Republic of the Philippines

Department of Education
REGION VI- WESTERN VISAYAS
SCHOOLS DIVISION OF ESCALANTE CITY
ESCALANTE NATIONAL HIGH SCHOOL
Brgy. Alimango, Escalante City, Negros Occidental

An entry to the

Division Scilympics 2023

Life Science

Utilization of Acalypha indica L. leaves for in vitro clotting mechanism of

human blood

Team Category

Feuna B. Banguiran
Cyrille Andrea P. Labiga
Sharl M. Tanguyan

Student Researchers

Christine R. Aquino

Research Adviser

Escalante National High School

Escalante City, Negros Occidental
Region VI
 EXECUTIVE SUMMARY

Acalypha indica, locally known as "sinko-sinko,”, a perennial weed found almost everywhere

in the country, has been found to exhibit potential pharmacological properties (J. Ethnopharmacol,

2017). The leaves of this plant are used by locals for their healing properties when used on open

wounds. There has been a lot of studies on the action of plant extract done on an In-Vivo set up,

local studies are, however, very limited, especially with the utilization of its leaves, on in- vitro blood

clotting mechanism.

A study using Gas Chromatography-Mass Spectrometry has shown the herb to contain

Kaempferol – a flavonoid which is said to inhibit thrombosis and platelet activation and, therefore

delay aggregation or clotting time (Choi JH , et al, 2015). This study is, therefore, carried out in order

to collect more scientific data on the use of the leaf extract of the aforementioned plant in external

blood clotting. This experimental study is designed to determine the level of effectiveness and

duration of Acalypha indica as an agent for blood clotting. The objective of this study is to specifically

examine the clotting or bleeding time of leaf extract from Acalypha indica. Acalypha indica is known

for its potent anti-inflammatory and antioxidant properties. This study will be able to accomplish its

goal by extracting the plant and using an in vitro procedure. Thus, Acalypha indica will be extracted

for this study, and its clotting and bleeding times will be examined through in vitro procedure. The

leaves will be dried up and pulverized. Then, ethanol will be added to the pulverized leaves of

Acalypha indica, and the ethanol will be removed by boiling. The extract will be sent to an accredited

laboratory for testing. 160 blood samples will be collected by the researchers for testing. A total of

40 blood samples—20 male and 20 female—will be taken for each of the four blood types (types a,

b, ab, and o). A medical technician must be present to supervise the process. As a result, the findings

of this study will benefit the field of medicine. The findings will help the community by enlightening

them about Acalypha indica's influence on external blood clotting in the human body.
 INTRODUCTION

Acalypha indica is a very common herbal plant found in abundance almost everywhere in

every community. Most often considered as a weed and disruptors of normal garden growths, the

plant has actually been source of traditional medicinal purposes since ancient times. Using parts of

the plant has many benefits for human medicinal needs. It is, in fact, widely used in traditional

medicine to treat inflammation, bacterial infection, diabetes, hyperlipidemia, wounds, cancer as well

as a diuretic.

A recent study using Gas Chromatograpphy-Mass Spectrometry by Choi JH. Et al (2015) has

shown the herb to contain Kaempferol – a flavonoid which is said to inhibit thrombosis and platelet

activation and delay aggregation or clotting time. This study, however was done in an In-vivo set-up

or experimentation and little is known on the effect of the plant extract under In-Vitro condition. It

is for this reason that this study will be done in order to fill the gap re: the action of Acalypha indica

extract on the In-Vitro clotting time of human blood. How Acalypha indica extract could mitigate

prolonged bleeding time or facilitate a faster clotting time will be the centerpiece of this research

work.

Blood clotting time is a very important hemo-physiologic mechanism in the human body. The

rapidity or the inability of the blood to aggregate and form a clot has tremendous relevance in the

field of medicine. Thrombosis and Dengue are but two of the medical conditions that result from

two opposite conditions of the blood. Thrombosis is defined as a medical condition that occurs

when a blood clot forms in the blood vessels and reduce bloodflow (is necessary for human life.

