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The Physiologic Effects of the Crude Concoction from Euphorbia Hirta (Tawa-Tawa) Using Staphylococcus Aureus for Anti-Bacterial Tests and Animal Inoculation Assay for Toxicity 1

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Chapter 1

INTRODUCTION

Background

Euphorbia hirta, belongs to the family of Euphorbiaceae which is a large family of dicotyledons, with about 300 genera and over 5,000 species. Here in the Philippines, the Euphorbia hirta, is commonly referred to as Tawa-tawa or Gatas-gatas in some provinces. It is also known as Asthma weed or Snake weed in the United States. The plants of 3 different species share Phoretic variations, these plants are: (1) Mutha (Cyperus rotundus), (2) Gatas-gatas (Euphorbia hirta) and (3) Botoncillo (Gomphena globosa). Tawa-tawa is usually very abundant in tropical regions such as the Philippines. A simple weed scattered in sunny lawns, waste places and open grasslands. It is pantropic in distribution. The plant is an annual, hairy herb, usually branched from the base, spreading up to 40 cm long. The stem is slender and often reddish and purplish in color, covered with yellowish bristly hairs especially in younger parts. The leaves are oppositely arranged, elliptical-oblong to oblong-lanceolate, 1 to 2.5 cm long, toothed at the edge, and blotched with purple in the middle. In the axils appear numerous involucres, purplish or greenish, dense, axillary, short stalk clusters or crowded cymes, about 1 mm long. The capsules are broadly ovoid, hairy, three-angled, about 1.5 cm. The small green flowers constitute the inflourescence characteristics of the euphorbias. The stem and the leaves produce white or milky juice when cut (Lind and Tallantire, 1971;Anonymous 2005). In some parts of Africa, extract of the plant are used in the treatment of asthma and respiratory tract inflammations (Kokwaro, 1993). The plant contains relatively abundant white latex. The white latex is capable of causing dermatitis (Oliver, 1960). The plant shows antibiotic activity (Sofowora, 1993). Upon reading some medical research studies on Euphorbia hirta revealed that the plant is very popular amongst its specie; Euphorbia hirta as an herbal medicinal plant has been presumed to possess therapeutic virtues, thus there is a need to determine its antibacterial potentials.

Objectives

The study about Euphorbia hirta (Tawa-tawa) is intended to determine the Minimum Inhibitory Concentration (MIC), anti-microbial properties using Staphylococcus aureus, toxicity and platelet count.

Specifically, this study singled out to answer the following questions:

1. What is the Minimum Inhibitory Concentration (MIC) of crude extract concoction of Tawa-Tawa roots? 2. Does Tawa-Tawa possess any anti-bacterial effect towards the test organism- Staphylococcus aureus? 3. Does the crude extract concoction of Tawa-Tawa roots possess any toxic effect when administered orally to the test organism-albino mice? 4. Can Tawa-Tawa affect the platelet production of albino mice induced with thrombocytopenic drugs such as aspirin?

Significance of the Study

Physician. This study would help medical practitioners especially doctors of medicine particularly hematologist who specialize in the diagnosis and treatment of Hemorrhagic Fever (H-Fever) or commonly known as Dengue Fever. Pathologist is still searching and discovering for treatment for the disease. Dengue Fever Patients. Dengue virus causes thrombocytopenia. This study would help patients suffering from dengue fever lessen the burden of having the disease. Health Workers. Health workers play an important role in information dissemination. Through the Health workers, information about the disease could be avoided and minimized. Community. The global burden of dengue has grown dramatically in recent decades, and thus increased number of cases affects the community especially the burden of the disease remains proportionately much greater in Asian and Pacific countries including Philippines. Future Researchers. This study would be of help to future researchers, especially in the field of drugs and medicine to come up with a treatment of Dengue fever. This would also serve as a guide to study other possible toxic, and anti-microbial properties of Tawa- Tawa. This work therefore, was undertaken to authenticate the activities of Tawa- Tawa on toxicity, anti-microbial properties, and amplification of platelet count.

Milieu

This study encompasses the potential pythochemistry of Euphorbia hirta as well as the anti-microbial activities using Staphylococcus aureus, toxicity, and animal inoculation assay. This study was conducted from November 2005 to March 2008 in the City of Bacolod, Negros Occidental. This experimental study was performed in the Biochemistry Laboratory in the University of Negros Occidental- Recoletos, under the supervision of Mr. Ronnie Gicana. The results of the research holds true only during the time the research was done. Any modifications and further study maybe conducted to obtain more accurate and precise results.

Chapter 2
Review of Related Literature

This chapter presents related concepts on the risk factors and daily practices lead to dengue fever. Important epidemiological measures, environmental factors and prevention and control are presented to provide basis that these factors contribute significantly to the occurrence of disease. The effects of Tawa-Tawa were cited in the study on MIC, Agar Diffusion, and Animal Inoculation Assays to identify their possible toxicity, anti-microbial property, environmental management and amplification of platelets are also reviewed.

Medicinal Plants Medicinal plants are a source of great economic value in the Philippine Island. Nature has bestowed on us a very rich botanical wealth and a large number of diverse types of plants grow in different parts of the country. Philippines is rich in all the 3 levels of biodiversity, namely; species, genetic diversity and habitat diversity. (Ahmad I, et.al, Ethnopharmacol, 1998). In Philippines, thousands of species are known to have medicinal value and the use of different parts of several medicinal plants to cure specific ailments has been in vogue since ancient times. Herbal medicine is still the mainstay of about 75-80% of the whole population, mainly in developing countries, for primary health care because of better cultural acceptability, better compatibility with the human body and fewer side effects. However, the last few years have seen a major increase in their use in the developed world. (Rajkot and Jamnagar, 2004). Nowadays multiple drug resistance has developed due to the indiscriminate use of commercial antimicrobial drugs commonly used in the treatment of infectious disease. In addition to this problem, antibiotics are sometimes associated with adverse effects on the host including hypersensitivity, immune-suppression and allergic reactions. This situation forced scientists to search for new antimicrobial substances. Given the alarming incidence of antibiotic resistance in bacteria of medical importance, there is a constant need for new and effective therapeutic agents. Therefore, there is a need to develop alternative antimicrobial drugs for the treatment of infectious diseases from medicinal plants and random screenin of active plants for active chemicals is important. (Davis J, Science,1995)

