IN VITRO 15-LIPOXYGENASE INHIBITION OF TARO (Colocasia Esculenta)

LEAF EXTRACT: A NON-STERIODAL ANTI-INFLAMMATORY TREATMENT

A Research Proposal Presented

to the Faculty of Pharmacy Department

San Pedro College, Davao City

In Partial Fulfillment

of the Requirements for the Degree

BACHELOR OF SCIENCE IN PHARMACY

By:

Alquino, Mary Kris Centeena

Concha, Kate Julia

Deita, Juanito Carlo VI

Dela Peña, Ysabella Eunice

Doyo, Kris Shiela

Magallona, Rio

Maquiling, Chelsea Kyle

Muhammad, Sha Aima

Saga, Vheronica
 CHAPTER I

INTRODUCTION

Inflammation, according to the study of Christian Nordqvist of Medical News Today, is a

defense mechanism in the body. The immune system recognizes damaged cells, irritants, and

pathogens, and it begins the healing process.

When inflammation occurs, chemicals from the body's white blood cells are released into

the blood or affected tissues to protect your body from foreign substances. This release of

chemicals increases the blood flow to the area of injury or infection, and may result in redness

and warmth. Some of the chemicals cause a leak of fluid into the tissues, resulting in swelling.

This protective process may stimulate nerves and cause pain. (Dr. David Zelman, 2016)

According to National Heart, Lung and Blood Institute, bronchitis is an inflammation in

which the bronchial tubes that carries air to your lungs, become inflamed. There are two main

types of bronchitis: Acute Bronchitis and Chronic Bronchitis.

Leukotrienes is produced by the production of the pro-inflammatory mediators through

the oxidation process of arachidonic acid pathway. The initiation of biological receptors in

inflammatory cells caused by leukotrienes will manifest bronchial disorder and allergic reactions,

therefore lipoxygenase enzymes such as 5,12 and 15-Lipoxygenases are produced which can be
found in the kidney, liver and adipose tissues. Inhibitions of these enzymes may obviate

inflammatory conditions.

Last 2011, the American Lung Association (ALA) stated that bronchial diseases like

Asthma, Acute and Chronic Bronchitis were present in more than 10million people. Center for

Disease Control and Prevention U.S. statistics state that ilast 2017, approximately 8.9 million are

now diagnosed with Chronic Bronchitis and 3.5 million with Emphysema. While in the

Philippines, COPD, a severe bronchial disease, is now one of the ten leading causes of death.

Taro is the common name for edible aroids which are important staple foods in many

parts of the world, particularly in Asia and the Pacific Islands. Within the family Araceae, there

is one ‘‘true taro’’, namely Colocasia esculenta Linn. Extract of Colocasia esculenta leaves has

been traditionally used for the treatment of various ailments in Ayurveda and Unani medicine.

Colocasia esculenta is traditionally used in various diseases such as high blood pressure, hepatic

disorder, rheumatic pain, pulmonary congestion, ulcer etc. The Colocasia esculenta has been

reported for anti-inflammatory, hypolipidemic, anti-cancer, antioxidant, and antibacterial

activities.

According to Yiao Li et al, quercetin, a polyphenol derived from plants, has a wide range

of biological actions including anti-carcinogenic, anti-inflammatory and antiviral activities.

Quercetin, a flavonoid found in fruits and vegetables, has unique biological properties that may

improve mental/physical performance and reduce infection risk. These properties form the basis

for potential benefits to overall health and disease resistance, including anti-carcinogenic, anti-
inflammatory, antiviral, antioxidant, and psycho stimulant activities, as well as the ability to

inhibit lipid peroxidation, platelet aggregation and capillary permeability, and to stimulate

mitochondrial biogenesis.

The leaves of Colocasia esculenta contains flavonoids such as vitexin, isovitexin,

orientin, isoorientin, schaftoside, isoschaftoside, luteolin, apigenin, vitamins A, B, and C,

thiamine riboflavin, niacin, oxalic acid, and minerals such as magnesium, calcium, phosphorus,

sodium, potassium, iron, zinc, copper, and boron. One of the reported use of the

phytoconstituents found on the extract is its anti-inflammatory activity.