Dengue on the other hand is described, by Mayo Clinic, to happen when your blood vessels become

damaged and leaky and the number of clot-forming cells (platelets) in your bloodstream drops. This

can lead to shock, internal bleeding, organ failure and even death.
 In vitro clotting procedure is a laboratory test done by medical professionals. According to

World Health Organization (2020), In vitro simply means ‘in glass’, meaning these tests are typically

conducted in test tubes and similar equipment, as opposed to in vivo tests, which are conducted in

the body itself. A human blood is an essential in an in vitro clotting procedure. In this test, the In-

Vitro clotting-bleeding time (CT-BT) procedure that will be used, using slides, lancets and the plant

extract. A bleeding time test determines how quickly your blood clots to stop bleeding (Elaine Luo

and Brian Karns, 2018). The procedure will be done under the supervision of a licensed Medical

Technologist.

Sampling method that will be employed in this study will be purposive sampling. The

sampling method is the method of choice in order to include the parameter of ABO Typing in

relation the blood clotting time under the influence of Acalypha indica extract.

Objective

Traditional medicinal plants prominently show a higher efficacy in the medical field, and one

of them is Acalypha indica. One of our key objectives is to find out whether, the application of the

extract will increase or decrease in-vitro clotting time, in relation to the amount of extract used.

Specifically, it aims to measure its impact on in-vitro clotting time in relation to:

1. ABO type

2. Gender
 Conceptual Framework

Blood Clotting
Amount of Acalypha indica Extract Time

Independent Variable Dependent

Variable

This research is anchored on the effectiveness of a traditional medical plant—Acalypha

indica—as a blood clotting agent in vitro. In addition, to test the duration of Acalypha indica as a

factor to prevent a low platelet count and aid hemostasis. This study will examine how the amount

of extract influences the clotting time and how the extract will affect the blood sample's clotting time

if it is added.

Acalypha indica is not only an ordinary plant that can be found somewhere in the backyard,

but it is also a herbal plant that can be used as a treatment for several illnesses. R. Seebaluck et al.

(2015) mentioned that almost every part of Acalypha indica can be used to aid or as a remedy for

several ailments.

According to Nor Syahiran Zahidin et al. (2017), Acalypha Indica is an herbal plant that is

used by the locals for several therapeutic treatments such as anthelmintic, anti-ulcer, bronchitis,

asthma, wound healing, anti-bacterial and other applications because Acalypha indica has properties

and has the capability to serve as anthelmintic, anti-ulcer, bronchitis, asthma, wound healing, and

anti-bacterial treatment.
 Acalypha indica has many benefits. The extract of Acalypha indica has anti-inflammatory,

analgesic, anthelmintic, antibacterial, anti-venomous, anti-ulcer, larvicidal, ovicidal, and antioxidant

properties. It also has the capacity to heal wounds (Helthy Leaf, 2021).
 REVIEW OF RELATED LITERATURE

What is Acalypha Indica?

Acalypha Indica, commonly known as Indian Acalypha, Indian Mercury, Indian Copperleaf,

Indian Nettle or Three-seeded Mercury which is classified as Acalypha belonging to the family

Euphorbiaceae. Acalypha Indica  is an herbal plant that grows in wet, temperate and tropical region,

primarily along the earth’s equator line (Zahidin et al., 2017). It is known for its health and medicinal

properties and it grows abundantly all ober the land.

Tradional Medicine

Acalypha Indica is used widely in traditional medicines. Acalypha Indica has a lot of benefits

for human health it is used specifically for the treatment of various diseases and health issues, and

research has proven the use of this herb. Acalypha Indica herbal plant is used in Powerful

pharmacological activities and traditional medicine such as anti-inflammatory, anti-bacterial, anti-

cancer, anti-diabetic, anti-hypertensive, anti-diabetic, and antihypertensive (Healthy Leaf, 2021). It is

a traditional medicine used by locals of some other countries around the world. This plant widely

used in the traditional medicinal system of India and many other countries have been reported to

possess anti-cancer, anti-diabetic, anti-oxidant, anti-bacterial, antifungal hepatoprotective, anti-

inflammatory, and also used to check anti-ulcers and wounds healing (Chekuri et al., 2020).

