ASSESSMENT OF THE CYTOTOXICITY EXHIBITED BY ACTIVE

COMPONENTS PRESENT IN CRUDE EXTRACTS TAKEN FROM THE

DIFFERENT PARTS OF COLOCASIA ESCULENTA (CORM, STEM, LEAF)
AGAINST PROSTATE CANCER

A RESEARCH PAPER

PRESENTED TO THE
CURRICULUM AND INSTRUCTION DIVISION
PHILIPPINE SCIENCE HIGH SCHOOL—CENTRAL VISAYAS CAMPUS
TALAYTAY ARGAO, CEBU

IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS IN STEMR1

BY

NICOLE CONRAD C. OBRERO

RAPHAELA J. REYES
JOHN CODY GERSON C. SESPENE

MAY 2021

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 CHAPTER 1

INTRODUCTION

Background of the Study

Prostate cancer is currently the second most common cancer among men and the fifth

leading cause of death globally (Rawla, 2019). In the world, incidence rates are 3‐fold higher

in transitioned than in transitioning countries (37.5 and 11.3 per 100,000, respectively),

whereas mortality rates are less variable (8.1 and 5.9 per 100,000, respectively). In the

Philippines, prostate cancer is ranked no. 5 in the number of cases (with 8 242 new cases) and

ranked no. 9 in the number of deaths (with 3 164) in 2020. The disease is clinically

characterized by a long latency period and slow tumor growth―thus, making it an ideal

candidate for chemopreventive interventions using bioactive components' cytotoxicity to

prostate cancer cells (Chhabra et al., 2018). Although PCa is treatable, it persists in troubling

people and making their lives arduous. Surgery, radiotherapy, and chemotherapy are the most

common treatments for PCa, and over the last few years, these treatments have become

increasingly aggressive, with negative side effects and increased toxicity. Thus, as a result,

non-toxic, highly efficacious against multiple cancers, palatable, and cost-effective cancer

therapeutics are urgently needed. Medicinal plants have piqued researchers' interest due to their

anticancer properties and lack of side effects (Desai et al., 2008).

Several agents isolated in edible plants have exhibited chemopreventive properties

responsible for selectively targeting cancer cells (Stan et al., 2007). Colocasia esculenta,

commonly known as taro, is an edible plant native to Southeast Asia and has been extensively

cultivated here. Taro is an abundant source of antioxidants, primarily phenolic compounds

distributed diversely and abundantly. Taro phytochemicals have immunomodulatory,

antioxidant, antitumoral, antimetastatic, antimutagenic, anti-hyperglycemic, and anti-

2 of 26
hypercholesterolemic bioactivities in addition to antioxidant properties (Pereira et al., 2020).

Furthermore, taro has a lower glycemic index than potatoes and may reduce the incidence and

prevalence of several diseases, including cancer (Simsek & Nehir, 2015; Foster et al., 2002).

Cytotoxicity refers to the toxicity caused due to the action of chemotherapeutic agents

on living cells. Cytotoxicity tests and assays are significant as they aid in the determination of

the biomedical use of certain living cells, specifically on cancer chemoprevention (Mukherjee

et al., 2019). The present study focuses on assessing the cytotoxicity exhibited by the active

compounds present in crude extracts taken from different parts (corm, stem, and leaves) of

Colocasia esculenta against prostate cancer.

Objectives of the Study

The main objective of the study is to assess the cytotoxicity of the active compounds

present in crude extracts taken from various parts (corm, stems, leaves) of Colocasia esculenta

in inhibiting prostate cancer. Specific objectives are as follows:

1. To assess the cytotoxic effects of the crude corm, stem, and leaf extracts prepared

from C.esculenta on the PC3 and DU145 prostate cancer cell lines;

2. To identify the bioactive compounds present in the crude corm, stem, and leaf

extracts prepared from C.esculenta on the PC3 and DU145 prostate cancer cell

lines;

3. To determine the half-maximal inhibitory concentrations (IC₅₀) of the crude corm,

leaf, and stem extracts at varying concentrations on the PC3 and DU145 prostate

cancer cell lines;

Significance of the Study

Plant metabolites constitute the most effective treatment for cancer chemoprevention

in many parts of the world. Although synthetic drugs have overshadowed the market of general

medicine in the past few decades, plant-based drugs are still widely adopted in cancer treatment

3 of 26
following tight legislation and strict surveillance (Katnoria et al., 2020). The study hopes to

benefit and contribute to the growing knowledge of the natural chemopreventive agents present

in Colocasia esculenta corm, stem and leaves, the parts of the ubiquitous backyard plant that

exhibits anti-carcinogenic properties. Moreover, this study could be deemed critical to the

following:

Pharmaceutical Companies. The development of new derivatives from bioactive

compounds of food origin has been a feasible method to diminish toxicity and elevate their

effectiveness against cancer (Ramawat & Goyal, 2008). With the discovery of the maximum

efficiency of natural cytotoxic agents present in Colocasia esculenta against prostate cancer

cells, the study may spur several medical types of research led by pharmaceutical companies

and eventually lead to the advent of non-invasive treatment strategies for prostate cancer.

