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Mechanisms of Corticosteroid Action in Immune Thrombocytopenic Purpura (Itp): Experimental Studies Using Itp-Prone Mice

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Chapter 1
The Problem and Its Setting
Introduction
Thrombocytopenia or abnormally low platelet counts in the body are typically caused by genetic disorder or fatal diseases. This condition may result from thromboembolic or hemorrhagic complications caused by heparin as well as from various diseases states such as chronic liver disease and dengue hemorrhagic fever. In most rural areas a health care institution is often inaccessible and most patients cannot afford high cost medication. Henceforth, herbal medicines are often used as an alternative treatment. Inadequacy of providing supportive treatment to correct the decrease of platelet is fatal to patients. Thus, several decoctions of herbal plants are purportedly used to improve platelet count.
Recently, the mortality rate in heparin – induced thrombocytopenia (HIT) has been reported as high as 20% (Greinacher & Lubanow, 2003). Retrospective data shows that up to 8% of heparinized patients will develop the antibody associated with HIT and that approximately 1–5% of patients on heparin will progress to develop HIT with thrombocytopenia suffering from venous and/or arterial thrombosis in at least one-third of cases (Warkentin et al., 1998). Heparin – induced thrombocytopenia (HIT) is a potentially devastating immune mediated adverse drug reaction caused by the emergence of antibodies that activate platelets in the presence of heparin. Despite thrombocytopenia, bleeding is rare; rather, HIT is strongly associated with thromboembolic complications such as death and amputation which involves in both arterial and venous systems (Franchini, 2005). One of the popular diseases in the tropical country that is responsible for the drastic decrease in platelet count (thrombocytopenia) could be Dengue as reported by the Department of Health in the Philippines; National Epidemiology Center in 2005, there were 17,934 dengue cases and 249 deaths reported from January to September 2005. Moreover, in the year 2006, there is 2, 125, 039 cases with reported 257 deaths in January to October 2006. But despite its notoriety and prevalence in such areas, in depth research into its nature and mechanism of infection and pathogenicity has been partially stifled by the complicated structural and physiological aspects of the virus once it has entered a suitable host. According to Agapito Hornido, Davao del Norte provincial health officer, said that dengue figures are usually greater in highly urbanized cities such as Davao City. At a press conference briefing on dengue held at the Big 8 Corporate Hotel in Tagum City, Hornido qouted ‘’ Davao del Norte’s dengue cases reached 2,930, with one recorded death, last year.’’ Stated by Ebol that 234 of that dengue cases were considered severe. Nowadays people are being bombarded with thousand of medications, the level of sensibility in front of diseases is very high and that's why the use of medicinal plants can represent the best solution. Practically in the Philippines, an old herbal medicine known as the Tawa-tawa plant ( Euphorbia hirta Linn.) - a common folkloric plant that also otherwise recognized as gatas-gatas or mangagaw (Quisumbing 1978 ). Practitioners of traditional medicines believe that Tawa-tawa is good for patient suffering from Dengue, which can cause thrombocytopenia – a persistent decrease in the number of platelets in the blood that is often associated with hemorrhagic conditions. Patients and professionals alike have continued to use of Tawa-tawa plant ( Euphorbia hirta Linn.), due to its claimed ability to increase platelet counts even without solid evidence to support its clinical use (Philippine Pharmaceutical Association, 2009) As evident from folklore uses of Tawa-tawa plant ( Euphorbia hirta Linn.) , they cause alleviation from number of diseases. Traditionally, one of the common remedial actions was for combating fall in platelet count and their influence on blood disorders. Even though modern method of treatment is successful in ameliorating the pains and discomforts of human being, there is still dearth of discovery for effective management of thrombocytopenia. In line with this scenario, the research envisaged is designed to explore the role of Tawa-tawa ( Euphorbia hirta Linn.) plant, as well as in combination on heparin - induced thrombocytopenia in albino mice.
Statement of the Problem
The researchers intended to determine the effectiveness of the Tawa-tawa ( Euphorbia hirta) on increasing the platelet count of thrombocytopenic mice. The researchers are intended to find out the extent of the effects of “Tawa-tawa” in increasing the platelet count in heparin – induced thrombocytopenic mice.
Specifically, this research seeks the answers of the following questions:
1. Is there a significant increase of platelet in heparin – induced thrombocytopenic mice?
2. Is there a possible difference on platelet count of heparin – induced thrombocytopenic mice on the following group:
a. Group A – Control Group (Distilled Water)

