Anticoagulant activity of Paragis Pudadera et. al PDF

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The Anticoagulant activity of (Eleusine indica) Paragis in Human blood Coagulation time

The Anticoagulant activity of (Eleusine indica) Paragis on Blood Types A, AB and O

An Official Entry to the

2019 School-based Research Fair Competition

LIFE SCIENCE GROUP CATEGORY (CELLULAR AND MOLECULAR BIOLOGY)

John Jeycee B. Mosquera, Joshua Klyne P. Pudadera and Vanessa M. Pira-an Student Researchers

Phillip Raymund R. De Oca Project Adviser

Bacolod City National High School Bacolod City, Negros Occidental Region VI, Western Visayas

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The Anticoagulant activity of (Eleusine indica) Paragis in Human blood Coagulation time

ABSTRACT

Blood is a major part of our body. As we get injured, blood may lose if not for the use of the coagulation cascade which is made by the body in order to regenerate and stop the bleeding, but some illnesses have some complications regarding the action of coagulation cascade which is brought by genetics, lifestyle and other more. Thus, the idea of fixing this problem was formulated by the researchers and could also be beneficial to the community. Several plants have active bioactive compounds which helps prevent the blood from coagulation. Paragis is a tropical, perennial grass which is widely spread and possess risks for some farms. The researchers used whole leaf and was dried for 3 days, macerated and then filtrated into a container. Using a micropipette, 250 μL, 500 μL and 1000 μL was separately put in Vacutainers. Blood was taken from three volunteers with A, AB and O blood types where every 30 seconds is check whether the blood clotted or not. Then, clotted bloods were destroyed and centrifugated in 15 minutes at 3000 rpm. ANOVA (Analysis of Variance) using Randomized Complete Block Design with the aid of Tukey’s HSD were used to determine significant difference in each group and to determine the means that are significantly different from different groups. At the end, the researchers conclude that Paragis has anticoagulant properties and is significantly different from each other with 1000 μL as the longest time of coagulation which is above 6-10 minutes. 2|Pag e

The Anticoagulant activity of (Eleusine indica) Paragis in Human blood Coagulation time

Table of Contents Page 1 2 3 4 5 6 10 12 18 25 26 27 28 29

Title of the Study` Abstract Table of Contents List of Figures List of Tables Research Plan Introduction Methodology Results Discussion Conclusion Recommendation Acknowledgements References

List of Figures Page Figure 1: Relationship between Dependent and Independent

13

Variable

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The Anticoagulant activity of (Eleusine indica) Paragis in Human blood Coagulation time Figure 2: Flowchart of General Procedures 14 Figure 3: Preparation of needed materials

32

Figure 4: Transfer of specific microliters of extracts subjected

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on different blood type

Figure 5: Preparation of blood samples

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Figure 6:

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Centrifugation of blood and disposal of used

needles Figure 7: Results of coagulation

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List of Tables Page Table 1: Coagulation time formed in 3 replicates when

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exposed to varying amounts of (Eleusine indica) Paragis extracts Table 2: Analysis of Variance using the Randomized Complete

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The Anticoagulant activity of (Eleusine indica) Paragis in Human blood Coagulation time Block Design Table 3: Determination of Anticoagulation activity between

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four (4) treatments with the use of ANOVA (Analysis of Variance) Table 4: Inferential Statistics in Determining the Significant

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Difference between groups

Table 5. Turkey HSD in Determining Significant Difference in

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the longest time of Coagulation between five (5) different treatments

RESEARCH PLAN Anticoagulant activity of (Eleusine indica) Paragis in Human blood Coagulation time (PT) with respective blood types Proponent: John Jeycee B. Mosquera, Joshua Klyne P. Pudadera, Vanessa M. Pira-an

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The Anticoagulant activity of (Eleusine indica) Paragis in Human blood Coagulation time A. Questions/Problems being addressed

