ANTHELMINTIC EFFECT OF MIMOSA PUDICA LINNE LEAVES EXTRACT ON NATIVE CHICKEN, GALLUS DOMESTICUS NATURALLY-INFECTED WITH GASTRO INTESTINAL

PARASITES

BRINGAS, JENIE VIE CABUNTALAN, SAHERA MEDINA, JAMES ARYL

Chapter 1 Introduction
Background of the study
The use of traditional plants for medicinal purposes is as old as man himself. Primitive man probably learned medicinal value from intuition and observations of the animals around him. One of these plants which nature in her beauty has endowed the Philippines is abundant in the Mimosa pudica Linne. The leaves Extract of M. pudica Linne makahiya has been used for centuries in the treatment of dysentery, dysmenorrhoea and administered as diuretic.

Chapter 1 Introduction
Objectives
General objective To determine if the M. pudica Linne leaves extract is viable to elicit an anthelmintic effect. Specific objectives 1. To prepare different concentration of leaves extract of M. pudica Linne. 2. To conduct an identification of intestinal worms expelled by G. domesticus after the administration of the M. pudica L. extract. 3. To find out the concentration of M. pudica L. extract that could exhibit anthelmintic effects to G. domesticus.

Chapter 1 Introduction
Statement of the problem 1. Does M. pudica Linne extract exhibit anthelmintic effect?
2. At what concentration would M. pudica L. extract be exhibiting anthelmintic effect? 3. What are the gastro-intestinal parasites are expelled after the administration of the extract?

Chapter 1 Introduction
Collection and identification

Experimental Framework
The following are the proposed flow of processes that will be conducted for the research experimentation.

of sample.

Washing, Drying and Extraction

Preparation of the extracts of M. pudica Linne according to concentration. Administration of the Extracts, commercial anthelmintic(control) and water to the G. domesticus.

Evaluation of the Feces

Date Gathering Data analysis
Figure 1: Experimental Framework

Chapter 1 Introduction
Hypothesis
A. Null (Ho) M. pudica Linne leaves extract does not exhibit anthelmintic activity in Native chicken. B. Alternative (Ha) M. pudica Linne leaves extract exhibits anthelmintic activity in Native chicken.

Chapter 1 Introduction
Significance of the Study
People living in cities and surrounded by modernized hospitals ignore the importance of herbal medicinal plants.
Reduce budget in poultry raising and in daily expenses for maintaining chickens. Finally, these serve as references for other researchers to develop an inexpensive and effective anthelmintic that easily available in large quantities in the Philippines.

Chapter 1 Introduction
Scope
The anthelmintic effect of M. pudica L. leaves Extract in Native Chicken naturally infected with Gastro-intestinal Delimitation Only ten samples of chicken are used in the experiment Flotation Method will be used as the method Identification of the chemical compound causing the anthelmintic activity will not included.

Chapter 1 Introduction
Definitions of Terms
Anthelmintic - drugs that expel parasitic worms (helminths) from the body, by either stunning or killing them. (Quisumbing,1952). Extract - As concentrated preparations of vegetable or animals drugs obtained by removal of the active constituents of the respective drugs suitable menstrual. (Troy,2006) Infection - An infection is the colonization of a host organism by parasitic species.

Chapter 1 Introduction
Gastrointestinal (GI) Parasites - Gastro refers to the stomach, intestinal refers to the small intestines (duodenum, jejunum, and ileum), and a parasite is any organism that lives on or within another living animal and uses it to survive. (Saunders,1999) Native chicken - Is a breed of chicken Gallus domesticus. They are a utility bird, raised for meat and eggs. They are popular choice for backyard flocks because of their egg laying abilities and hardiness. (Raymond,2000)

Chapter 2 Review of Related literature

SCIENTIFIC NAME: Mimosa pudica LINNE FAMILY NAME: FABACEAE LOCAL NAME: MAKAHIYA

Local Literature

Historical Background
M. pudica linne is very common and abundant throughout the Philippines in open, waste places at low and medium altitudes in settled areas. It was introduced from tropical America and is now a pantropic weed.