This study was initiated by the researchers in hopes of decreasing the impediments of

diagnosed patients. The researchers also aim to find alternative treatments and solutions for

chronic bronchial inflammation like Bronchitis, Asthma and Chronic Obstructive Pulmonary

Disease (COPD). Lastly, this study aims to draw attention to respiratory diseases as mentioned

above for awareness and early prevention as needed.

 CHAPTER II

RESEARCH QUESTIONS

This chapter includes different literatures and studies that will assist the researchers in

doing their research. It covers the basic information of our plant, Colocasia esculenta (Taro), its

taxonomic hierarchy and description, phytochemical components of Colocasia esculenta (Taro)

that has the anti-inflammatory activity, the biologic activity common in Araceae family, some

information about inflammatory disease and the established medical agents that are widely used

for prevention and treatment for various inflammatory diseases.

II A. Review of Related Literature

I. Taxonomic Classification of Colocasia esculenta L. Schott

Taxonomic Hierarchy

Kingdom Plantae

Superdivision Embryophyta

Division Tracheophyta

Class Magnoliopsida (Dicotyledons)

Order Alismatales

Family Aracea

Genus Colocasia

Species esculenta
 .

https://garden.org/plants/view/75965/Elephant-
Ear-Colocasia-esculenta//

II. Taxonomic Description of Plan

Colocasia esculenta L. Schott, commonly known as Gabi or Taro, is a herbaceous

plant that measures for about a two meters. It has a swollen shallow root system (Corm)

that contains high amounts of starch. Structurally, the shape of the corm is usually

cylindrical and it measure 30 centimeters in height and 15 centimeter in diameter but they

vary in their size, shape and colour.

III. Phytochemical Components of Colocasia esculenta L. Schott

 According to Bruno, V.E. et al, Colocasia esculenta contains flavonoids,

β-sitosterol and steroids. It is also a good source of calcium, phosphorus and iron. Major

constituents on ethanol extract showed alkaloids, flavonoids, saponins and tannins.

Amino acids are found in the tubers. Anthocyanins perlargonilin, 3-glucoside, cyaniding

3-rhamnoside and cyaniding 3-glucoside. Ethyl acetate and n-butanol fractions of leaves

yielded 9 compounds: orientin; isoorientin; vitexin; isovitexin; luteolin-7-O-glucoside;

luteolin-7-O-rutinoside; rosemarinic acid; 1-O-feruloyl-D-glucoside; and 1-O-caffeoyl-

D-glucoside are found in the Corm.

A study done by Boots, A.W et al concluded that the constituent quercetin, the

most commonly occurring flavonoid, has anti-inflammatory effect, although further

clinical research is needed.

IV. Biological Activity of Araceae Family

According to Roy, S. et al, Many plants of family Araceae have been reported to

have antibacterial activities and numerous researches doing screening for pure

compounds that is responsible for this antibacterial activity. Other plants of this family

exhibit hypoglycemic potential by by the inhibition of α- Amylase and α- Glucosidase

enzyme. A recent study concluded that Anchomanes difformis that belongs to Araceae

family exhibited some degrees of inhibition against albumin induced inflammation at a

later phase in rats while also showing significant activity in formalin induced nociception

in rats although the exact mechanism of this plant in initiating anti-inflammation and

anti-nociception in rats could be further investigated

 V. Disease

There are dozens of inflammatory disorders, it occurs mostly when there are

injured tissues from the rest of the body. Such as inflammation of the joints in rheumatoid

arthritis, acute bronchitis describes the inflammation of the bronchi from common

infection of the lower respiratory tract. In chronic lung diseases such as asthma, several

studies describe those high levels of 15- LOX (Lipoxygenase) in airway of patients with

severe asthma.15- LOX is an enzyme that resolution and results to inflammation.

Colocasia esculenta has its activity to inhibit 15-LOX preventing certain inflammation.

VI. Medicinal Agents

There are agents that inhibits 15-LOX enzyme, which inhibit the 15-LOX enzyme

responsible for inflammation of bronchioles. Those where leukotriene receptor antagonist

like Montelukast (Singulair) to relieve the symptoms of asthma. Other agents like

zileuton, afirlukast, and pranlukast treat chronic asthma. These agents used to prevent and

manage asthma symptoms and to relieve the symptoms of seasonal allergic rhinitis or hay

fever inflammation in the nose.