Blood is one of the major factors in which we sustain our daily lives. It performs many functions and it is also considered as the river of life. But what happens when blood is lost in our body? Veins ruptures due to some factors which worsens the blood. This is where homeostasis or simply called blood coagulation goes in, a complex biochemical reaction in which the body inhibits the anticoagulants to stop bleeding. There are also conditions in which the low level of clotting factors in your blood may cause heavy or unusual bleeding into the joints, such as Hemophilia A and B which is mostly a rare occurrence. In normal operations, blood clotting only occurs when there is a damage in a blood vessel. However, these conditions may cause blood clots to form even though there is no active bleeding. When this happens, a person may be at risk of thromboembolic diseases. Therefore, when a person is at risk of these conditions, they are given supplements called oral anticoagulants such as warfarin, a long-term anticoagulant. The use of herbal medicines was also approved by the World Health Organization since there were no or less side effects compared to artificially made medicines. (Eleusine indica) Paragis on the other hand, is an invasive weed found from all over the world. When animals such as cats and dogs have an upset stomach, they it the plant and gets better. Paragis is also identified as antibacterial, anthelminthic, anti-oxidative, cytotoxic,

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The Anticoagulant activity of (Eleusine indica) Paragis in Human blood Coagulation time anti-diarrheal, diuretic, anti-diabetic, anti-malarial, anti-inflammatory and many other functions were identified by past researches. B. Objectives/Expected outcome/Hypothesis In general, this study wants to find out if there is an anticoagulation activity of Paragis on different sets tested in their coagulation time. To be specific, this study wants to find:



What is the Anticoagulant activity of (Eleusine indica) Paragis on different sets of blood samples taken from one of the researchers? The different sets including the negative control (unaffected), blood samples with 250µL of Paragis, blood samples treated with 500µL of Paragis, blood samples treated with 1000µL of Paragis and lastly,

blood

samples

treated

with

EDTA

(Ethylenediaminetetraacetic acid). 

Is there a significant difference in the level of Anticoagulant potential on different sets of blood samples, including the negative control, blood samples treated with 250µL, 500µL, 1000µL Paragis extract and EDTA (Ethylenediaminetetraacetic acid).

C. Hypothesis The hypothesis will be formulated based on the objective of the study: There is no significant difference in the anticoagulation activity of (Eleusine indica) Paragis on the different sets of blood including 250µL,

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The Anticoagulant activity of (Eleusine indica) Paragis in Human blood Coagulation time 500µL, 1000µL Paragis extract, EDTA (Ethylenediaminetetraacetic acid), and negative control. D. Procedures D.1 Collection of Materials The researchers will gather the (Eleusine indica) Paragis in their local backyard since Paragis is mostly common in grassy areas. Other materials such as the vacutainer and the filter paper were bought from the medical pharmacy store. D.2 Maceration of Paragis The maceration of Paragis was conducted by the one of the 9researchers in their home. First, the Paragis grass and flowers were dried 3 days to remove the moisture content. Then, the researcher put 500mL of water into the blender and start blendering for (3) times. Next, the researcher put the extract in a secure lid covered with filter paper to ensure that the extract is pure. Lastly, the extract was stored in a temperature not less than 37° or remained to be in a normal room temperature. D.3 Extraction of blood from the Donor Blood will be taken from one of the researchers. Samples were labeled based on different blood types and amount of the extract. The extraction of blood will be held in Q Diagnostic Laboratory with the assistance of a licensed Medical technologists. 2mL of Blood from the Median basilic

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The Anticoagulant activity of (Eleusine indica) Paragis in Human blood Coagulation time vein will be taken and to be tested. After the blood collection, it will be stored in Vacutainers in order to prevent the blood from contact in external factors. D.4 Coagulation analysis in different sets of blood, control & EDTA After the blood was collected and set in different amounts of the time and recorded prior to the extraction. After all the extraction was finished, the recording stopped, and the Vacutainers will be subjected to centrifugation and set to 15 minutes and separate the PPP (Platelet Poor Plasma). D.5 Statistical Analysis The results of the coagulation analysis will be encoded in excel and will be expressed in seconds. One-way ANOVA test will be used in determining significant difference in the coagulation time of ( Eleusine indica) Paragis aqueous extract. The Stastical analysis will be analyzed using an SPSS program (Statistical Package for Social Sciences).