b. Group B –Experimental Groups (Aqueous Extract of Euphorbia hirta) On Different Concentrations:

i. 100mg/ml ii. 150mg/ml iii. 200mg/ml Null Hypothesis
1. There is no significant effect in increasing platelet in Aqueous Extract of Tawa – tawa (Euphorbia hirta) plant.
2. Is there a possible difference on platelet count of heparin – induced thrombocytopenic mice on the following group:
a. Group A – Control Group (Distilled Water)
b. Group B –Experimental Group (Aqueous Extract of Euphorbia hirta) On Different concentrations:
i. 100mg/ ml ii. 150mg/ml iii. 200mg/ml

Theoretical Framework The oldest remedies known to mankind are herbal medicines.Tawa-tawa (Euphorbia hirta L. ) in the Philippines is one of them. It has been considered as a folkloric herbal remedy for Dengue that causes faster recovery compared to the body’s natural restoration of health. . Practitioners of traditional medicines believe that decoction of tawa–tawa leaves can reverse viral infection and prevent the fever from moving into critical stages, although there are no scientific studies proving its effectiveness. More recently, concerns from local authorities have been raised in the reported rampant indiscriminate use of E. hirta in the management of dengue patients. Despite, warnings from health authorities that there is not enough efficacy and safety data to support its clinical use, patients and health professional continued to use E. hirta.
The confidence of the general public in the effectiveness of E. hirta in treating dengue cases was solely based on the results of animal studies showing that it exerts a platelet count-increasing activity, and testimonials from patients or health professionals who claimed to experience clinical benefits from its use. To safeguard the public, more detailed information should be provided to let both the patients and the health professionals fully understand the potential dangers of using E. hirta in dengue patients or in any other medical conditions that concerns in increasing platelet activity (National Drug Information Center, Philippines).
Conceptual Framework The researchers will use this to conceptualize the theoretical data gathered. It is to identify the thrombogenic activity of Euphorbia hirta. The illustration below shows the dependent and independent variable of data. The dependent variable is the Platelet Count Activity of Heparin – Induced Thrombocytopenic mice. The independent variable is the Ethanolic Extract of Tawa-Tawa (Euphorbia hirta) Plant.