Medicinal Properties and Active Ingredient

The medicinal property of Acalypha Indica is what makes it a potential source of traditional

medicine. It is rich in properties that can prove that it can be used widely as medicine. The

biochemical constituents of Acalypha indica are tannins, flavonoids, cyanogenic glucoside

anaglyphic, acalyphamide, aurantiamide, succinimide, and the pyranoquinolinone alkaloid flindersin

( Dineshkumar et al., 2010). The chemical constituents od of this plant has defensive and protective
properties. The medicinal Properties of this plant are derived from its different parts. The plant

contains phenolic compounds, flavonoids, steroids, Tannins, and Saponins. All these compounds

provide anthelmintic, laxative, expectorant, antioxidant, detox agent, anti-inflammatory, and other

beneficial properties (Akshaya Venkatesan, 2019). Research also shows that Acalypha Indica has

ethanol leaf extract which can be used for wound regeneration. According to Laut et al., (2018), In

conclusion, a medicated ointment containing an extract of A. indica leaves exhibited wound healing

process in the incision wound models by enhanced wound contraction and shortened epithelization

period. The active ingredients and medical properties of Acalypha indica and its chemical institute

investigations prove its importance as a valuable medicinal plant.

What is external blood clot/ hemostasis?

Blood clotting is part of a larger process called hemostasis. Hemostasis is a mechanism

where blood is obstructed to flow further in order to initiate healing of wounds and prevent further

blood loss. According to Dave, et. Al, (2022), there are four stages of hemostasis:  1) Constriction of

the blood vessel. 2) Formation of a temporary “platelet plug." 3) Activation of the coagulation

cascade. 4) Formation of “fibrin plug” or the final clot. The inability of the body to perform and

maintain such mechanism when it is encountered to injuries leads numerous kinds of diseases and

disorders. Failure to achieve and maintain adequate hemostasis can result from hereditary defects in

platelet and coagulation proteins; failure to regulate hemostasis, because of defects in inhibitory

components and damage to blood vessels that do not threaten blood loss, can result in thrombosis

in peripheral veins and coronary arteries and can lead to stroke and pulmonary embolism Essentials

of Medical Biochemistry (Second Edition), 2015. 

Acalypha Indica extract

Acalypha Indica contains extracts and constituents that can be used as blood clotting agent.

Studies says that the extracted elements can be used for blood clotting. It was highlighted that the

A.indica leaf extracts affects the intrinsic pathway of coagulation cascade and thus prolongs the

clotting time, hence this plant can be used in the management of blood clotting diseases (Sivaji Asha,

2017). Study shows that Acalypha Indica has a blood clotting agent. According to the Deparment of

Biochemistry (2017), the first time attempted to see the anti-coagulant properties of different

extracts of leaves of A.indica. From the findings it was found that this plant may act as an promising

alternative in the treatment of thrombotic disorders.

Benefits of Acalypha Indica:

Anti-inflammatory properties

The Indian Acalypha herb contains powerful anti-inflammatory properties. External

application of these plants can significantly reduce inflammation. To reap the benefits of this plant, it

is usually applied as a poultice. It quickly heals injuries and reduces inflammation.

Analgesic properties

It has a high level of analgesic activity, which implies it reduces pain,  it is also used as a

poultice on wounds to help reduce pain and inflammation. This herb's methanol extract has

excellent analgesic properties.

Antibacterial Property

The aforementioned plant has both antifungal and antibacterial properties which makes it

one of the best remedies for skin problems. The oil can be prepared and used to treat many skin

problems like bronchitis, eczema etc. Acalypha powder can be used in face packs for the treatment

of various skin problems.

Anthelmintic properties
 It has anthelmintic properties. Traditionally; the juice or decoction made of Acalypha leaf is

consumed to get rid of intestinal worms. Research has proven the uses of this herb.

Antivenom properties

  It is abundant in anti-toxic properties. Extraction of the leaves of the plant effectively kills

the venom of Russell Vipers. The leaves are usually boiled in water and given to people who are

bitten by the Russel Viper.

Antioxidant properties

Indian Acalypha plant has antioxidant properties. Extract of this herbal plant helps to remove

free radicals from the human body which causes premature ageing.