Euphorbia hirta or (Tawa- Tawa)

Description Euphorbia hirta is a terrestrial, annual, erect herb, up to 60 cm tall. It is taproot white or brown. The stem is round, solid, hairy, with abundant milk sap. Stipules are present. The leaves are simple, not lobed or divided, opposite, sessile or stalked, elliptic, less than 2 cm long/wide, hairy on both sides, denser pilosity along the veins in the lower face, more scattered on the upper side; leaf base asymmetric, margin finely dentate, apex acute, base acute, 3-veined not to the top. Flowers are unisexual, solitary or grouped together in an axillary cyme, stalked, and petals are absent. It is distributed in pan tropical, or partly sub-tropical areas and found in roadsides and wastelands as cultivated areas. (Le Bourgeois T., et. al., 2001)

Medicinal Uses

Euphorbia hirta has traditionally been used in Asia to treat bronchitic asthma and laryngeal spasm, though in modern herbalism it is more used in the treatment of intestinal amoebic dysentery. It should not be used without expert guidance, however, since large doses cause gastro-intestinal irritation, nausea and vomiting.
It is anti-asthmatic, antipruritic, sedative, antidote, diuretic, purgative and homostatic. The aerial parts of the plant are harvested when in flower during the summer and can be dried for later use. The stem, which is taken internally, is famed as a treatment for asthma, bronchitis and various other lung complaints. The herb relaxes the bronchioles but apparently depresses the heart and general respiration. It is usually used in combination with other anti-asthma herbs such as Grindelia camporum and Lobelia inflata. It is also used to treat intestinal amoebic dysentery.
The whole plant is decocted and used in the treatment of athlete's foot, dysentery, enteritis and skin conditions. It has been used in the treatment of syphilis.
The sap is applied to warts in order to destroy them. The treatment needs to be repeated 2 - 3 times a day over a period of several weeks to be fully effective. (http://www.ibiblio.org/pfaf/cgi-bin/arr_html?Euphorbia+hirta)

Cultivation details

Euphorbia hirta prefers a light well-drained moderately rich loam in an open sunny position. It is not very tolerant of frost, though it can probably be grown successfully in this country as a spring-sown annual. Hybridizes with other members of this genus. The ripe seed is released explosively from the seed capsules. Members of this genus are rarely if ever troubled by browsing deer or rabbits. This genus has been singled out as a potential source of latex (for making rubber) for the temperate zone, although no individual species has been singled out. (http://www.ibiblio.org/pfaf/cgi-bin/arr_html?Euphorbia+hirta)

Propagation

Euphorbia hirta is propagated in seed - sow mid to late spring in situ. Germination usually takes place within 2 - 3 weeks at 20°c. It might be best to sow the seed in a cool greenhouse in early March. When they are large enough to handle, prick the seedlings out into individual pots and plant out the seedlings in late May. This will give the plants longer to grow and mature. (http://www.ibiblio.org/pfaf/cgi-bin/arr_html?Euphorbia+hirta)

Biologic Activity
Minimum Inhibition Concentration. In microbiology, is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. Minimum inhibitory concentrations are important in diagnostic laboratories to confirm resistance of microorganisms to an antimicrobial agent and also to monitor the activity of new antimicrobial agents. MICs can be determined by agar or broth dilution methods. Commercial methods are the E-test or Oxoid MICE valuator method using strips of a gradient of antibiotic concentration. E-test strips create ellipses of microbial inhibition. The point at which the MIC is taken within the ellipse of inhibition is the point where the bacterial growth crosses the strip. Clinically, the minimum inhibitory concentrations are used not only to determine the amount of antibiotic that the patient will receive but also the type of antibiotic used, which in turn lowers the opportunity for microbial resistance to specific antimicrobial agents. (www.wikipedia.com)
Toxicity.
The degree to which a substance can harm humans or animals. It can be acute, sub-chronic, or chronic: Acute toxicity involves harmful effects in an organism through a single or short-term exposure. Sub-chronic toxicity is the ability of a toxic substance to cause effects for more than one year but less than the lifetime of the exposed organism. Chronic toxicity is the ability of a substance or mixture of substances to cause harmful effects over an extended period, usually upon repeated or continuous exposure, sometimes lasting for the entire life of exposed organism. (http://www.medterms.com/script/main/art.asp?articlekey=34093)
Anti-microbial.
Clinical substance produced either by a microorganism or by a synthetic means that is capable of killing or suppressing growth of microorganisms. Antimicrobial agents play a central role in the control and management of infectious diseases, some understanding of their mode of action and the mechanisms microorganisms deploy to circumvent antimicrobial activity is important, especially because diagnostic laboratories are expected to design and implement tests that measure a pathogen’s response to antimicrobial activity. Several key steps must be completed for an antimicrobial agent to successfully inhibit or kill the infecting microorganism. First, the agent must be in an active form. This is ensured through the pharmacodynamic design of the drug, which takes into account the route through which the patient will receive the agent (e.g. orally, intramuscularly, intravenously). Second, the antibiotic must also be able to achieve sufficient levels or concentrations at the site of infection so that it has a chance to exert an antibacterial effect (i.e., be in anatomic approximation with the infecting bacteria). The ability to achieve adequate levels depends on the pharmakokinetic properties of the agent. (Bailey & Scott’s Diagnostic Microbiology, Eleventh Edition)