Figure 1: Conceptual Framework of the Study
Significance of the Study
This study is conducted by the researchers to observe, seek facts and results to prove Tawa-Tawa plant ( Euphorbia hirta L.) can increase the platelet level in heparin – induced thrombocytopenic mice. Upon knowing its results, this study will be beneficial to the following: Pharmacists; This study will help the pharmacists to formulate a drug product that is a platelet enhancer.
Pharmacy students. This study will help the students to be aware and enhance the use of Euphorbia hirta and use it as a fundamental knowledge for future studies.
Department of Agriculture. This study will help the Agricultural sector to recommend the local farmers to propagate Euphorbia hirta and supply the market with locally produced raw materials that can be exported internationally.
Farmer. This study will give the farmers alternative source of income by propagating the plant and supplying the market.
Food and Drug Administration. This study is vital to the FDA in providing a criteria in formulating condition in the causality and the control of variance to provide accurate assessment of cause and effect relationships between dependent and independent variables.
Client. This study provide probable solution for patients suffering dengue fever or other thrombocytopenia illness.
Society. This study will help to lessen the case of death caused by Dengue fever. For their information and knowledge so that they will be more cautious in using traditional medicines with maintenance drugs.
Davao Del Norte locality. They will be informed first hand of the result, interpretation, and recommendations of this study.
Department of Health. The discovery of therapeutic use of Tawa-Tawa plant (Euphorbia hirta Linn.) which will help them in promoting innovative drugs that are available locally and can be exported internationally.
Scope and Delimitation This study is centered on the thrombogenic activity of Tawa-Tawa plant (Euphorbia hirta).Thus, the study provides concrete background about platelet production and the processes involved and the plant itself, and its briefly explained relevant composition. Moreover, the study will definitely include the procedures necessary for Euphorbia hirta aqueous extract induction. In addition, the study will require fundamental knowledge on how to handle a test animal. Blood sampling of the test animals is also a part of the study. The test animals to be used-mice will be bought in Carmen, Davao Del Norte. The researchers will use the whole ‘’tawa-tawa’’ plant and the collection of plant material will be done in Tagum City Only. Heparin is administered to lower the platelet count of the test experiment-rats. Platelet Count will be conducted in City Medical Laboratory located at Lapu-Lapu Street, Tagum City . This experiment procedure will be performed haphazardly in Tagum Doctors College Inc. Mahogany St., Rabe Subd.,Visayan Village, Tagum City. However, the study does not cover the molecular aspects of the reaction nor does cover any other adverse reactions the plant may produce to the test animals-rats change other than the platelet count.
Definition of Terms
1) Decoction -an extract obtained by decocting
-The act or process of decocting
-Decoction of Euphorbia hirta was prepared by boiling 100 grams of fresh whole plant with 500 mL distilled water for 15 minutes. After filtration, a clear yellowish brown solution with a concentration of 556.7 mg/ml.
2) Platelet -Thrombocyte; a small round thin blood cell that helps blood to stop flowing from a cut by becoming thick and sticky.
Review of Related Literature
Euphorbia hirta The plant Euphorbia hirta is a common medicinal plant used not only here in the Philippines but also in Australia, India, Mexico and some parts of Africa. It is commonly known as gatas-gatas or tawa-tawa in the native tongue. It is under the family: Euphorbiaceae (see under figure 2) and is known to the English world as Australian asthma weed, snake weed and cat’s hair (Quisumbing, 1978). Although the commonly accepted scientific term is Euphorbia hirta, other scientific names exist. These are Euphorbia pilulifera, Euphorbia capitata, and Chamaesyce hirta (Plants for a Future, 2007). The plant is abundant in most parts of the archipelago. It is an annual weed that grow almost anywhere in the country and was coined pantropic (Quisumbing 1978). In comparison to its close relative, Steinmann was able to study the morphological differences of E.hirta with close relative of the family Euphorbiaceae designated as Eiphorbia nocens. It was concluded that both were distinguishable from each other through several morphological differences such as varied sizes (Steinmann 2003).The plant can grow up to 40 centimeters in length with a purplish shade. It is a hairy herb with “elliptic-oblong” leaves arranged oppositely (Quisumbing, 1978). It is also jagged at the leaves end and its involucres are vivid (see figure 3).
Euphorbia hirta Scientific classification