Anti-ulcer properties

The extraction of Indian Acalypha has amazing anti-inflammatory properties. The use of this

plant’s extract has been shown to significantly reduce ulceration, gastric emptying and HDT.

Synthesis:

On this page, the definition of Acalypha indica and its benefits are discussed. This also

discussed hemostasis and its definition. These related works provide knowledge and information

about the utilization of Acalypha indica leaves for blood clotting by in vitro procedure.

There are similar studies that use Acalypha indica's extract as an ointment for cutaneous

wound healing in mice (M. Laut et al., 2019). However, these differ in methodology and method of

testing. This study will be tested through in vitro mechanisms. All of the data in these related works

are relevant to the present research and therefore useful as a foundation for the interpretation and

discussion of the study's conclusions.

 METHODOLOGY

General Procedure:

Extraction of Acalypha indica leaves 

A. Collection

 Gathering of Acalypha indica Leaves

Green-colored, unwilted, fresh, and healthy leaves of Acalypha Indica will be collected using

gloves at 148 A, East Avenue, Barangay Balintawak, Escalante City, Negros Occidental, and they will

be placed in a container.

 Gathering of Other Supplies

The gloves and masks will be purchased at Mercury Drugstore, located at Barangay Balintawak,

Escalante City, laboratory materials that will be used in clotting time, bleeding time testing will be

provided by the laboratory.

B. Preparation Acalypha Indica Leaves

 Washing Process

Using a strainer, the leaves will be washed under running water until each leaf is clean and free

from any remnants.

 Drying and Pulverization of Acalypha indica

Leaves will be placed in a tray lined with parchment paper and will be sun dried until it is completely

dry, then it will be pounded using mortar and pestle until it is coarsely ground.

 Maceration of the leaves

The maceration process will utilize ethanol as a solvent, the pulverized Acalypha indica will be

soaked in ethanol in an airtight container for 3 days then it will be filtered using a filter paper. The

ethanol will be then removed from the potent via evaporation through boiling.
C. Testing/Application

After the extraction, the substance will be sent to a laboratory at for testing for its clotting time

and bleeding time test.

Sampling Method

Purposive Sampling will be used in this experimental research. One Hundred and Sixty blood

samples will be collected from 160 individual volunteers with no history of oral contraceptive or

anticoagulant therapy. It will be categorized according to abo typing; type A, B, AB, and O. 40

samples of type “A” blood will be drawn which will be distributed equally to 20 female and 20 male;

40 samples of type “B” blood will be drawn, 20 female and 20 male; 40 samples of type “AB” blood

will be drawn, 20 female and 20 male; samples of type o blood will be drawn, 20 female and 20

male.

CT-BT test procedure

1. Collect drops of blood from a finger of a participant and directly place one drop on each of

three slides

2. Add a drop of varying concentration on each of the 3 slides

3. Scoop regularly every 15 seconds the mixture of blood and extract using the pointed tip of

the used lancet

4. Observe for the formation of fibrin or thread-like substance every time you scoop with the

lancet.

5. Stop the timer (take the time time) the moment a thread –like formation happens.

6. Do the same to set up (slide) 2 and three using a different concentration of the extract.

7. Do the same procedure to the rest of the 160 participants.

Expected Outputs and Potential Impacts

Expected outcome of this study will:

a. Compare whether the Indica extract will have the same impact with that of the In-vivo
set-up

b. Find out if the extract will Speeding up or delaying of clotting time, specifically on the
basis of:

1. ABO type
2. gender

This study will contribute to the field of medicine especially in its application in the field of

medicine. It hopes to further add inputs into how Medicinal plants like Acalypha indica can be used

for treatment, specifically in external procedures and hemostasis.

This study will further widen understanding of alternative sources of medicine specifically in

remote areas where traditional western medicine are not easily available.