Staphylococcus

Staphylococcus is group of bacteria that can cause a multitude of diseases as a result of infection of various tissues of the body. Staphylococci that are associated with infections in humans are colonizers of various skin and mucosal surfaces. Because the carrier state is common among the human population, infections are frequently acquired when the colonizing strain gains entrance to a normally sterile site as a result of trauma or abrasion to the skin or mucosal surface. Staphylococci are also transmitted from person to person. Upon transmission, the organism may become established as part of recipient’s normal flora and later be introduced to sterile sites by trauma or invasive procedures. Alternatively, a surgeon or nurse may directly introduce the organism to normally sterile sites, such as during surgery. Person-to-person spread of staphylococci, particularly those that have acquired antimicrobial resistance, most notably occurs in hospitals. Over 30 different types of Staphylococci can infect humans, but most infections are caused by Staphylococcus aureus. Staphylococci can be found normally in the nose and on the skin (and less commonly in other locations) of 20%-30% of healthy adults. In the majority of cases, the bacteria do not cause disease. However, damage to the skin or other injury may allow the bacteria to overcome the natural protective mechanisms of the body, leading to infection. (Bailey & Scott’s Diagnostic Microbiology, Eleventh Edition).

Pathogenesis of S. aureus Infections Staphylococcus aureus causes a variety of suppurative (pus-forming) infections and toxinoses in humans. It causes superficial skin lesions such as boils, styes and furunculosis; more serious infections such as pneumonia, mastitis, phlebitis, meningitis, and urinary tract infections; and deep-seated infections, such as osteomyelitis and endocarditis. S. aureus is a major cause of hospital acquired (nosocomial) infection of surgical wounds and infections associated with indwelling medical devices. S. aureus causes food poisoning by releasing enterotoxins into food, and toxic shock syndrome by release of superantigens into the blood stream. (http://www.medicinenet.com/staph_infection/index.htm).

Antibiotic Resistance

Methicillin-resistant staphylococcus aureus (MRSA), is a type of Staphylococcus aureus that is resistant to the antibiotic methicillin and other drugs in the same class, including penicillin, amoxicillin, and oxacillin. . Just as S. aureus can be carried on the skin or in the nose without causing any disease, MRSA can be carried in this way also. Stretomycin is an antibiotic drug, the first of a class of drugs called aminoglycosides to be discovered, and was the first antibiotic remedy for tuberculosis. It is derived from the actinobacterium Streptomyces griseus. Streptomycin stops bacterial growth by damaging cell membranes and inhibiting protein synthesis. (http://www.medicinenet.com/staph_infection/index.htm).

Laboratory Mice

Most of the mice used in laboratories are white albino house mice (Mus musculus). The house mouse, a member of the rodent family, originated in ancient Asia and later spread throughout Europe. In the 1500s, they traveled to North and South America on English, French and Spanish ships. Today house mice appear both in the wild and commensally (nesting within human dwellings) in nearly all parts of the United States. The mouse has been used in biomedical research since the early 20th century. Today, more than 3,000 genetically defined strains of lab mice are used for research purposes. The primary supply source of laboratory mice is commercial breeders. Relatively few are captured from the wild or bred in research laboratories, although the latter practice is becoming more common with some genetically engineered mice. Several characteristics have led to the increased use of mice in research. Scientists say that mice are genetically similar to humans (at least 80 percent of DNA in mice is identical to that of humans). They are also used because of their small size, short lifespan and reproductive cycle, low maintenance in captivity, and mild manner. For these reasons, house mice constitute the majority of mammals used in research, testing, and education. More than ten million mice are used each year in U.S. laboratories alone, in tests of new procedures and drugs as well as in research involved in the production of biological products such as vaccines. (http://www.hsus.org/animals_in_research/species_used_in_research/mouse.html).

Mouse Biology There is no question that mice feel pain. In fact, mice are used in pain research studies to help determine the role genes play in pain sensitivity. Pain may not be readily noticeable in mice, as they instinctively attempt to hide illness and injury because predators in the wild target weak or sick animals. However, mice in significant pain or distress, or that are seriously sick, typically display a lack of activity, sunken eyes, ruffled fur, and an arched back or huddled position.

Mouse Behavior The natural habitat of mice is forests and grasslands, although nests may be found in nearly any undisturbed place with a nearby food source. In the wild, mice live in single, male-dominated groups called colonies. A typical colony consists of a dominant male, several females, and their young. Occasionally, males may share territories. Females are less aggressive than males but may establish their own hierarchy within a territory. Mice kept in a confined environment, such as a lab setting, are usually more territorial than wild mice. There is rarely fighting among female mice groups but grouped male mice can inflict serious injuries on each other. To show their dominance over other mice in their territory, males may exhibit a behavior termed "barbering" in which they chew distinct areas of fur, usually around the muzzle or whiskers, from subordinate mice, leaving a bald patch. (http://www.hsus.org/animals_in_research/species_used_in_research/mouse.html).

Of Mice and Humans Despite their bad reputation, not much about the average mouse's appearance could be considered imposing or threatening. Adult mice generally weigh less than one ounce and range in length from two-and-a-half to three-and-a-half inches, minus the tail. A mouse's tail is usually only slightly shorter than the animal's body. While the mild-mannered mouse is ill-suited for confrontation and nearly always prefers flight to fighting, a single mouse can incite a near panic among people simply by scurrying across a room. Their lightning speed most likely puts people off guard. Albino house mice, the same kind used in laboratories, are the most commonly kept pet mice. "Fancy mice," originally bred for show purposes, are also kept as pets. Fancy mice are larger than other mice, have longer tails and larger ears, and occur with a wide variety of colors, markings, and coats.
(http://www.hsus.org/animals_in_research/species_used_in_research/mouse.html).

Dangerous Medicine Toxicity testing performed on rat’s exposes them to extremely painful experiments such as single dose acute toxicity tests, which are used to determine the lethal dose of a chemical. Besides the animal cruelty inherent in such tests, drugs found safe through toxicity testing in rats in fact have caused severe, permanent side effects and even death when taken by humans. (Daniel D Price, MD, 1998).

