IN-VITRO EVALUATION OF THE ANTHELMINTIC

1

IN-VITRO EVALUATION OF THE ANTHELMINTIC ACTIVITY PRESENT FROM THE EXTRACT OF GUYABANO LEAVES (Annona muricata) ON ROUNDWORMS (Ascaris Lumbricoides)

John Paulo D. Mendoza

JRLMHS, Science Deparment Gapan City, Nueva Ecija

2

ABSTRACT Anthelmintic drugs like Albendazole and Mebendazole are currently in rapid demand due to the increasing dense host population of parasites. Despite the prevalence of parasitic worms, anthelmintic drug discovery is the poor relation of the pharmaceutical industry that could also bring physiological side effects. This study evaluates the in-vitro bioassay test evaluation of the anthelmentic activity present from the extract of Guyabano (Anona Muricata Linn) leaves and its potential as an alternative medicine for deworming. Guyabano leaves were finely cut into smaller pieces then pulverized by blending. It was soaked in a 100 mL of 95% ethyl alcohol for about 48 hours to obtain the extraction. The extract was brought in the Philippine Rice Institute to be subjected in Rotary Evaporation process. The obtained volume of Guyabano leaves gave the exact 43.75% yield. Five treatments were prepared for the experimental design. Five roundworms were subjected into petri plates and poured each with various concentrations of Guyabano extract. The reaction of the roundworms were observed and recorded by minutes of the time of paralysis and death. It reveals that the Treatment 3 (100 ml of GL extract) with the least amount of time expended (D= 37.02) displayed anthelmintic acivity which is almost 17 minutes comparable to its expulsion in the Combantrin (D=20.7) as the positive control. It can be stated that extract of Guyabano Leaves has a potential to endure anthelmintic activity on Ascaris Lumbricoides. It also establishes a significant difference between the comparable effectiveness of each treatments in terms of the parameters used, Time of Paralysis (P) and Time of Death (D). Based on the published phytochemical screening of Guyabano leaves, its amorphous alkaloid and substance of flavonoids which are bio-active compound used for textiles and ceramics can also display chemical properties like anthelmintic activity.

3

ACKNOWLEDGEMENTS In line with this study, we would like to thank God for helping this study reach its attainable results to better contribute for the expanding industry of Medicine. We would like to express our deepest gratitude to those who helped us, from the very beginning and the end of this project, to Mrs. Leonora Alcantara and Ashley Joy Alcantara for providing us the time and place of study. To our research adviser, Mrs. Alice Y. Caymo, for approving this research and certain approval requests and providing us necessary tools for our study. To Mr. Jerone Mejia, Mr. Jeffrey Sta. Ines and Mrs. Wajavina Catacutan, for guiding and teaching us the importance of conducting a research with their full-pledge logical and moral support And to all of the proponents of this study, Albert Mendoza, Mark Razon, Eric Paolo Velasquez, Jillian Lomaad, Kane Rayah Delos Santos, Rinoah Rivera, John Vincent Tecson, Micaela Nicolas, Jazzel V. Young, Pamella Ordonez, John Carlo A. Alcantara, Charmane G. Chuidian, Marjorie Manalo, Rosalyn Carreon, Marijean Benitez, Joshua Mendoza. I, John Paulo Mendoza, leader of this research, profoundly gave my knowledge, time and effort to expand the growing industry of medicinal plants. Allysa Faith B. Milad, for giving us the idea of this study and Jamal Omar. S. Sarangani, for giving us the courage and determination to push through this research

4

TABLE OF CONTENTS Title Page……………………………………………………………...………… ..1 Abstract................................................................................................................. 2 Acknowledgement................................................................................................. 3 Table of Contents……...........................................................................................4 CHAPTER I – INTRODUCTION……………………………………………..5-8 A. Background of the study...............................................................................5 B. Objective of the Study...................................................................................5 C. Statement of the Problem..............................................................................6 D. Significance of the Study..............................................................................6 E. Scope and Limitations...................................................................................7 F. Time and Place of Study………………………………………………..……...8 CHAPTER II - REVIEW OF RELATED LITERATURE…………………8-13 A. Research Literature/Publications.........................................................8-13 CHAPTER III - METHODOLOGY.............................................................13-17 CHAPTER IV –RESULTS AND DISCUSSIONS………………………..18-22 A. Production and Chemical constituents of Guyabano leaf extract…………………………………………..……………….….19 B. Data acquired from the experiment………………………………...19 C. Graphical Presentation of Data.....................................................21 D. Statistical Analysis of the treatments………………………….......22 CHAPTER - V SUMMARY, CONCLUSION, AND RECOMMENDATION A. Conclusions and Recommendations…………….……………………… B Bibliography……………………………….………………………………..