Chapter 2 Review of Related literature

M. pudica linne can be grown in most soils as long as it drains well. They are good for locations that are deficient in nitrogen, since they are able to convert nitrogen from the atmosphere into a usable form. Most plants do not share this ability.

Figure 2: Makahiya

Figure 3: Makahiya in its Native Habitat

Chapter 2 Review of Related literature

Chemical constituents M. Pudica Linne contains the toxic alkaloid mimosine, which has been found to also have antiproliferative and apoptotic effects.

The extracts of M. pudica Linne immobilize the filariform larvae of Figure 4: Structure of Strongyloides stercoralis in less than one Mimosine hour. Makahiya contain has high tannins which has been use as dewormer in goats (Dr. Tomas Fernandez,2004)
The roots contain tannin 10 percent (Nadkarni and Dymrock, 1951)

Chapter 2 Review of Related literature

Figure 5: Native Chicken

Figure 6: Native Chicken in its Native Habitat

Darag is a general term used of the Philippine native chicken strain indigenous to and most dominant in Western Visayas. This chicken was evolve from the Gallus, galls Red Jungle fowl.(Robinson RD,1990)

Chapter 2 Review of Related literature

Ascaridia galli

Figure 6: Ascaridia galli

The A. galli is a nematode parasite that causes ascariasis, or worm infection, in poultry.
Life Cycle

Eggs have thick, albuminous shell that are resistant to desiccation and persist for a long time in the environment. Larvae do not hatch but moult inside the eggs until it reaches the L3(third) stage.

Chapter 2 Review of Related literature Anthelmintic Study on M. pudica Linne

Some medicinal research has looked into its other possible uses , including as an anthelmintic or a medicine against intestinal parasites (Mr. Tan,2010 )

Makahiya (M. pudica L.) can be used as anthelmintics or dewormers in treating parasitism in goats , these plants are rich in bioactive compounds that are critical in killing, lyzing, and expelling adult worms, their eggs and larvae (Dr. Tomas Fernandez,2004)

Chapter 2 Review of Related literature Toxic effect of M.pudica Linne

Mimosine rumen microbes convert into a toxic compound that interferes seriously with thyroid gland function and causes hair loss other toxic effects at low Concentration (30-50%)

death (>60%) particularly to ruminants, rats, mice, pigs and poultry

Safest dose per concentration 3-8mL and toxic dose >8mL. It can cause poisoning of cattle in Papua New Guinea, especially when cut and dried and has caused stunted growth in chickens in Indonesia.

Chapter 2 Review of Related literature

Foreign Literature

Figure 7: Different Species of M. pudica From left to right: Mimosa pudica Linnaeus, M. bimucronata de Candolle, and M. diplotricha de C. Mimosine, Mimosine, tannins, Mimosine, tannins, Chemical tannins, alkaloids alkaloids alkaloids constituents

Part use MOA

Root, flower, leaves,seeds

Root, flower, leaves,seeds

Root, flower, leaves

Critical in killing, lyzing, and expelling adult worms, their eggs and larvae

Chapter 2 Review of Related literature

In West and East Africa, M.pudica Linne is used in treatment of constipation, hepatitis, malaria fever, gastro-intestinal parasites.

Six Jamaica medicinal plant, M. Pudica L. could inhibit the larvae within 1 hour of exposure by in vitro bioassay of aqueous extracts methanol (AME) of the green leaves of mimosa ( M. pudica L).

Chapter 3 Methodology
Source of Stock
G. domesticus freely ranged in the backyard in Laguna was used in this study.
They were examined if they are positive of internal parasites through fecal analysis using flotation method. G. domesticus found positive of parasites were placed into five separate groups of labeled cages three days before the administration of the extract.

Chapter 3 Methodology
Collection and preparation of Samples
Collection and rearing of test samples including preparation of extract according to concentration of M. pudica Linne were done upon testing. Fresh leaves of M. pudica L. were collected from Laguna. leaves was washed thoroughly Dry and weigh about 300g Placed in mortal and pestle The pulverized leaves were placed in a clean cheese cloth and extracted to beaker.