Handling Techniques

In handling laboratory mice, there are several things that must be kept in mind. You must be firm but gentle and always handle the animal in the same way. A mouse is best handled by picking it up by the base of the tail, then gently grasping a pinch of lose skin over the shoulder area between the thumb and forefinger (Figure 1). Figure 1

The mouse is restrained in such a way can be easily manipulated. If the mice are excitable and moving around in the cage so that grasping the base of the tail is difficult, cup the hand over the top of the mouse and then grasp the tail gently at the base with a thumb and forefinger. Care must be taken not to attempt to grab the tail other than close to the base because this may result in slippage of the skin and subcutaneous tissue from the bone leading to necrosis, infection, and sloughing of the tail area where the skin has been pulled off. (http://www.fau.edu/research/ovs/VetData/mouse.php)

Husbandry Mice should be kept in rooms with the temperature set at about 70F and humidity at 50%. Lights should not be too bright since most white mice are albinos and too much light hurts their eyes. They are diurnal which means they need about 12 hours of light and 12 hours of darkness each day. Their bedding should not be wood shaving since some wood emits toxic fumes to mice. They should have fresh mouse or rat food and water available at all times. Their bedding should be changed 2 or 3 times a week to prevent the buildup of urea. (http://www.fau.edu/research/ovs/VetData/mouse.php)

Table 1. Physiological and General Data of Albino Mouse

|COMMON NAME |MOUSE |
|ORDER |RODENTIA |
|FAMILY |MURIDAE |
|GENUS |MUS |
|SPECIES |MUSCULUS |
|BLOOD PLATELETS 103/mm3 |160-410 |
|FEED DAILY gm/kg |15g/100g |
|WATER |15ml/100g |
|ROOM TEMPERATURE C |25 |
|WEIGHT ADULT MALE gm |20-40 |
|WEIGHT ADULT FEMALE gm |25-40 |
|LIFE SPAN YEARS |1.5-3 |
|ROOM TEMPERATURE °C |25 |

Thrombocytopenia A common clinical disorder and results from one or any combination of mechanisms, including production, and splenic sequestration. Thrombocytopenia diminishes the effectiveness of hemostasis, and bleeding may result. The severity of bleeding is related to the degree of thrombocytopeniam. (Clinical Hematology, Principles, Procedures, Correlations) The causes of thrombocytopenia are often divided into three major causes of low platelets: low production of platelets in the bone marrow, increased breakdown of platelets in the bloodstream (intravascular), and increased breakdown of platelets in the spleen or liver (extravascular). The disorders that involve the breakdown of platelets include: immune thrombocytopenic purpura (ITP), drug-induced immune thrombocytopenia, drug-induced nonimmune thrombocytopenia, thrombotic thrombocytopenic purpura, disseminated intravascular coagulation (DIC), hypersplenism. Symptoms of thrombocytopenia include bruising, nosebleeds or bleeding in the mouth and rashes. Other symptoms may be present as well, depending on the cause of the condition. Mild thrombocytopenia can occur without symptoms. (http://en.wikipedia.org/wiki/Thrombocytopenia)

Dengue Fever

Overview Dengue fever is an infectious disease carried by mosquitoes and caused by any of four related dengue viruses. This disease used to be called "break-bone" fever because it sometimes causes severe joint and muscle pain that feels like bones are breaking, hence the name. Health experts have known about dengue fever for more than 200 years.

Cause

Dengue fever can be caused by any one of four types of dengue virus: DEN-1, DEN-2, DEN-3, and DEN-4. You can be infected by at least two if not all four types at different times during your lifetime, but only once by the same type.
Transmission
You can get dengue virus infections from the bite of an infected Aedes mosquito. Mosquitoes become infected when they bite infected humans, and later transmit infection to other people they bite. Two main species of mosquito, Aedes aegypti and Aedes albopictus, have been responsible for all cases of dengue transmitted in this country. Dengue is not contagious from person to person.
Symptoms
Symptoms of typical uncomplicated (classic) dengue usually start with fever within 4 to 7 days after you have been bitten by an infected mosquito and include high fever, up to 105ºF, severe headache, retro-orbital (behind the eye) pain, severe joint and muscle pain, nausea and vomiting, and rashes.

Diagnosis

Your health care provider can diagnose dengue fever by doing two blood tests, 2 to 3 weeks apart. The tests can show whether a sample of your blood contains antibodies to the virus. In epidemics, a health care provider often can diagnose dengue by typical signs and symptom.

Treatment

There is no specific treatment for classic dengue fever, and most people recover within 2 weeks. To help with recovery, health care experts recommend getting plenty of bed rest, drinking lots of fluids, and taking medicine to reduce fever.