Introduction Blood is one of the major biological structures that help us survive and function as a human being. Blood is the basic unit of life which composes of cells that maintains the required supplement the body needs. The blood which composes of erythrocytes, lymphocytes and thrombocytes which functions together to keep the body safe and healthy. Blood may be lost due to some clotting factors are missing or damaged, few platelets, or malfunction of platelets

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The Anticoagulant activity of (Eleusine indica) Paragis in Human blood Coagulation time (C.O. Ikese et al., 2015). These disorders are sometimes linked in genetic, caused by illness or treatment from specific drugs and even some herbal remedies (Cordier et al., 2012). The body uses a built-in process called coagulation in which the blood changes from liquid to solid to help clot the wound faster. Bleeding disorders often develop when the blood can’t function properly. For the body to clot, blood proteins and platelets binds together to form at the damaged blood vessel. It then forms fibrin which then clots the platelets and prevent blood form flowing (Karim et al., 2013). Treatment of bleeding disorders comes in the form of anti-coagulants. These are used in hypercoagulable states or thrombophilia where the blood clots when there are no cuts, thus having an increased risk of forming blood clots in the arteries which may then cause buildup of arterial plaque and reduces blood flow and increases the risk of developing cardiovascular diseases (Mengome et al., 2014). In these situations, medications including anticoagulants and antiplatelets prevents the blood to clot. In the case of antiplatelets, it interferes the binding of platelets that starts the blood clot. Anticoagulants interferes in the clotting factors of the blood involved in the process. Clotting disorders mainly lacks function or malfunction of the blood protein which may affect the blood cascade. To lessen the impact of the effect, natural medications in traditional Chinese medicines have been used by various researchers using Chinese herbal medicines, acupuncture, and Tai chi quan significantly reduced the effects of atrial fibrillation. Anticoagulant herbs which contains coumarin and salicylate phytochemicals help the body prevent blood from coagulating, also called blood thinners that delay formation of blood clots.

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The Anticoagulant activity of (Eleusine indica) Paragis in Human blood Coagulation time These chemicals interfere in the coagulation cascade that produces certain chemicals which helps reduce or lessen the blood clot (Anthony, 2011) (Eleusine indica L., Gaertn) Paragis on the other hand, also called goosegrass is an annual and perennial summer grass. It usually grows on all parts of the world were summer water are concurrent and does not grows in nonirrigated deserts. It is said to be vulnerable to viruses such as the one causing sugar cane mosaic. Seed germination of Paragis, showed proof of dormancy since its caryopses is soluble in water and aggravating the scarification which breaks the caryopses and thus spreading its germination (Vargas et al., 2013). Paragis is said to be diuretic, antihelmintic, anti-inflammatory, antiplasmodial, antidiabetic,

phytoremediative,

pancreatic

lipase

inhibitory,

antioxidant,

antileishmanial, antioxidant, cytotoxic, anticonvulsant, and antibacterial. In coastal Guyana, it is said to apply it in wounds to stop the bleeding (Clayton et al., 2014). Paragis is said to be dominant and mostly invasive in farms since they have low resistance on glyposphate thus germination is easily spread by air and other factors (Vargas et. al, 2012) Recently, herbal medicine has been a trend as confirmed by the WHO (World Health Organization). In China, the use of traditional herbal medicines treating SARS (Severe Acute Respiratory Syndrome) had been prominent. In other words, the use of herbal medicine is much better than artificial since it is low cost and has much fewer side effects (Tilburt and Kaptchuk, 2008). Thus, the researcher wants to use these plants to benefit the community and learn its possible implications. The researchers want to prove that even these invasive 11 | P a g e

The Anticoagulant activity of (Eleusine indica) Paragis in Human blood Coagulation time species could benefit from the medicinal value and improving the living conditions of the community by reducing the development of having these further complications. The result of this study will be beneficial to science and technology field since additional knowledge and new concepts along this area is created. This study could use as a basis for students especially in academic institutions. This also could change the course of medicine since herbal technology is now used all around the world and it can benefit patients suffering from blood disorders including thrombophilia, DVT or venous thromboembolism, and arterial thrombosis. METHODOLOGY Research Design This research study employed (2) two different research designs, the descriptive research design and the experimental research design. In the descriptive research design, the researchers described the varying amounts of anticoagulation potential (Eleusine indica) Paragis aqueous extract on human blood coagulation time (PT). In the experimental research design, the researchers followed the specific procedures on blood extraction and application of treatment to the blood samples to determine the anticoagulation property of the varying amounts of anticoagulation potential ( Eleusine indica) Paragis aqueous extract on human blood coagulation time (PT).

