1 In Vitro Antiplatelet Activity of Tridax procumbens

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of alkaloids, carotenoids, flavonoids (catechins and flavones), fumaric acid, ... catarrh, dysentery, diarrhoea and to prevent falling of hair promotes the growth of ...
In Vitro Antiplatelet Activity of Tridax procumbens Linn.

In partial fulfillment of MSPH 602L Advanced Pharmacology Lab

Jean Balatero Ganub, RPh Janine Lou Matuod Monsales, RPh

First Semester SY 2016-2017

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Chapter 1 Introduction

Rationale of the Study

Drugs that inhibit platelet function are currently used to decrease the risk of occlusive arterial events in patients with atherosclerosis. They include: (1) cyclooxygenase-1 (COX-1) inhibitors, such as aspirin; (2) antagonists of the P2Y12 receptor for adenosine diphosphate (ADP), such as the thienopyridines (ticlopidine, clopidogrel and prasugrel) and the direct inhibitor ticagrelor; (3) glycoprotein IIb/IIIa antagonists, such as abciximab, eptifibatide and tirofiban. All these drugs are used during coronary interventions and in the medical management of acute coronary syndromes, while aspirin and thienopyridines only are used in long term prevention of cardiovascular and cerebrovascular events. In the last years, the issue of resistance to anti-platelet agents, in particular aspirin and clopidogrel, has been largely emphasized in the medical literature. Although several studies have been published on this issue, its definition, diagnosis, causes and clinical consequences are still uncertain. According to the result of the study of Sy et al in 2012 on prevalence of atherosclerosis- related risk factors and diseases in the Philippines, the said prevalence were higher in 2008 than in 2003 and in present. These findings indicate a need for active collaborative intervention by medical societies and pharmaceutical organizations in the Philippines in relation to this health issue. Tridax procumbens Linn (Compositae) is a weed naturalized in Asia and found throughout the Philippines. Local people know it as “Kanding-kanding” and popularly known in English as “coat buttons”. The phytochemical screening revealed the presence of alkaloids, carotenoids, flavonoids (catechins and flavones), fumaric acid, fl-sitosterol, saponins and tannins. It is richly endowed with carotenoids, saponins, oleanolic acid and ions like sodium, potassium and calcium. Luteolin, glucoluteolin, quercetin and isoquercetin have been reported from its flowers. It has known for its number of pharmacological activities like hepatoprotective activity, antiinflammatory, wound healing, antidiabetic activity, hypotensive effect, immunomodulating property, bronchial

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catarrh, dysentery, diarrhoea and to prevent falling of hair promotes the growth of hair, and antimicrobial activity against both gram-positive and gram-negative bacteria. The leaf juice possesses antiseptic, insecticidal and parasiticidal properties, as a remedy against conjunctivitis and is used also to check haemorrhage from cuts, bruises and wounds insect repellent. It is also used as bioadsorbent for chromium (VI). (Mundada & Shivhare, 2010) The sulphated polysaccharide from the widespread Tridax procumbens plant was studied for the anticoagulant, antiherpetic and antibacterial activity. The anticoagulant activity was determined by the activated partial thromboplastin time assay. The sulphated polysaccharide from T. procumbens represented potent anticoagulant reaching the efficacy to heparin and chondroitin sulphate. Sulphated polysaccharide exhibited detectable antiviral effect towards HSV-1 with IC50 value 100–150 μg/ml. Furthermore, sulphated polysaccharide from T. procumbens was highly inhibitory against the bacterial strains Vibrio alginolyticus and Vibrio harveyi isolated from oil sardine. (Naqash & Nazeer, 2011) Thrombous formation inside the blood vessels obstructs blood flow through the circulatory system leading hypertension, stroke to the heart, anoxia and so on. The complete deprivation of oxygen and infarction is a mode of cell death. Crude biologicals and their components possessing anti-thrombotic activity have been reported before. This study was aimed to investigate thrombolytic activity of ethanol extracts of four traditionally used medicinal plants. For this an in-vitro thrombolytic study was carried out along with streptokinase, and ethanol was taken as reference standard and negative control, respectively. The ethanol extracts (5 μg/μl) of Alpinia conchigera, Lannea grandis, Aglaonema hookerianum and Tridax procumbens 24.50%, 13.31%, 11.18%, and 8.70% clot lysis, respectively. Preliminary chemical group identification revealed the presence of alkaloids, glycosides, steroids, terpenoids, tannins and reducing sugars important secondary metabolites.(Sultana et al, 2012) The above mentioned literature stimulated the investigation of the antiplatelet activity of the extract from Tridax procumbens Linn leaves using Giemsa Microplate Assay and Spectrophotometric Assay. This study could contribute and can be a gateway to the development of a new antiplatelet medication.

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Objectives of the Study

This study will aim to examine the antiplatelet activity of Tridax procumbens Linn using Giemsa Microplate Assay and Spectrophotometric Assay. The specific objectives are the following: 1. To obtain the crude extract of Tridax procumbens Linn by soxhlation using 95% ethanol as solvent. 2. To detect an all-or-none response indicated by absence of Giemsa-stained violet gels or platelet aggregates on the microplate wells. 3. To measure the optical density using spectrophotometer. 4. To determine the antiplatelet activity of Tridax procumbens Linn based on the results from the Giemsa Microplate Assay and Spectrophotometric Assay.