Prevention The best way to prevent dengue virus infection is to take special precautions to avoid being bitten by mosquitoes. Several dengue vaccines are being developed, but none is likely to be licensed by the Food and Drug Administration in the next few years. Since, Aedes mosquitoes usually bite during the day, be sure to take precautions, especially during early morning hours before daybreak and in the late afternoon before dark.
Complications
Most people who develop dengue fever recover completely within 2 weeks. Some, especially adults, may be tired and/or depressed for several weeks to months after being infected with the virus. The more clinically severe dengue hemorrhagic fever and dengue shock syndromes can result in vascular (blood vessel) and liver damage, and can be life threatening. (www.eMedicine.com)
Aspirin, or acetylsalicylic acid (ASA) A salicylate drug often used as an analgesic (to relieve minor aches and pains), antipyretic (to reduce fever), and as an anti-inflammatory. It also has an antiplatelet ("anti-clotting") effect and is used in long-term, low doses to prevent heart attacks and blood clot formation in people at high risk for developing blood clots. Low dose of aspirin (160 mg) may also be given immediately after an acute heart attack; these doses may inhibit the synthesis of prothrombin and therefore produce a second and different anticoagulant effect, although this is not well understood. Aspirin was the first-discovered member of the class of drugs known as non-steroidal anti-inflammatory drugs (NSAIDs), not all of which are salicylates, although they all have similar effects and most have some mechanism of action which involves non-selective inhibition of the enzyme cyclooxygenase. (www.wikipedia.com)
Veterinary uses. Aspirin has been used to treat pain and arthritis in veterinary medicine, primarily in cats and dogs, although it is not particularly recommended, as there are better medications available. Also, dogs are particularly susceptible to the gastrointestinal side effects associated with salicylates. Horses have also been given aspirin for pain relief, although it is not commonly used due to its relatively short-lived analgesic effects. Horses are also fairly sensitive to the gastrointestinal side effects. Nevertheless, it has shown promise in its use as an anticoagulant, mostly in cases of laminitis. Aspirin’s use in animals should only be done under the direct supervision of a veterinarian. (www.wikipedia.com)
Resistance. For some people, aspirin does not have as strong an effect on platelets as for others, an effect known as aspirin "resistance" or insensitivity. One study has suggested that women are more likely to be resistant than men and a different, aggregate study of 2,930 patients found 28% to be resistant. These results may be contested, however, as there is currently no accepted method of determining who is and who isn't resistant. (www.wikipedia.com)
Toxicity. The toxic dose of aspirin is generally considered greater than 150 mg per kg of body mass. Moderate toxicity occurs at doses up to 300 mg/kg, severe toxicity occurs between 300 to 500 mg/kg, and a potentially lethal dose is greater than 500 mg/kg. [60] This is the equivalent of many dozens of the common 325 mg tablets, depending on body weight. However children cannot tolerate as much aspirin per unit body weight as adults can. (www.wikipedia.com)

Table 2. Dosage of Aspirin

|ACETHYLSALICYLICATE ACID (ASPIRIN) |120-300 MG/KG PO OR 100-150 MG/KG PO q4h |NONSTEROIDAL ANTI-INFLAMMATORY, ANALGESIC|
| |(14) // 25 IP/20 SC/120 PO MG/KG Q4H(2) | |

Chapter 3

MATERIAL AND METHODS

This chapter presents the research design, procedure, and materials that were used in the study.

Research Design The experimental design of the study was carried out under strict laboratory conditions. Equipment, facilities, and glasswares of the Biochemical Laboratory of the University of Negros Occidental- Recoletos were utilized in the research with proper permission. The University of Negros Occidental- Recoletos Biochemical Laboratory has been put up to facilitate Unorians in the different laboratory course and to cater free laboratory services.

Sample Preparation and Extraction Procedure Fresh roots of Euhorbia hirta (tawa-tawa) were collected within the campus of the University of Negros Occidental- Recoletos. It was identified by Mr. Hermie Cordero, a B.S. Agriculture major in Agronomy. The fresh roots were air-dried and cut finely into pieces. One hundred grams (100g) of the finely cut roots were weighed into 1000ml of distilled water in a beaker. This was boiled using Bunsen burner, and then allowed to evaporate and reduced half of the volume of the distilled water (500ml), stirred from time to time. The 500ml solution, which is the concentrated one, is cooled to 40˚C and filtered using Whatman filter paper into 500ml volumetric flask. The filtrate was sterilized using autoclave. Figure1 illustrates the schematic presentation of the sample preparation and extraction procedure.

Inoculum Preparation Using Luria Broth and Luria Agar Slant Broth and agar cultures of the test organism (Staphylococcus aureus) were prepared 24 hours before the procedure was commenced. The microorganism is from University of the Philippines, Los Baños.

Luria Agar Slant Culture Direct laboratory method was used to grow bacteria on Luria Agar Slant. Peptone, yeast, NaCl, and agar are all the ingredients that were weighed and computed by ratio and proportion to make a Luria Agar Slant. Table 1 shows the components of Luria agar. In 1000 ml distilled water, all ingredients were mixed and diluted in appropriate amount of distilled water in an Erlenmeyer flask with cotton plug, and then placed the media into the water bath for heating until clear. The media is then sterilized at 115 psi for 15 minutes, and allowed to cool to 40˚C before dispensing it to sterile test tubes with cotton plugs and allowed to satisfy in a slant manner. The agar slant was cultivated by using Staphylococcus aureus and was streaked into the agar slant aseptically. The Luria agar with the culture was incubated for 18-24 hours at room temperature and good for 72 hours consumption. Figure 2 illustrates schematic diagram of the Luria agar inoculum preparation.

Luria Broth Culture

Direct laboratory method was used to grow Staphylococcus aureus on Luria broth to used as working culture, same ingredients were used with the same ratio and proportion as to the composition of Luria agar except for the agar. All ingredients were weighed and computed as well, mixed and diluted in distilled water in an Erlenmeyer flask with cotton plug. The solution was heated in a water bath until clear, sterilized at 115 psi for 15minutes, allowed to cool to 40˚C a loopfull of inoculum from the Luria agar slant was taken and was aseptically transferred to the Luria broth. The Luria broth with the culture was incubated at room temperature for 18-24 hours good for 24 hours consumption.

Experimental methodology

The materials and methods for this study are divided into three experiments. The first experiment is the establishment of MIC, which investigates the inhibition of Staphylococcus aureus on the different concentrations of the crude extract of tawa-tawa, while the second experiment is an investigation of the anti-bacterial property of the tawa-tawa, against Staphylococcus aureus. The last experiment investigates the effect of tawa-tawa to the platelet count of mice using albino mice as the model specie.

Mixture of all ingredients computed
By ratio and proportion peptone and water yeast, NaCl
.

Minimum Inhibition Concentration

The first experiment of the research investigates the minimum inhibition concentration that will give or show no observable growth after 24 hours of incubation. Serial dilutions technique was used using 11 tubes assay. Luria broth is a general media that does not cause microorganisms to grow in flocks or flogs and is used as a diluting agent together with the crude extract of tawa-tawa to get the desired concentrations. The tawa-tawa extract was dispensed in 11 test tubes aseptically from 0% to 100% concentrations. Table 1:shows the concentration of serial dilutions of Tawa-Tawa root extract. Then from the working culture, dispensed 0.1 ml into the 11 test tubes. The tubes were incubated for 24 hours in 37˚ C or room temperature, and then results were recorded. The experiment was done in three trials with triplicates in every trial. Figure 3: Schematic diagram showing the methodology in conducting MIC.