Kingdom: Plantae

Order: Malpighiales

Family: Euphorbiaceae

Genus: Euphorbia

Species: E. hirta
Figure 2: Euphorbia hirta Plant Taxonomy

Figure 3: Euphorbia hirta
Constituents
Aqueous extract composition was limited to the qualitative chemical identification and thin-layer chromatographies of different substances as gallic and catechic tannins, flavonoid compounds, organic acids, amino acids, glycosides, saponins and reducing compounds, according to the literature (Cabalion et al.). Some chemical composition was pointed out, such as: gallic acid, quercetin (C15H10O7), phenolic substance (C28H18O15), triacontane, ceryl alcohol, euphosterol, phytosterol, phytosterolin, jambulol (C16H3O4(OH)) and melissic acid in Quisumbings book (Quisumbing, 1978). E. hirta contains glycosides.
The action of Euphorbia hirta on the increase platelets of patients infected by Thrombocytopenia is unknown yet. Consequently, it is safe to hypothesize that its action may be on the platelet production, with or without Dengue infection. Thus, it is also imperative to tackle in detail the processes that involve the thrombocytes in mice (the research’ specimen).
The Platelets and Mus Musculus
Platelets are an essential component of the blood. It is the smallest formed cell in the blood (Fischbach 1980). They are nucleated, with flattened oval shapes. They are essential for blood clotting (Fischbach 1980). It prevents hemorrhage by forming “hemostatic plug at sites of vascular injury” and thus, stops further unnecessary blood lost. They are also partly responsible during the formation of thrombus. Furthermore, they are said to be involved in tissue injury, inflammation, and wound healing. If for any reason, blood platelet count falls below normal, the condition is called thrombocytopenia. These are possible through the attraction leucocytes and its binding afterwards (Dignat-George). With that said, decrease in platelet count could lead to complications such internal hemorrhage or condition that involves the bone marrow, such as leukemia and severe types of anemia could lead to a reduction in the number of platelet produced. One of the popular diseases in the tropics that are responsible for drastic decrease in platelet count or thrombocytopenia could be Dengue. But despite its notoriety and prevalence in such areas, in-depth research into its nature and mechanisms of infection and pathogenicity has been partially stifled by the complicated structural and physiological aspects of the virus once it has entered a suitable host. Other illnesses related to thrombocytopenia are Leukemia, Lymphoma, yellow fibrosis, HIV AIDS, Myelofibrosis and Hemolytic-uremic syndrome (Beers et al. 2003). In the recent events, botanicals have been a major source for alternative medicine that led to the establishment of Philippine Institute of Traditional and Alternative Health Care (PITAHC) through the approval of Republic Act 8423. This is due to the development of resistance by pathogens to drugs has led to the search for bioactive compounds within botanicals. This is coupled with diseases that have no chemotherapeutical cure. With that, the search encompasses the field of ethno-pharmacology (Hansel and Lagare 2005). The use of medicinal plants is an essential aspect in maintaining health and treating disease according to the World Health Organization (WHO 2000). Today, one of the current known ethno-botanicals in the Philippines is the plant Euphorbia hirta or commonly known as tawa-tawa in the Tagalog dialect.
According to Berndt’s review of megakaryocyte lineage, the production of circulating blood platelets occurs through the distinct megakaryocyte bone marrow cells. The initial stage of megakaryocyte differentiation is the formation of the burst-forming unit (BFU-meg). Following this developmental stage is the megakaryocyte colony-forming unit (CFU-meg). Shortly after, the proliferative capacity and formation of colonies of the mature megakaryocyte progenitors is lost due to the gradual disappearance of the expression of the CD34 marker of primitive haemopoiesis. Once the mitotic phase is complete, the megakaryocytes undergo nuclear endoreduplication, which is also referred to as polyploidization. Although this process is poorly understood, it was distinguished by having several successive rounds of DNA replication and endomitosis without the stage of cytokinesis. The stage wherein endomitosis is involved with occurs during the “promegakaryoblast” phase where cells are distinguishable by their platelet peroxidase in the endoplastic reticulum and perinuclear space. The structure of the promegakaryoblasts is identified with diameter of 7-15µm and the production of surface glycoprotein (GP) IIb/IIa, cytoplasmic platelet factor 4 and von Willebrand factor (vWF). Moreover, rodent cells’ acetylcholine esterase activity is considered to be the most commonly employed marker of the megakaryocyte lineage (Berndt 2000). An intricate meshwork of demarcation membrane developed by mature megakaryocytes in the cytoplasm was presumed to be invaginations of plasma membrane. The membranes were considered to have defined platelet fields where formed platelets would be released from after fragmentation (Berndt 2000). The structure of platelets was characterized as being small, anucleate effector cells that are integral to the proper homeostasis of a particular organism. Also, platelets participate in the fibrinolytic cascade and mechanisms maintaining vascular integrity/tone in addition to their production of factors that are present in angiogenesis and would recovery. As such, the deficiency of the platelets in performing their tasks from either quantitative or qualitative means would relatively increase the chances of bleeding and hemorrhage (Berndt 2000). The increase