Theoretical Framework

All the variables (both dependent and independent) involved within the scope of this study are being represented using the succeeding illustration. The whole scheme of this study shall be based on the relationship between these variables. Independent Variables The Antiplatelet Activity of Tridax procumbens Linn Leaves Crude Extract using: 



Geimsa Microplate Assay; and Spectrophotometric Assay

Dependent Variables Actual results on the: 



Detection of an all-or-none response in Giemsa Microplate Assay; and Optical density data in Spectrophotometric Assay

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Significance of the Study

The antiplatelet activity screening of Tridax procumbens Linn can give a contribution to the following: Pharmaceutical Industry. In the last few years, the concept of resistance to antiplatelet agents has been largely emphasized in the medical literature, although its definition is still uncertain. The real prevalence of resistance to aspirin appears to be rather low. In contrast, resistance to clopidogrel (a P2Y12 inhibitor), which is mostly due to inefficient metabolism of the pro-drug clopidogrel to its active metabolite, is a rather frequent condition. (Cattaneo, 2011) This study could be a solution to the problem of clopidogrel resistance by the discovery of a new antiplatelet medication. Pharmaceutical Researchers. This study will provide additional body of knowledge to other researchers and can also promote continued research operations that will improve the competence of the pharmaceutical scientific community.

Scope and Delimitation

The study will be conducted at the Pharmaceutical Chemistry Laboratory Room and Instrumentation Room of USC-TC SHP Building and will be limited to the examination of the antiplatelet activity using Giemsa Microplate Assay and Spectrophotometric Assay of Tridax procumbens Linn leaves crude extract obtained from soxhlation. The Giemsa Microplate Assay will be limited to the detection of an all-or-none response at a set concentration selecting for potent inhibitors that is indicated by absence of Giemsa-stained violet gels or platelet aggregates on the microplate wells, while the Spectrophotometric Assay will be limited to the measurement of the optical density and the data obtained will be statistically treated using the calculation of mean and standard deviation.

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Chapter 2 Materials and Methods

The study will be conducted at the Pharmaceutical Chemistry Laboratory Room and Instrumentation Room of University of San Carlos – Talamban Campus SHP Building. The following materials will be used: 95% ethanol, PRP-human type O, Tyrode’s buffer containing 0.25% bovine serum albumin, normal saline solution, aspirin, Giemsa dye, and collagen.

Plant Materials

Tridax procumbens Linn plant will be collected at Manga District, Tagbilaran City. The leaves will be gathered and the other plant parts will be discarded. The leaves will then be washed with running water to remove dirt and other foreign particles. The washed leaves will be air-dried for 24 hours to prepare for comminution and extraction.

Preparation of Extract

The dried leaves will be comminuted using a blender and will then be subjected to soxhlation for 8 hours at 60oC with 95% ethanol as solvent. The solvent of the resulting extract will initially be removed using the rotary evaporator set to 60 rpm at 60oC for 30 minutes. The resulting extract will then be subjected to further removal of solvent using the vacufuge machine.

Specimen

Platelet rich-plasma, human type O, will be obtained in the Blood Center. Residual red blood cells will be removed through centrifugation at 1800 rpm for 5 minutes. These will then be stored in the prepared vials, set ready for the following antiplatelet assays.

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Microplate Assay Platelets will be washed with Tyrode’s buffer (pH 7.4) and will be centrifuged at 1800 rpm for 10 minutes (Kuo et al., 1990). Platelets will then be resuspended 1:1 (vol/vol) in EDTA-free Tyrode’s buffer containing 0.25% bovine serum albumin (Sigma). The platelet suspension (192 μl) will be transferred to wells in a 96-well microtiter plate and will be incubated at 37°C for 5 minutes. Crude extracts of Tridax procumbens Linn leaves that will previously be dissolved in NSS (8 μl of 50 mg/ml stock; final concentration in the microplate, 2 mg/ml) will be added in duplicate and will be incubated at 37°C for 30 minutes. Aspirin (Sigma, 2 mg/ml) will be used as positive control. NSS will be used as negative control. Platelets will be induced to aggregate by addition of 0.25 M CaCl2 (4 μl) (Born and Cross, 1963). The plate will be incubated at 37°C for 30 minutes. Giemsa dye (4 μl) will be added to each well and will be incubated for 5 minutes. The microtiter plate will then be inverted, will be tapped gently, and will be washed with distilled water. Each well will be observed for the presence or absence of violet gels.

Spectrophotometric Assay

1 ml of PRP will be taken in each tube. First two tubes will be taken as positive and negative control and test samples will be added in increasing amount in consequent tubes and final volume of each tube will be made up to 2 ml by adding normal saline. All tubes will be incubated for at 37°C for 3 minutes and then 0.2ml of collagen (0.2mg/dl) will be added in each tube. Aggregation will be induced under continuous stirring at 1000 rpm for 3 minutes and the aggregation will be monitored under spectrophotometer at 400 nm λ. In tube no. 2 normal saline and tube no. 1, 0.4ml aspirin (0.9mg/dl) will be added in place of test sample (Ansari et al, 2016).

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Collection of Tridax procumbens Linn leaves

Soxhlation of Tridax procumbens Linn extract

Procurement and preparation of the specimen PRP-human type O

Microplate Assay

Spectrophotometric Assay

Antiplatelet Activity of Tridax procumbens Linn

Figure 1. Research Design

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