Table 1
Concentration of Serial Dilutions of Tawa-Tawa Root Extract

|Tubes |Concentration |Tawa-tawa Extract |Luria Broth |Staphylococci Culture |
|1 |0% |0 ml |10 ml |0.1 ml |
|2 |10% |1 ml |9 ml |0.1 ml |
|3 |20% |2 ml |8 ml |0.1 ml |
|4 |30% |3 ml |7 ml |0.1 ml |
|5 |40% |4 ml |6 ml |0.1 ml |
|6 |50% |5 ml |5 ml |0.1 ml |
|7 |60% |6 ml |4 ml |0.1 ml |
|8 |70% |7 ml |3 ml |0.1 ml |
|9 |80% |8 ml |2 ml |0.1 ml |
|10 |90% |9 ml |1 ml |0.1 ml |
|11 |100% |10 ml |0 ml |0.1 ml |

Antibacteriocity

Staphylococcus aureus were reisolated and the pure cultures subcultured on Luria agar and Luria broth. They were stored and incubated at 37˚ C or room temperature for 24 hours. The agar diffusion method was adopted for the study. Base and Top Agars composition were computed by ratio and proportion. All ingredients for Mueller Hinton Agar Media include beef extract, cassein, starch and agar powder and were diluted in distilled water. Top agar composition includes peptone, agar, and Sodium Chloride (NaCl) diluted in distilled water. Table 2: Shows the composition of Top and Base Agars with their ratio and proportion.

Table 2

Components of Top and Base Agars in 1000ml Distilled Water

|Composition |Base agar |Composition |Top agar |
|Agar |6g |Agar |70g |
|Starch |53g |Peptone |30g |
|Beef Extract |4.5g |NaCl |50g |
|Cassein |51g | | |

All ingredients of base and top agars were computed by ratio and proportion, sterilize at 115ppm for 15 minutes, allowed to cool to 40˚C. For base agar was dispensed on a petridish aseptically and then allowed to solidify. Top agar was added with 1.0 ml of 1x108 CFU/ml of bacteria, swirled gently to ensure uniform distribution of the microorganism. Dispensed top agar on the petri dish with base agar, overlay and solidify, within 2 hours, 4 cu cylinders were placed in the plates (about 5.0 mm dm) using a sterile force and an equal volume of 1ml of the extract for the 2 test controls, streptomycin ampule for the positive control and sterile distilled water for the negative control were transferred into the holes using micropipettor. These plates were allowed to stand for 1 hour for prediffusion of the extract to occur and were incubated at 37°C for 24 hour. Figure 4: Shows schematic diagram of Antibacteriocity and Sensitivity. Three trials were done and three petri dish are made for every trial. At the end of incubation, zones of inhibition that developed were measured and the average of zones of inhibition was calculated.

Figure 4: Schematic diagram of Antibacteriocity and Sensitivity.

Toxicity Initial LC50 studies carried out were used to determine the maximum concentration that did not produce an increase in the platelet count in albino mice. Four groups of albino mice each comprising 10mice, randomly selected. They were placed in different cages. Based on the LC50, studies, concentrations of the crude extract of tawa-tawa are 100%, 80%, 60%, 0% were orally administered into each group of the mice respectively, the administration of 1ml of tawa-tawa extract was carried out on daily basis for 2 days. The control group was orally administered with distilled water. Food and water were provided. The albino mice were first drawn blood samples through tail vein checked for their normal platelet count on the 2nd day, they were induced with thrombocytopenic drugs such as aspirin for a day and platelet count were checked. On the third day, tawa-tawa extract were administered to them and blood samples were analyzed for any effect on their platelet count.

Chapter 4

Results and Discussion

Results

This chapter presents the data collected using the research methodology, and the analysis and interpretation of data using the prescribed statistical tool for the experiment design. The first experiment investigates the highest concentration of the extract that does not produce significant reversion on the test organism- Staphylococcus aureus. Table 1: Shows the MIC of the Euphorbia hirta extract against Staphylococcus aureus.

Table 1: MIC of the Euphorbia hirta extract against Staphylococcus aureus

|Concentration of Tawa-tawa extract g/ml |Staphylococcus aureus growth |
|0% |( + ) |
|10% |( + ) |
|20% |( + ) |
|30% |( + ) |
|40% |( + ) |
|50% |( + ) |
|60% |( + ) |
|70% |( + ) |
|80% |( - ) |
|90% |( - ) |
|100% |( - ) |

With 3 trials in every 10 serial dilutions used per trial for this experiment, in triplicates, the minimum inhibitory concentration was produced against Staphylococcus aureus with a concentration of 80%.

The antibacterial screening of the extract was established using the cap cylinder method with the test isolates- Staphylococcus aureus where 90% of the concentration were used for the test control. The results show that the extract did not inhibit growth of Staphylococcus aureus at 90% concentration used. Streptomycin was used for the ( + ) control and showed zone of growth inhibition of 3.59 mm in circumference as a total mean value of the 3 trials, in triplicates, was exhibited. Table 2: shows the zones of inhibition for the 3 trials.