II B. Related Studies

The following studies and concepts are used to justify the framework of this study.
 According to the study of Boot, A.W. et al entitled The anti-inflammatory activity

of quercetin, they concluded that quercetin increases the antioxidant capacity in vivo and

displays anti-inflammatory effects in vitro, but not in vivo or ex vivo, in the blood of

healthy volunteers.

According to Tongol, K. and Querequincia, J, inhibition of 15-LOX can be used

as a in vitro assay model for lipoxygenase inhibition by novel agents synthesized or

extracted from natural sources such as medicinal plants.

According to Li, Y. et al, Quercetin possess unique biological properties that may

improve mental/physical performance and reduce infection risks. In vitro, quercetin

exhibits a broad range of biological actions including anti-cancer, anti-inflammatory,

antiviral activities.

III C. Statement of the Problem

The main problem of the study is to determining the anti-inflammatory potential

of Colocasia esculenta leaf, 15-lipoxygenase inhibition in particular by identifying its

efficacy in terms percent inhibition in comparison to the positive control – ibuprofen.

Specifically, it sought to answer the following subproblems:

1. Are polyphenols present in the Ethnolic Extract of Colocasia esculenta leaf extract.

2. How much is the Phenolic and Flavonoid Content of the Ethanolic Extract of

Colocasia esculenta leaf extract.

3. Is there a significant difference in the in vitro anti-inflammatory activity between the

Colocasia esculenta Leaf extract and ibuprofen using 15-Lipoxgenase Inhibitory Assay.

4. What is the 15-Lipoxygenase capacity of Colocasia esculenta Leaf extract using IC50.

Chapter 3

METHODOLOGY

This chapter covers detailed discussion of the methodologies that will be used by the

researchers in the process of collecting data; the research locale, instruments, detailed procedures

and statistical treatment that will be applied in the study.

III. A. Materials and Methods

1.) Research locale

The plant sample of Colocasia esculenta will be collected at Norala, South

Cotabato. The plant product will be locally authenticated and verified at the

Biology Laboratory of Ateneo de Davao University, Jacinto Street, Davao City.

The plant drying and extraction of the plant substance will be performed in the

laboratories of San Pedro College. The phytochemical screening, microbiology

test, toxicology test, as well as assay in the in-vitro anti-inflammatory activity of

Colocasia esculenta extract will also be performed in the laboratories of San

Pedro College.
 2.) Research Measures

In this study, the researchers will be using equipments and apparatus that are

relevant in plant extraction, phytochemical screening, in-vitro assay as well as other

methods that will be used. Desiccator will be used in plant extraction.

Spectrophotometer will be used in phytochemical screening and in in-vitro assays.

III B. Mode of Analysis

Plant Sample Collection and Preparation

Collection of fresh plant will happen at Norala, South Cotabato. Running

water will be used to clean and wash the leaves of the fresh samples. The samples

will be air dried for approximately 7 days.

Plant Authentication

The authentication of the leaves of Colocasia esculenta was done at

Ateneo de Davao University – Biological Collections in the Biology Department.

Plant Extraction

The leaves of Colocasia esculenta were shade-dried and grinded into fine

powder. Powder will be soaked in Ethanol for 24 h. It was sieved with a muslin

cloth and filtered with Whatman No. 1 filter paper. The filtrate was evaporated to

dryness via rotatory evaporation. The dried extract yield was scrapped out of the
 stainless bowl and 10 g of it was dissolved in 100 ml of distilled water to get a

concentration of 100 mg/ml for use.

Determination of Moisture Content of the Pulverized Leaves

Cut fine pieces of dried taro leaves and put them into the moisture analyzer. Make

sure that the moisture content analyzer will not exceed by 10%.

Iodoform Test

Dissolve 1 pinch of the extract to 1 ml of water then add 0.25 ml of sodium

hydroxide to the mixture. Add another 0.25ml of water and 5 drops of potassium triiodide

solution to the mixture. Mix and stand for 10 mins. If yellow precipitate occurs ethanol is

present.

Phytochemical Screening

Two trials using two TLC plates were prepared. Using a pencil and as drawn at

the top and bottom of the TLC plate with a margin of 0.25cm, respectively. Using a

capilliary tube filled with the Taro extract, apply three spots of extract into the bottom

line of the TLC plate and let it dry for 15 minutes. Prepare the chamber for the mobile

phase by using 250 mL beaker filled with 7 mL – 10 mL of solvent system then soak a ¼

piece of cut filter paper, cover it with a watch glass then place the spotted TLC place
inside the chamber. Wait until the solvent will reach the top line of the TLC plate. The

presence of spots indicates that the solvent system is the correct system used. Compute

for the RF value.