5

INTRODUCTION Background of the Study The Philippines presents as remarkably rich plants. Approximately 8,000 different species are known in the Archipelago. Many of them were investigated for used as our medicinal Herbs. According to world health organization (WHO), greater than 80% of the total world’s population depends on the traditional medicines in order to satisfy their primary health care needs. It also suggested in improving the technologies for cultivation of medicinal plants6. The chemical substances of the medicinal plants which have the capacity of exerting a physiologic action on the human body were the primary features. So far, in some places outside the Philippines like America, they make further studies on how to make use of such plants. They make such processes by their high technology, eventually producing thousands and thousands of medicinal products. As we can see, they are now in liquid form, tablets, syrups, powders, capsules and many other. Objectives The principal aim of the study is to evaluate the anthelmintic activity of the extract of Guyabano leaves on Ascaris Lumbricoides. Specifically, it intends to; 1. evaluate the anthelmintic activity present from the extract of Guyabano Leaves on AscarisLumbricoidesusing different treatments through In-vitro Biological Assay test. 2. synthesize and determine the presence of different active compounds from the extract of Guyabano leaves that provide its various pharmacological activities.

6

3. determine the effects/reactions of AscarisLumbricoides concocted with various extract concentrations/treatments testedin the assay, which involved determination of time of paralysis (P) and time of death (D) of the worms.. Statement of the Problem In line with this project, it was conducted to answer the following question regarding the anthelmintic activity present from the extract of Guyabano Leaves: 1. How may the extract from Guyabano leaves be produced? 2. What are the bioactive constituents present from the extract of Guyabano Leaves provide its ability to display anthelmintic activity? 3. How may the effectiveness of different concentrations of Guyabano leaves extract on Ascaris Lumbricoides be described in terms of: a. Time of Paralysis b. Time of Death 4. Is there a significant difference between the effectiveness of concentrations of Guyabano leaves extract on Ascaris Lumbricoides in terms of: a. Time of Paralysis b. Time of Death 5. Is the Guyabano leaf extract comparable to the positive control, in terms of Time of Paralysis (P) and Time for Death (D) of worms? Significance of the Study The study has been helpful to the researchers for it had come up to their realization how important utilizing the herbs and it helps improve their knowledge about the different medicinal values of all plants especially Guyabano Leaves extract which can be used as a substitute for other synthetic anthelmintic drugs. Thus helping people who cannot afford to buy too expensive drugs to cure such ailments.

7

The Pharmacy Students, this study would best help the Pharmacy students for it will add more information on these kind of plants. And through this study they can have some choice in choosing a drug for deworming which is more economical. Community People, to the people of the community information gained from the result of this study would help and encourage them to plant medicinal plants right in their own backyard knowing the uses of the medicinal plants. To the Leaders, Based on the result of this study the community leaders could help disseminate the use of local plants in treating common diseases better than synthetic or commercial drugs. To the Manufacturers, This information can be used as a step for more interesting research on Philippines Medicinal plants. The findings of this study can be used as another source of medication and provide lesser budget for costly imported raw materials. Scope and Limitations This study was only focused in evaluating the Anthelmintic Activity present from the Guyabano leaves extract. It only focuses on the pointers mentioned in the objectives and statement of the Problem. The number Ascaris Lumbricoides on each petri plates also affects the factor and results of this research, futher utilization of worms is advised. This study only focuses on the use of only one nematode. Other nematodes might give off different results compare to this study.