Table 2: Zones of inhibition for the 3 trials.
Sensitivity
|First trial |Streptomycin |Distilled water |T1 |T2 |
|A. |4.0 |0 |0 |0 |
|B. |3.8 |0 |0 |0 |
|C. |3.4 |0 |0 |0 |
|Total mean |3.73 |0 |0 |0 |

Sensitivity

|Second trial |Streptomycin |Distilled water |T1 |T2 |
|A. |4.0 |0 |0 |0 |
|B. |3.0 |0 |0 |0 |
|C. |3.5 |0 |0 |0 |
|Total mean |3.50 |0 |0 |0 |

Sensitivity

|Third trial |Streptomycin |Distilled water |T1 |T2 |
|A. |3.5 |0 |0 |0 |
|B. |3.7 |0 |0 |0 |
|C. |3.4 |0 |0 |0 |
|Total mean |3.73 |0 |0 |0 |

The results of the platelet count analysis of the blood samples of mice administered with different concentrations of the extract were shown in Table 3. In general, although the values obtained for platelet count in determining the effects of tawa-tawa on thrombocytopenia, differed. They were not significantly different (α=0.05) from the values obtained from the control for this parameter. For the values obtained from the mice before treatment of aspirin and extract, there was no significant difference from those mice treated from control. But there was significant difference from the value obtained from those mice treated with other concentrations. This experiment yields the decrease in platelet count after administering aspirin and the increase in platelet count after administering tawa-tawa extract at different concentrations plus a control of which distilled water was used. Figure 1: Platelet Count Curve for Albino Mice Administered to different Concentrations of Extract, before taking Aspirin and tawa-tawa Extract, After Aspirin and After Extract.

Table 3
Platelet Count Analysis

| |Concentration of Extract |Before Aspirin and |After Aspirin |After Tawa-tawa |
| | |Tawa-tawa | | |
| | | | | |
| |0% | | | |
|Tail Blue | |57 |40 |36 |
|Left Ear Blue | |51 |50 |48 |
|Stomach Red | |181 |35 |12 |
|Total mean | |96 |42 |32 |

| |Concentration of Extract |Before Aspirin and |After Aspirin |After Tawa-tawa |
| | |Tawa-tawa | | |
| | | | | |
| |60% | | | |
|Right Ear Red | |202 |23 |31 |
|Left Foot Blue | |48 |21 |87 |
|Stomach Red | |44 |35 |50 |
|Total mean | |98 |26 |56 |

| |Concentration of Extract |Before Aspirin and |After Aspirin |After Tawa-tawa |
| | |Tawa-tawa | | |
| | | | | |
| |80% | | | |
|Head Red | |116 |25 |69 |
|Right Hand Blue | |74 |29 |33 |
|Neck Blue | |50 |28 |86 |
|Total mean | |80 |27 |63 |

| |Concentration of Extract |Before Aspirin and |After Aspirin |After Tawa-tawa |
| | |Tawa-tawa | | |
| | | | | |
| |100% | | | |
|Right Ear Blue | |48 |21 |72 |
|Right Ear Black | |85 |42 |55 |
|Right Foot Red | |255 |24 |30 |
|Total mean | |129 |29 |92 |

This experiment investigates the toxicity of the extract of different concentrations to Albino mice as the test organisms. Three concentrations of extract plus a control were used in order to know the test concentration lethal to the Albino mice. This experiment yields the survival/ mortality curve for Albino mice, as well as the test solutions median lethal concentration. Table 4: Shows the result of mortality exhibited after 24 hours of exposure to the extract.

Table 4
Result of mortality exhibited after 24 hours of exposure to the extract

| | |Number of Test Organisms Dead at |
|Concentration of Extract |Number of Test Organism | | | |
| | |8 hour |16 hour |24 hour |
|0% |10 |0 |0 |0 |
|60% |10 |0 |1 |1 |
|80% |10 |0 |0 |0 |
|100% |10 |0 |0 |1 |

Establishment of LC50 is very important for this experiment because it is from this concentration that lethal effects of the extract on Albino mice survival after prolonged exposure were determined. With ten mice used per treatment for this experiment, in triplicates, the number of survivors left after 24 hours of continuous exposure to extract, were counted.

Discussion

In this study, the results obtained indicated that the crude extract of Euphorbia hirta does not inhibit the Growth of the test isolates- Staphylococcus aureus. This therefore shows that the extract contains a substance that does not inhibit the growth of some microorganisms. Other researches have also shown that extract of some parts of the Euphorbia hirta inhibits the growth of various microorganisms at different concentrations. (Akujobi et al., 2004). Crude extract of Euphorbia hirta was inactive against the gram-positive bacteria, i.e. Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus subtilis (Chanda S., Turk J Biol, 2005). Zones of inhibition showed no growth exhibited by the extract against Staphylococcus aureus does not justify their use by traditional medical practitioners in the treatment of sores and open wounds. (Braude,1982). The MIC exhibited by the extract against Staphylococcus aureus is of great significance in the healthcare system, since the result could be of basis if extract could be used as an alternative treatment of infection caused by microbes, if it could develop resistance to known antibiotics. The inability of the extract to inhibit Staphylococcus aureus maybe that it possesses a mechanism for detoxifying the active principles in the extract. Some bacteria are known to possess mechanisms by which they convert substances that inhibit their growth to non- toxic compounds. For instance, Staphylococcus aureus produces the enzyme penicillinase, which converts the antibiotic penicillin to penicillinoic acid, which is no longer inhibitory to its growth. (Singleton, 1999). Statistical analysis revealed that for platelet count there was no significant (α= 0.05) differences between concentrations of the extract and the control orally administered to the mice. Then, the extract did not induced production or destruction of platelet. These results indicate that the extract is less toxic haematogically, at least to the mice, at the different concentration administered. Euphorbia hirta is commonly used traditionally in the production of platelet count (Igoli et al, 2005) however, more research studies need to be carried out to determine the chemistry of the particular active principle and the effect at these concentration.

From the proponents’, the results obtained forms a good basis for further phytochemical and pharmacological investigation.

Summary, Conclusions and Recommendation

Summary

This study was conducted to determine the MIC, Antibacterial properties, and Toxicological and platelet count effects on the Albino mice of the crude extract of the roots of Euphorbia hirta. The study utilized an experimental design using Serial Dilutions Technique, Sensitivity test, and Animal Inoculation Assay. The data were then analyzed using One- Way Anova.