in platelet numbers was also assumed to be regulated by the release of a thrombopoetic factor although none have been isolated so far to prove its mechanisms. The effect of such regulators were seen in the reduction of endomitic rates, producing megakaryocytes of lower ploidy in contrast to a scenario of lower platelet concentration where higher ploidy megakaryocytes were formed (Beeler et al. 1994). The estimated lifespan of platelets is around 4.8 days according to the in vivo biotinylation of C3H mice platelets by Manning et al. Reports of unusually low blood platelet counts in murine samples taken from two recent publications in Blood were examined by a correspondence initiated by Jack Levin that had come up with several responses from other studies. The “normal” platelet count was said to be around 1000 x 109/ L or 1,000,000/ mm3 under phase contrast microscopy. Yet, their observations had shown that the lower platelet counts in normal mice were subject to the procedures used to extract the blood as well as the method of counting the platelets themselves. It was noted that the measures taken in using automated platelet counts were important in ensuring that the count was done accurately and was able to cover the entire population of the given sample. Another key aspect was that of the dilution factor involved in the collection of whole blood in contrast to platelet-rich plasma samples (Ebbe et al.). In a journal article where mice were induced with hypoxia, the platelet count changed, increasing in short-term hypoxia and decreasing in long-term hypoxia. It is essential to point out the duration of platelet increase, which is 1 to 3 days. As such, one can infer that mice platlet production could range in this duration(Clift, 1978).
Borelli and Zucker (1954) were able to observe that platelet circulating in the blood stream took the form of thin discs with a few possessing processes upon examination under an electron microscope. Moreover, thin preparations under darkfield illumination was a able to detect platelets as “spindleforming” bodies. Devine and Maurer-Spurej (2001) were also able to conclude that aggregation of platelets are not dependent on shape change and shape change is not necessarily proceeded by aggregation. They stress that the role of platelet shape change of individual platelets covers other functions of its physiology.
Synthesis
The above related literatures and studies were being sorted from primary, secondary, and tertiary references. Facts attained were fixed accordingly and were utilized by the researchers to support the theory of the study. Havin g these facts presented in paper can provide the readers a background information to completely understand what the case is all about.
Euphorbia hirta is a folkloric herbal remedy that is used to increase platelet in patient suffering from thrombocytopenia. In urbanized areas, it is purpotedly used to alleviate thrombocytopenic patients. And thus theoretically can restore the patients platelet count than the normal body’s health restoration. It contains a phytochemical constituent that has an increasing activity. E. hirta contains glycosides which can cause hemolysis in blood. Platelets are an essential component of the blood since it prevents hemorrhage by forming haemostatic plugs at site of vascular injury and thus stops further unncessary blood lost. Platelets are also responsible during the formation of thrombus. Furthermore they are said to be involve in wound healing.
Related Studies
In an Australian database (Australian, 2007), the plant is used against asthmatic conditions and other related lung problems. Moreover, it is said to good against syphilis. It is said also to be used also for “intestinal amoebic dysentery”. In addition, some use it against athlete’s foot and other skin conditions such as warts (Plants for a Future 2007). In a chemical investigation, the n-hexane and its triterpines of the unsoiled parts of the plant showed a high anti-inflammatory effect of the both if combined (Vazquez et al. 1999). Also, powderized yethanolic E. hirta leaf extract was said to inhibits of E. coli, S. aureus, P. aeruginosa and B. subtilis in zones of inhibition (Anyanwu1et al, 2007).
In a journal article, the plant was said to contain high percentage of inorganic molecules and the plant contains relatively “high soluble extractives” in different solutions (Garcia et al. 2003). With that, another article experimented on the different chromatographic factors that were introduced to the rat serum that yielded different effect on the proteins. Six fractions were divided through their polarity. An analysis was done and resulted to a statistically significant increase in the total protein within the serum when administered by each fraction. Examples of these proteins are albumin and globulin; however, cells like thrombocytes are not specified (Adedapo et al. 2005). Antiplasmodial compounds were found to be present in the whole plant extract of E. hirta in a study by Koler et al (2002).
According to a journal article, aqueous extract composition was limited to the qualitative chemical identification and thin-layer chromatographies of different substances as gallic and catechic tanins, flavonoid compounds, organic acids, amino acids, glucides, saponosides and reducing compounds, according to the literature (Cabalion et al.). Some chemical composition was pointed out, such as: gallic acid, quercetin (C15H10O7), phenolic substance (C28H18O15), triacontane, ceryl alcohol, euphosterol, phytosterol, phytosterolin, jambulol (C16H3O4(OH)) and melissic acid in Quisumbings book (Quisumbing, 1978).
In a study by Adedapo et al. (2004), E. hirta was observed to have caused a significant reduction (P

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