8

Time and Place of Study The experiment was conducted on the last weeks of July up to the first week of September. A clear, clean and room temperature area provided by Mrs. Alcantara on Sto. Nino, Gapan City and necessary tools and equipments were borrowed and bought from DOST and National Book Store. The worms were obtained fromt the Lab Seventeen Diagnostic Center at Gapan City, Nueva Ecija

REVIEW OF RELATED LITERATURE

Guyabano and its Family

9

Guyabano(AnnonaMuricata Linn.)tree, or soursop in English, is a small tree, usually about 5 to 7 meters high. Guyabanois a fruit bearing tree, broadleaf, flowering, and evergreen that is native to Central America, the Caribbean and South America. Guyabano can be found in Mexico, Colombia, Brazil, Peru, and Venuzuela. Guyabano or Soursop is also native in sub-Saharan African countries. Guyabano or Soursop in adaptable to tropical climate and are currently cultivated for its fruit in most Southeast Asian countries such as Malaysia, Indonesia and Philippines (Vijayalakshmi, 2011) Annona muricata L. belongs to the family of Annonaceae also known asthe custard apple family, of flowering plants consisting of trees, shrubs, or rarely lianas, with 2106 accepted species and more than 130 genera, it is the largest family in Magnoliales. Several genera produce edible fruit, most notably Annona, Anonidium, Asimina, Rollinia, and Uvaria. It is a widespread small tree and has its native in Central America.The fruit of Annona Muricata Linn.is found to be edible in Yunnan province of China and their fruits is used commercially for the production of juice, candy and sherbets. Intensive chemical investigations of the leaves and seeds of this species have resulted in the isolation of a great number of acetogenins (Rajeswari, 2011) Guyabano leaves are oval shaped with smooth shiny texture. Guyabano leaves are 7 to 20 centimeters in length, pointed on both ends, with petioles about 5mm long.

Medicinal and Nutritional Value of Guyabano Research revolving around guyabano’s healing properties is lacking in the scientific world, but so far researchers have been studying guyabano for its ability to protect against cancer

10

and reduce side-effects of chemotherapy. The amorphous alkaloid found in its leaves has aso the ability to expel intestinal worms. Since 1976, over 20 independent labs researched Guyabano’s anti-cancer effects following initial research carried out by the National Cancer Institute. The National Cancer Institute found that guyabano’s “leaves and stems were found effective in attacking and destroying malignant cells. After the 1976 findings, that were apparently never released to the public, other research studies came out with similar conclusions: One study published in the Journal of Natural Products found that one chemical in Graviola was 10,000 times more potent than a chemotherapy drug called Adriamycin. The Catholic University of South Korea reports that guyabano is not only a threat to cancer cells, but also leaves healthy cells alone. This is not the case with chemo, which target all the cells – much like antibiotics indiscriminately destroying all gut bacteria, good and bad. Purdue University found that leaves from the guyabano tree are “killed cancer cells among six human cell lines”. The researchers also found that the leaves were particularly effective for prostate and pancreatic cancers. Parasitic Worms and Nematodes Members of phylum Nematoda, the roundworms, are found almost everywhere. This group includes some free-living forms which live in soil and water. However, most nematodes are parasites. At least fifty species are parasites of human alone (Oram, 2005). Ascaris is a parasite which can live in the intestines of humans and most other mammals. It “robs” the host of digested food, and, if enough worms are present, they may block the intestine, which could kill the host. Unlike hookworms, which attaches to the wall of the intestine and sucks blood. This loss of blood may lead to a lack of energy in the host. Outside the