This study singled out to answer the following questions and in this study these were answered:

1. What is the Minimum Inhibitory Concentration (MIC) of crude extract concoction of Tawa-Tawa roots? 2. Does Tawa-Tawa possess any anti-bacterial effect towards the test organism- Staphylococcus aureus? 3. Does the crude extract concoction of Tawa-Tawa roots possess any toxic effect when administered orally to the test organism-albino mice? 4. Can Tawa-Tawa affect the platelet production of albino mice induced with thrombocytopenic drugs such as aspirin?
Conclusion
Using the statistical analysis of the data, these are the results of the study: 1. Minimum Inhibition Concentration of crude extract concoction of Tawa- Tawa roots is 80%. 2. Crude extract concoction of Tawa- Tawa roots does not posses any anti-bacterial effect towards Staphylococcus aureus. 3. Crude extract concoction of Tawa- Tawa roots, haematologically, is less toxic, atleast to the mice, when administered orally with different concentration. 4. Results show that there is no significant difference in the crude extract concoction of Tawa- Tawa roots, thus does not affect the platelet count.

Recommendation Based on the findings and conclusions of this study, the proponent recommends the following studies should be made:

1. Further testing of the test substance should be made, this time utilizing other strains of test organisms such as E. coli, P. aeruginosa, B. subtilis and S. tyhi. 2. Mutagenicity should be done using Ames Test to determine any mutagenic effects of the test substance. 3. Conduct a research by using the other parts of Euphorbia hirta to be extracted and analyzed. 4. Studies should be conducted on other disease that could be treatable by Euphorbia hirta. 5. Investigate other serum biochemical parameters in mice subjected to studies on Euphorbia hirta.

Literature Cited

Forbes, B. A., et al. (2002). Anti microbial Principle. Staphylococcus. ELESEVIER SCIENCE Publisher, Philippines.
Lotspeich- Steininger, C. A., et al. (1992). Thrombocytopenia. Educational Publishing House, Philippines.
Lind and Tallantire. (1971). Description of E. hirta.
Kokwaro. (1993). Treatment of asthma.
Oliver. (1960). Latex cause dermatidis.

Sofowora. (1993). Antibiotic activity.

Ahmad I, et.al. (1998) Ethnopharmacol. Medicinal plants are a source of great economic value.
Rajkot and Jamnagar . (2004). Herbal medicine is still the mainstay of about 75-80% of the whole population.
Davis J, Science. (1995). Alternative antimicrobial drugs.

Le Bourgeois T., et. al. (2001). Description

http://www.ibiblio.org/pfaf/cgi-bin/arr_html?Euphorbia+hirta. Medicinal Uses, Cultivation details, propagation.

http://www.medterms.com/script/main/art.asp?articlekey=34093. Toxicity.

www.wikipedia.com. MIC. http://www.medicinenet.com/staph_infection/index.htm. Pathogenesis of S.aureus, and antibiotic resistance. http://www.hsus.org/animals_in_research/species_used_in_research/mouse.html-laboratory. Mice.

Daniel D Price, MD. (1998). Dangerous Medicine. http://www.fau.edu/research/ovs/VetData/mouse.php. Handling Techniques. http://www.fau.edu/research/ovs/VetData/mouse.php. Physiological and General Data of Albino Mouse, Husbandry.
Clinical Hematology, Principles, Procedures, Correlations. Thrombocytopenia. www.eMedicine.com. Dengue fever.
Akujobi et al. (2004). Euphorbia hirta inhibits the growth of various microorganisms.
Chanda S., Turk J Biol. (2005). Crude extract of Euphorbia hirta was inactive against the gram-positive bacteria.
Braude. (1982). No growth exhibited by the extract against Staphylococcus aureus.
Singleton. (1999). Staphylococcus aureus produces the enzyme penicillinase.
Igoli et al. (2005). Euphorbia hirta is commonly used traditionally in the production of platelet count.

-----------------------
Euphorbia hirta
(Tawa-Tawa)
Roots 100g

Air dry, cut into fine pieces

Weigh into 1000ml-distilled water in a beaker

Boil for 25-30 min until reduced to 500ml distilled water

Cool for 40Ú[?]C, filter in volumetric flask

Sterilized at 115 psi for 15 min

Store at ambient temperature

Figure1: Schematic diagram showing the sCool for 40˚C, filter in volumetric flask

Sterilized at 115 psi for 15 min

Store at ambient temperature

Figure1: Schematic diagram showing the sample preparation and extraction procedure.

Luria broth

Luria agar

Figure2: Illustrates schematic diagram of the Luria agar inoculum preparation.

Incubate at room temperature for 18-24 hours (may last for three days)

Incubate at room temperature for 18-24 hours (good only for 24 hours consumption)

Transfer to Luria broth

Culture the bacteria

Get a loopful of culture

Dispense in test tubes.
Allowed to solidify.

Sterilize for 15min at 115ppm

Heat in water bath

Luria
Broth
(1)

Extract of tawa-tawa (2)

Working culture
(3)

Add aseptically

Dispense

Dispense

Dispense 0.1ml

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Sterilize

Cool at 40° C

140

Read

Figure 3. Schematic diagram of MIC.

Add aseptically

Add aseptically

Incubate for 24 hours at room temperature

20

40

60

80

100

120

0

0[pic]%

60%

80%

100%

42

96

Incubate upright for 24-48 hours

Dispense 100 цl of (+) and (-) controls in each cylinder & the other 2 for the aseptically test substance

Put 4 glass cylinder within 2 hours

Overlay on base agar & solidify

Add bacterial working culture 1ml of 1 x 108 CFU/ml

Cool at 40° C

Sterilize at 115 psi for 15 minutes

Top Agar
All ingredients mixed & computed by ratio & proportion.

Solidify

Dispense to petridish aseptically

Cool to 40° C

Base Agar
All ingredients mixed & computed by ratio and proportion

Figure 1: Platelet Count Curve for Albino Mice Administered to different Concentrations of Extract, before taking Aspirin and Tawa-tawa Extract, After Aspirin and After Extract.

80

129

32

56

26

63

27

29

92

98

Extract Concentration (% v/v)

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