Negative Control, 250µL (Eleusine indica), Paragis 500µL of (Eleusine indica) Paragis 1000µL (Eleusine indica)

Coagulation Time

ge

lant activity of (Eleusi

od me

Fig. 1 Presents the relationship between the Dependent variable and the Independent variable of the study

Locale and Duration of the Study The plants used in this study were all gathered from the backyard of one of the researchers. The flowers of Paragis will be used in the extraction. The gathered specimens will be dried naturally in the sun for 3 days so that the moisture content will be removed. After the Drying process, the dried plant will undergo into maceration. It is first finely chopped and put in a blender added with 250 mL of water. After three blender process, the substance will be filtrated using a filter paper for (3) so that there is no remaining residue. Moreover, the test on the measure of coagulation time of different sets will be done in Riverside College with the aid of a licensed medical technologist. Materials and Equipment

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The Anticoagulant activity of (Eleusine indica) Paragis in Human blood Coagulation time The materials used in this study were ( Eleusine indica) Paragis grass, blood samples, mortar and pestle, small jar, Vacutainers (Suremed™), Ethylenediaminetetraacetic acid (EDTA), rubber tourniquet, stopwatch app, pipette, and piece of cloth. While the equipment to be used is a Centrifuge (Digisystem Laboratory Instruments™, DSC-200T with AR-1508) and Nu Life Extraction of varying extracts using a micropipette Needle Burner and Syringe Destroyer (Scoova Exim™). Fig. 2 Flowchart of General Procedures Collection of Materials Coagulation analysis of varying amounts of Paragis (Eleusine indica) and EDTA

Drying Process

Maceration of Paragis

Extraction of Blood Samples from Each Donor

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The Anticoagulant activity of (Eleusine indica) Paragis in Human blood Coagulation time

General Procedures

C.1 Collection of Materials The Paragis plant will be collected at one of the researcher’s residence in Brgy. Vista Alegre, Arao Relocation Site. The test tubes were bought at Tabletas 5 Pharmacy. Other materials and equipment were provided by Q Statistical Analysis Diagnostic Laboratory such as the micropipette, needle burner and centrifuge machine. C.2 Maceration of Paragis The maceration of Paragis was made by one of the researchers in their local garden. The leaves and flowers were cut into 3cm and was dried under the sun for (3) three days. After drying, it was finely powdered using a mortar and pestle. It was then blended with 100mL of water and left standing below room temperature. Next, it was filtered using a tissue and damp cloth and was stored in a jar and again stored below room temperature prior to the experiment.

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The Anticoagulant activity of (Eleusine indica) Paragis in Human blood Coagulation time C.3 Phlebotomy of Blood The venipuncture of blood was held in Q Diagnostic Laboratory in Corner Locsin-Burgos Streets, Brgy. II, Bacolod city. To ensure safety practice, a well-maintained area must be followed. The donor was instructed by a licensed Medical technologist on following procedures and prevent complications. The site of venipuncture was located at the median basilic vein and rubbed alcohol to prevent further infection. After hygienic procedures, the tourniquet was applied on the arm to stop bleeding. Then, the needle was injected thoroughly to collect blood with a specific ratio at 2mL. After blood collection, it was stored in a vacutainer and slowly pull out the needle C.4 Coagulation Analysis For the first batch, after each blood was taken and put on vacutainers, using a stopwatch app, the time was recorded. The vacutainers were tilted upside down to check if the blood coagulated or not. After checking all blood, the vacutainers were mixed again and was subjected to centrifugation. A centrifuge (Digisystem Laboratory Instruments™, DSC-200T with AR-1508) was used by a licensed medical technologist. At 15 minutes, it was set to 3000 rpm at 60Hertz. When the 1st batch finished, another set of vacutainers were prepared and again set to 3000 rpm at 60Hertz. C.5 Proper Disposal After the drawing of blood, the syringe was immediately prepared for disposal. A needle burner and syringe destroyer (Scoova Exim™) was used by the licensed medical technologist. The needle was inserted in a

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The Anticoagulant activity of (Eleusine indica) Paragis in Human blood Coagulation tim...