11

bodies of their hosts, hookworms need warm soil temperatures. Therefore, they are common in southern parts of the United States. They can enter the human body through bare foot (Atkins, 2007). Parasitism and the Epidemiology of Ascariasis Parasitism can be a limiting factor. In a parasitic relationship, one organism lives in or on another organism, which is usually harmed by the parasite (Oram, 2005). This limitation is density-dependent. Parasitism usually causes death only when the parasites are in very dense host populations. The possibility that parasites will be passed from one organism to the next greater in a dense host population. (Hummer, Smoot et al, 2005). Ascariasis is the most common helminthic infection, with an estimated worldwide prevalence of 25% (0.8-1.22 billion people). Usually asymptomatic, ascariasis is most prevalent in children of tropical and developing countries, where they are perpetuated by contamination of soil by human feces or use of untreated feces as fertilizer. Ascariasis is most common and intensive in children, who are more likely than adults to be symptomatic. In children, intestinal obstruction caused by heavy worm burden (≥60) is the most common manifestation of disease. An estimated 2 per 1000 infected children develop intestinal obstruction per year. Among children aged 1-12 years who presented to a Filipino hospital with abdominal emergencies between 1958-1962, symptomatic Ascaris lumbricoides infection was responsible for 12.8% of cases, with 68% of those due to intestinal obstruction, usually at the terminal ileum. The peak incidence was at age 2 years in a series from Colombia and age 4.8 years in a series from Turkey. (Cunna, 2005) The chemical constituents of Guyabano leaves

12

Phytochemical screening of the Guyabano leaves extract showed the presence of alkaloids, carbohydrates, coumarins, flavonoids, glycosides, phenolic compounds, phytosterols, proteins, quinones, saponins, steroids and terpenoids. The ethanolic leaf extract shows high phosphorous and iron content of 128mg% and 1.075mg% respectively. (Greenberg, 2005) Most acetogenins are white waxy derivatives of long chain fatty acids (C32 or C34), and the theterminal carboxylic acid is combined with a 2-propanol unit at the C-2 position to form a methylsubstituted –unsaturated lactone. Anthelmintics Anthelmintics are drugs that are used to treat infections with parasitic worms. This includes both flat worms, e.g., flukes and tapeworms and round worms, i.e., nematodes. They are of huge importance for human tropical medicine and for veterinary medicine. The World Health Organization estimates that a staggering 2 billion people harbour parasitic worm infections (http://www.who.int/wormcontrol/statistics/). Parasitic worms also infect livestock and crops, affecting food production with a resultant economic impact. Also of importance is the infection of domestic pets. Indeed, the companion animal market is a major economic consideration for animal health companies undertaking drug discovery programmes. Despite the prevalence of parasitic worms, anthelmintic drug discovery is the poor relation of the pharmaceutical industry. The simple reason is that the nations, which suffer most from these tropical diseases, have little money to invest in drug discovery or therapy. It comes as no surprise therefore that the drugs available for human treatment was first developed as veterinary medicines. There is thus a pitifully small repertoire of chemotherapeutic agents available for treatment. In some respects, this situation has been exacerbated by the remarkable

13

success of ivermectin over the last twenty years (Geary, 2005), which has decreased motivation for anthelmintic drug discovery programmes (Geary, Sangster and Thompson, 1999). This prompts concern, as anthelmintic resistance has been widely reported in livestock and it may only be a matter of time before this phenomenon occurs in parasites of humans. Broad-spectrum anthelmintic are effective against parasitic flat worms and nematodes. However, the majority of drugs are more limited in their action, e.g., praziquantel and combantrin, a drug used in the treatment of schistosomiasis and thought to act by disrupting calcium homeostasis (Greenberg, 2005), has no activity against nematodes. For the purpose of this review, we will focus on drugs used in human and veterinary medicine to treat parasitic nematode infection.

METHODOLOGY

14

Collection of Guyabano Leaves

Guyabano leaves were collected in Guyabano trees of Gapan City, Nueva Ecija. The leaves were brought The collected leaves were subjected for 48 hours of air drying in a well-ventilated area. Pulverization of Guyabano Dried Leaves

15

The dried Guyabano leaves were finely cut into smaller pieces and pulverized for 3 minutes with the use of a blender. The pulverized leaves were subjected for air drying for 24 hours. Extraction of Guyabano Leaves

16

The pulverized leaves were weighed; 5 grams of pulverized leaves were obtained and soaked at 100 mL of 95% ethanol for 48 hours.