Chapter 3 Methodology
Undergo filtration to remove impurities. Extract were diluted in three separated 250ml beaker. beakers were labeled 75%, 50%, and 25% concentration of the extract. First beaker - contains 25ml of distilled water +75ml of leaves = 75% concentration Second beaker - 50ml of distilled water + 50ml of extract =50% concentration.

Chapter 3 Methodology
Third beaker - 75ml of distilled water + 25ml of extract = 25% concentration. For the control (Sagupaan Hammer) the 5 mL of the solution was mix to a 1 liter of the distilled water 15 mL of distilled water for Untreated Sample.

Chapter 3 Methodology
Administration of the treatment
Fifteen samples of G. domesticus were divided into three groups. First group composed of nine G. domesticus which divided into three groups composed of three G. domesticus to be treated orally using 5ml gastric gavage with 75%, 50% and 25% concentration extract. Second group composed of three G. domesticus and untreated (negative control) distilled water. The third group (positive control) is composed of three G. domesticus to be treated orally with commercial anthelmintic (Sagupaan Hammer) as the positive control.

Chapter 3 Methodology
Collection of Feces Samples
G. domesticus were properly observed after the administration of dewormer.
Feces excreted were immediately collect and placed in a clean labeled fecal cup examined in the Parasitology Laboratory in Department of Agriculture, Quezon city.

Chapter 3 Methodology
Determination of Anthelmintic Effects
The parasites expelled were counted to determine which treatment is most exhibiting anthelmintic effect.

Classification of Gastro- Intestinal Parasites

Collected parasites were identified as gastro-intestinal parasites. Ascaridia galli, Capillaria annulata, Strongloides spp., Hymenolepsis spp.and Coccicidia spp

Chapter 3 Methodology
Further classified as: Nematodes (roundworms), and Cestodes (Tapeworms ), protozoa(additional). Data Gathered
The data gathered are as follows. Frequency counts of gastro-intestinal parasites eggs expelled before and after administration of the different extract.

Treatments Treatment 1- 5mL of 30% extract per 3 months old (for Trial 1 and 2) Treatment 2 - 5mL of 50% extracts per 3 months old (for Trial 1 and 2) Treatment 3 - 5mL of 70% extract per 3 months old (for Trial 1 and 2) Treatment 4 - 5mL of hammer sagupaan (+ Control) per 3 months old (for Trial 1 and 2) Treatment 5 - 5mL of Purified Water (- control) per 3 months old (for Trial 1 and 2)

Chapter 4 Results and Discussion

Result for Normality
TABLE 2: Shapiro wilk test-test for normality
Treatment 1 (25%) 2(25%) 3 (50%) 4 (50%) 0.04588 0.04588 0.4588 0.00070 P-value 0.04588

Since the outcome eggs is not normally distributed the researchers cannot use one-way ANOVA. Instead, KRUSKALWALLIS test had been used, which is a non-parametric test.

5 (75%)
6 (75%) 7 (+ctrl) Sagupaan Hammer 8 (+ctrl) Sagupaan Hammer 9 (-ctrl) Distilled water 10 (-ctrl) Distilled water

0.09318
0.04588 0.04588 0.00109 0.00860

Chapter 4 Results and Discussion

Result for Kruskal-Wallis equality-of-populations rank test
Table 3: Kruskal Wallis Test

treatment 25% 25% 50% 50% 75% 75%

(+ctrl) Sagupaan Hammer (+ctrl) Sagupaan Hammer (-ctrl) Distilled Water (-ctrl) Distilled Water

obs Rank sum 5 120.00 5 114.50 5 117.00 5 95.00 5 142.00 5 150.00 5 95.00 5 117.00 5 150.00 5 117.00

From the result of kruskal wallis test giving the p-value of 0.7806 and 0.4018 is greater than alpha (0.05), it means that there have no sufficient evidence to reject the null hypothesis.
Therefore, the result shows that all of the 10 population medians have no significant difference between the median numbers of eggs.