*Test for Flavonoids

Add few drops of 10% conc. H2SO4 to 1 ml of extract then add 1

ml of ammonia afterwards. Formation of greenish yellow precipitate will

indicate that flavonoids are present.

*Test for Polyphenols

Add alcohol and few drops of FeCl3 solution to 1 ml of extract.

Formation of greenish yellow will signify that polyphenols are present.

Determination of Polyphenol Content

Preparation of Folin-Ciocalteau Phenol Reagent

Dissolve 10 grams sodium tungstate and 2.5 grams sodium molybdate in 70 ml water.

Add 5ml 85% phosphoric acid and 10ml concentrated hydrochloric acid. Reflux for 10

hrs. Add 15 grams of lithium sulfate, 5ml of water and 1 gtt. Bromine. Reflux for 15

minutes. Cool to room temperatue and bring to 100 ml with water.

Procedure:

Get 5g of extracted polyphenols and add 5mL of acetonitrile. Then add water to a

50 mL mark of the volumetric flask. The solution will be diluted 100 times with water

and will be made to react with dilute Folin-Ciocalteau phenol reagent (FC) and sodium
carbonate. It will be compared with a blank solution of Folin-Ciocalteau reagent and

sodium carbonate. Let the blank solution as A and the polyphenol solution as B. To

indicate the presence of polyphenol in the solution, solution B should yield greenish blue

color. Solution B will be further tested in the UV Spectrophotometer. UV

spectrophotometer indicates the optical density of the substance which is used for the

calculation of total polyphenol content.

Determination of Flavonoid Content

Preparation of Serial Dilution:

Dissolve 100mg of Quercetin into 100 mL of Ethanol. This will be considered as

the stock solution. Get 50 mL from the stock solution then add another 50 mL of ethanol

to make 0.5mg/mL concentration. Get 50 mL from 0.5 mg/mL then add another 50 mL of

ethanol to make 0.25 mg/mL concentration. Get 50 mL from 0.25mg/mL then add 50 mL

of ethanol to make 0.125 mg/mL concentration. Lastly, get 50 mL from 0.125 mg/mL

then add 50 ml of ethanol to make 0.0625 mg/mL concentration.

Procedure:

Add 0.1 mL of standard/sample solution to 0.3 mL of 5% sodium nitrate. Let it

stand for 5 minutes. After 5 minutes, add another 3 mL of 10% aluminum chloride to the

mixture. Let it stand for 6 minutes. After 6 minutes, add only 2 mL of diluted sodium

hydroxide. Absorbance must be at 500nm.

 Toxicological Tests

The Taro leaves extract was submitted to the Mediskin Clinic for the limit test of

heavy metals, Mercury (Hg) and Lead (Pb).

Microbiological Test

The Taro leaves extract was submitted to San Pedro Hospital – Laboratory

Department for the limit test of Staphylococcus aureus and Escherichia coli.

In vitro 15-Lipoxygenase screening assay

Methanol crude extract will be diluted to know its concentration according to

Cayman's literature. Ibuprofen will be used as the positive control. Cayman’s literature

regarding Chemical Company Lipoxygenase inhibitor screening assay kit will be used as

the protocol for reagent and enyzme preparations. Absorbance readings of the enzymatic

reactions will be done using a SH-1000 Corona microplate reader (Hitachi, Japan) at

490nm.

III C. Statistical Treatments

Statistical analysis results are expressed as Mean SD. The difference between

experimental groups could be compared by One-way Analysis of Variance (ANOVA)

followed by post-hoc test.

III D. Limitations of the study

1. This study only measures the anti-inflammatory activity of Colocasia esculenta plant

through an in-vitro measure using an assay kit.

2. The nature of the plant and its constituents may vary from different environments thus

affecting the characteristic and the percentage content of the constituent present in the

plant Colocasia esculenta.

 Chapter IV

Results

Presented in this section are the results of the data gathered during the whole duration of

the experiment on the determination of the in vitro 15-lipozygenase inhibitory activities

Colocasia esculenta leaves extract.

Corresponding tables and figures are presented on the proceedings to give clarity on the

data presentation, analysis and interpretation.