The

extract of

the sample

were

collected

using

nylon filtration method and brought in the Philippine Rice Institute to undergo Rotary Evaporation. Preparation of Treatments

17

Four roundworms were obtained from Lab Seventeen Diagnostic Center affiliate laboratory in Gapan City, Nueva Ecija.These will be subjected into petri plates and pour each with various concentrations (25,50 and 100 mL) of Guyabano extract, Combantrins as a positive control and 0.9% of Normal Saline Solution as negative control. In-vitro Bioassay Test

18

Bioassay test on the extract of Guyabano leaves was done primarily to evaluate its anthelmintic activity to determine the efficiency extract to expel parasitic worms. Combantrin (+) control and 0.9% Normal Saline Solution as (–) control were used to determine the differences of time in paralyzing and expelling Ascaris Lumbricoides. The reaction of Roundworms was technically observed. Each petri plates containing different treatments were timed using a timer with nanosecond time frame. Minutes of the time for paralysis and death were recorded.

RESULTS AND DISCUSSIONS Production of pure Guyabano leaves One Hundred Seventy-five ml of pure extract from Guyabano (Anonna Muricata Linn.) leaves were collected from the 20 grams of dried Guyabano leaves. The obtained volume of Guyabano leaves gave the exact 43.75% yield. This implies that

Guyabano leaves can be

soaked in ethanol to recover

its pure extract. The

product can be a promising

raw material of new

anthelmintic drugs.

19

The result of the bioassay test was determined by the time recorded on each treatments containing one Ascaris Lumbricoides on each treatments. Description of Guyabano leaves extract The phytochemical analysis from different published studies reveals that the Guyabano leaves extract contains alkaloids which is responsible for inhibiting the neuromascular transmitters of Ascaris Lumbricoides resulting in paralysis, either spastic or flaccid paralysis of an intestinal helminth (Geary, 2005) and Flavonoids which works by keeping the worm from absorbing sugar (glucose), so that the worm loses energy and dies. (Greenberg, 2005) Table 1. In-vitro evaluation of the anthelmintic activity average time of Paralysis (P) in and Death (D) in minutes on each treatments (both concentrated and control groups) TIME FOR TREATMENTS

DOSAGE

PARALYSIS (P) (min)

TIME FOR DEATH

T1

25 ml of GL extract

38.2

(P) (min) 58.05

20 T2

50 ml of GL extract

31.07

42.3

T3

100ml of GL extract

23.9

37.07

T4 (+)

10 ml of Combantrin

11.03

20.7

T5 (-)

10 ml of Natural Saline

0.00

0.00

Solution

The table shows the recorded time for respective treatments. Treatment 4 as the (+) positive control obtains the shortest span of time for Ascaris Lumbricoides to paralyze and expel. The concentrated treatments also showed minutes of expulsion, yet its more on longer span of time while the (-) negative control showed no reaction or anthelmintic activity in the process. Thus, In-vitro bioassay test gives off attainable results to evaluate and prove that there is anthelmintic activity present from each effective treatments, with respect to our dependent variable which is the extract of Guyabano Leaves, based on corresponding amounts of dosage. Figure 2- Average Presentation of the recorded time for Paralysis (P) and Death (D) on each treatments

21

The graph on the other hand, shows the columned changes done in the In-vitro bioassay test with the used of bar graphs showing the corresponding time for paralysis and death of each treatment. Resulting as, Treatment 4 with the fastest time expended in anthelmintic activity out of the five, followed by the concentrated extracts which also has the presence of anthelmintic activity with longer span of time. This result reveals that the Treatment 3 (100ml of GL extract) with the least amount of time expended (D= 37.02) displayed anthelmintic acivity which is almost 17 minutes comparable to its expulsion in the Combantrin (D=20.7) as the positive control. It can be stated that extract of Guyabano Leaves has a potential to endure anthelmintic activity on Ascaris Lumbricoides.

Anova: Single Factor SUMMARY Groups Data1 Data2 Data3 Data4 ANOVA Source of Variation Between Groups Within Groups Total

 Count 5 5 5 5

SS

0.05 Sum Average Variance 191.25 38.25 155.3 31.06 115.4 23.08 55.15 11.03

df

2041.047 0.0514 2041.098

MS

F

3 680.3488 211781.7

0.0125 0.00025 5E-05 5E-05

P-Value

F crit

0.000 3.238872

16 0.003213 19

Statistical Analysis for the Time of Paralysis (P)

22

The statistical data tool used was the One-way ANOVA. The calculated p.value of 0.0001 is