Chapter 4 Results and Discussion

Result for Friedmans Test (Nematodes)
Table 4: Friedman test for Nematodes

Treatment

p-value(alpha 0.05) 1.0000 0.6547 0.6547 0.6547 0.0

1 (25%) 2(50%) 3 (75%) 4 (+ctrl) Sagupaan Hammer 5 (-ctrl) Distilled Water

The data tested shows that the median samples 1 to 4 have a p-value greater than alpha 0.05 and it means of there is no sufficient evidence to reject the null hypothesis.
On the sample median no. 5 the p-value is less than the alpha 0.05 meaning that it has a sufficient evidence to reject the null hypothesis.

Chapter 4 Results and Discussion

Table 5: Number of eggs per treatment day for Nematodes-Friedman test

Sample 0 1(25%) 2(50%) 3(75%) 200 0 50 0 0 500 0 100 1 0

Day 3 10 50 0 0 0 150

11
0 0 0 0 5800

Sample no. 3(75%) is the concentration exhibiting the most anthelmintic effect sample no. 2(50%) is the concentration which possesses the same effect of sample no. 4(+ctrl). Therefore, as the concentration increase the effect exhibiting also increase.

100 3200 100 0

4(+ctrl)SagupanHammer 0 5(-ctrl) Distilled Water 500

Chapter 4 Results and Discussion

Result for cestodes
Table 6: Presence of Cestodes eggs per treatment group Treatment 0 1 (25%) 2(25%) 3 (50%) 4 (50%) 5 (75%) 6 (75%) 7 (+ctrl) Sagupaan Hammer 8 (+ctrl) Sagupaan Hammer 9 (-ctrl) Distilled Water 10 (-ctrl) Distilled Water (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 1* (+) (+) (+) (+) (-) (-) (+) (+) (+) (+) Day 3 (+) (-) (+) (+) (-) (-) (-) (-) (+) (+) 10 (+) (+) (-) (-) (-) (-) (-) (-) (+) (+) 11 (-) (+) (-) (-) (-) (-) (-) (-) (+) (+)

* day of application of treatment

Chapter 4 Results and Discussion

Result for protozoa (additional)
Table 7: Presence of Protozoans eggs per treatment group Treatment 0 1 (25%) 2 (25%) 3 (50%) 4 (50%) 5 (75%) 6 (75%) 7 (+ctrl) Sagupaan Hammer 8 (+ctrl) Sagupaan Hammer 9 (-ctrl) Distilled Water 10 (-ctrl) Distilled Water (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 1* (-) (-) (-) (-) (+) (-) (-) (-) (-) (-) Day 3 (-) (-) (-) (-) (-) (-) (-) (+) (-) (-) After 10 (-) (-) (-) (-) (-) (-) (-) (-) (+) (-) 11 (+) (+) (+) (+) (+) (-) (+) (+) (+) (+)

Chapter 4 Results and Discussion

Table 8: Classification of Intestinal parasites Nematodes Ascaridia Classificat galli ion of Strongloid gastro es spp. intestinal Capillaria parasites spp. Cestodes 20 Protozoa Coccicidia 56 oocyst spp.

14,100 eggs/gram Hymenole 600 psis spp. eggs/gram 6,800 eggs/gram

Chapter 5 SUMMARY, CONCLUSION AND RECOMMENDATION

In the study conducted, findings have given significant results showing that M. pudica Linne leaves extract possesses anthelmintic properties as shown by the series of tests, experimentation and validation procedures done. M. pudica Linne leaves extract was used for the anthelmintic effect and have given 75% (5mL/3 mos.old) concentrations showing its anthelmintic profile to G. domesticus as descriptive analysis used have shown to be not lethal for G. domesticus consumption unless a marked amount of extract (>5mL) is ingested.

Chapter 5 SUMMARY, CONCLUSION AND RECOMMENDATION

On the other hand, M. pudica Linne leaves extract at different concentrations with the positive and negative control showing that p-value for the Kruskal-Wallis and Friedman test is greater than alpha (0.05) meaning of there is no sufficient evidence to reject the null hypothesis. Also shows that all of the 10 population medians have no significant difference. Recommendation The utilized experimentation procedures employed were only few and limited for which the researchers highly recommend . further tests to be done with the M. pudica leaves extract using more comprehensive extraction procedures to further enhance the characterization, determination the property of the extracts Large number of sample to be tested.

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