100.00a
T8 (132) 100.00a
T9 (133) 100.00a
T10 (211) 100.00a
T11 (212) 100.00a
Treatments Mean
T12 (213) 100.00a
T13 (221) 98.04a
T14 (223) 98.60a
T15 (231) 100.00a
T16 (232) 98.96a
T17 (233) 98.15a
T18 (311) 100.00a
T19 (312) 99.02a
T20 (313) 98.99a
T21 (321) 100.00a
T22 (322) 99.12a
T23 (323) 100.00a
T24 (331) 100.00a
T25 (332) 100.00a
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Means with different letters are statistically
significant (plt0.01) by DMRT
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Plant Cocktail by Petroleum Ether-Ethanol
Extraction
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Plant Cocktail by Ethanol Extraction
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PHASE II ACTIVITIES
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Pharmacologic Studies of Plant Cocktail

• LD50 of plant cocktail

• ED50 of plant cocktail

Studies on Drug Forms
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• Studies on the appropriate binders for tablet
  forms of plant cocktail

• Studies on the appropriate capsules for plant
  cocktail

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Pharmacologic Studies of Plant Cocktail

• LD50 of plant cocktail

• ED50 of plant cocktail

Studies on Drug Forms
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• Studies on the appropriate binders for tablet
  forms of plant cocktail

• Studies on the appropriate capsules for plant
  cocktail

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Bioactive Compounds Elucidation
Plant Extracts
Isolate
Flavonoids Tannins Anthraquinone Alkaloids
Animal Model
Monoclonal Antibodies
Inject
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Isolate
Bioactive Compounds.
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Bioactive Compounds Elucidation
Solid Phase
Ligand
Antibody
Antigen
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Bioactive Compounds Elucidation
Antigen (bioactive compounds)
Inoculate
Hybridoma/E. coli
Testing
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Purified Anthelmintic
Testing
Final Product
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