WORK PLAN AND TARGET DELIVERABLES

Activity Done Date Adviser’s Signature

Pre - Implementation
I. Introduction January 30-31,2023

a. Rationale January 30,2023

b. Significance January 30, ,2023

c. Theoretical Framework January 31,2023

d. Objectives January 31,2023

II. Review of Literature January 31,2023

Implementation

III. Methodology February 1-3, 2023

a. Variables or parameters to be February 2-3, 2023

measured and analyzed

b. Treatments to be used and February 3, 2023

their layout

c. Experimental procedures and February 3, 2023

design

d. Evaluation Methods February 3, 2023

Post - Implementation

IV. Expected Output and February 3, 2023

Potential Impact

V. Workplan and Target February 4, 2023

Deliverables
VI. References February 4, 2023

VII. Executive Summary February 6, 2023

VIII. Cover Page February 6, 2023

REFERENCES
Ashwini U, Asha S.(2017). A Preliminary Novel Findings on Invitro Anti-Coagulant Activity of

Acalypha Indica Linn Leaf Extracts. International Journal of Pharmaceutical and Clinical Research

2017; 9(5): 425-429. http://impactfactor.org/PDF/IJPCR/9/IJPCR,Vol9,Issue5,Article15.pdf

Zahidin, N. S., Saidin S., Zulkifli, R. M., Muhamad, I. I.,Ya’akob, H. Nur, H.,. (2017). A review

of Acalypha indica L. (Euphorbiaceae) as traditional medicinal plant and its therapeutic potential.

Journal of Ethnopharmacology Volume 207, 31 July 2017, Pages 146-173.

https://doi.org/10.1016/j.jep.2017.06.019

SouthWorld.net. (2017). Herbs & Plants. Acalypha indica. A useful source of medicine.

https://www.southworld.net/herbs-plants-acalypha-indica-a-useful-source-of-medicine/

Dineshkumar, B; Vigneshkumar, P; Bhuvaneshwaran, S P; Mitra, Analava. (2010). Phyto-

pharmacology of Acalypha indica: A Review. International Journal of Biosciences, Alternative and

Holistic Medicine; Coimbatore Vol. 1, Iss. 2,  (2010): 27-32.

https://www.proquest.com/docview/1232077011

Auuvriplus.com. (2019). Acalypha Indica (குப்பைமேனி) Benefits.

https://auvriplus.com/acalypha-indica-benefits/

Seebaluck, R., Gurib-Fakim, A., Mahomoodally, F. Ethnopharmacol. 2015. Medicinal plants from the

genus Acalypha (Euphorbiaceae)--a review of their ethnopharmacology and phytochemistry.

National Library of Medicine. https://pubmed.ncbi.nlm.nih.gov/25446604/

Healthyleaf. 2021. Acalypha – Uses and Benefits Medicinal Qualities and Side Effects of Kuppaimeni /

Acalypha indica. https://helthyleaf.com/benefits-and-uses-of-acalypha/

MacGill, M. & Rowden, A. 2023. What are the causes of a low platelet count? Medical News Today.

https://www.medicalnewstoday.com/articles/314123

 Chekuri, S., Lingfa, L., Panjala, S., Sai Bindu, K. C., & Rani, R. (2020). Acalypha indica L. - an Important

Medicinal Plant: A Brief Review of Its Pharmacological Properties and Restorative Potential.

European Journal of Medical Plants.

https://www.researchgate.net/publication/343397844_Acalypha_indica_L_-

_an_Important_Medicinal_Plant_A_Brief_Review_of_Its_Pharmacological_Properties_and_Restora

tive_Potential

Laut, M. et al. 2019. Cutaneous wound healing activity of herbal ointment containing the leaf extract

of Acalypha indica L. on mice (Mus musculus). Journal of Physics: Conference Series 1146 012025.

https://iopscience.iop.org/article/10.1088/1742-6596/1146/1/012025/pdf

Elaine K. Luo & Brian Krans. (2018). Bleeding Time Test: Purpose, Procedure and Risks.

https://www.healthline.com/health/bleeding-time

World Health Organization. (2022). In vitro diagnostics. https://www.who.int/health-topics/in-vitro-

diagnostics#tab=tab_1

https://www.researchgate.net/figure/GCMS-analysis-bioactive-compounds-Acalypha-indica-

Ethanolic-leaves-extract_tbl2_349761868

https://pubmed.ncbi.nlm.nih.gov/26073152/#:~:text=Kaempferol%20completely%20attenuated

%20phosphorylation%20of,assay%20of%20collagen%2Fepinephrine%2Dstimulated