Prostate Cancer Patients. The findings of this study will benefit prostate cancer

patients as it will shed light on non-invasive treatments for prostate cancer. Moreover, the study

may lead to the production of medicines derived from the natural properties studied from the

corm, stem and leaves of Colocasia esculenta that can be affordable and sustainable for people

with prostate cancer.

Medical Oncologists. With the findings gathered from this research, new potential

treatments for prostate cancer that are safer and more natural than traditional chemotherapy

and medications may be produced. Medical oncologists will benefit from this as this would

lower the risk of their patients experiencing side effects from medication. They can access a

more comprehensive range of cure strategies for prostate cancer.

Future Researchers. The notions and data procured in the study may serve as

referential information for future researchers who wish to explore more on the efficiency of the

natural cytotoxic properties (to PCa cells) present in Colocasia esculenta corm, stem, and

4 of 26
leaves. Moreover, they may also employ the data presented in the study to test the credibility

of their methods and findings.

Scope and Limitations

Data collection will be conducted by experimenting on PCa cancer cell lines,

specifically PC3 and DU145 cell lines, which the researchers will acquire from hospitals and

cancer centers in Cebu City. Acquiring these cell lines will take time since obtaining them from

hospitals/medical institutions will be significantly challenging. Cell lines that are not of

prostate cancer patients are not within the scope of this paper. The groundwork will be done

through maceration, thin layer chromatography, and GC-MS from taro at a nearby laboratory

complete with all the needed equipment. Since Colocasia esculenta is a common backyard

plant, the researchers will not have a problem with its availability. Communication restraints

also add to the matter. The researchers live on different islands, and with the current situation

of the COVID19 pandemic, the development of the research will be slower than anticipated.

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 CHAPTER 2

REVIEW OF RELATED LITERATURE

In recent decades, prostate cancer (PCa) has become one of the leading diseases in

modern times, particularly in the Western world. Contemporaneous therapeutic strategies for

PCa have gradually become bodily-demanding and aggressive with an elevating degree of

adverse side effects. It has been observed that PCa incidence is considerably lower in Asia than

in the West. Cohort studies have suggested that this may have been the result of different diets

consumed between the two regions, while others propose this may be partly the product of the

inadequate practice and lack of accessibility of pharmaceutical prostate-specific antigen (PSA)

screening. Thus, multiple studies have emerged concerning the potential of specific

micronutrients present in usual food against various kinds of cancer to seek a cost-effective

and environmental-friendly treatment. This literature review will thoroughly examine the

cytotoxicity of natural agents, found in aqueous extracts taken from different plant parts of

Colocasia esculenta (corm, stem, leaves), against prostate cancer. Moreover, it will dig deeper

on what prostate cancer is, its epidemiology and pathophysiology and weighs the efficacy of

the current treatment strategies for PCa and the chemopreventive properties of specific dietary

agents.

Prostate Cancer

A. Epidemiology

Prostate cancer is a heterogeneous genetic disease characterized by a slower growth

rate and progression, mostly affecting the male population in their later years. Prostate cancer

(PCa) is the second most frequently diagnosed cancer in men, responsible for an approximated

1.41 million cases and 375,304 deaths in 2020 worldwide and 8,242 new cases and 3,164 deaths

in the Philippines alone (Sung et al., 2021). Large variations have been found to exist between

geographical regions, with a 13-fold difference between regions of highest rates and lowest

6 of 26
rates. Higher incident rates have always been observed in most parts of the West, while several

studies have noted that PCa incidence in Asia is gradually rising but still is significantly lower

than that of Western countries (Ha Chung et al., 2019; Sung et al., 2021). While first-world

countries in the West showed stabilizing and decreasing mortality trends over the past five

years, mortality rates vary differently in many regions of Asia. Results from Chen et al. (2012)

based on Singapore’s PCa mortality data have observed a gradual elevation in mortality rates

from 1968 to 1992 followed by a decline from 1993 onwards. Katanoda et al. (2015) also found

that mortality rates in Japan have decreased since 2004. Data from Kimura & Egawa (2018)

and Sung et al. (2021) established that mortality rates have generally increased in most Asian

countries. The reasons for the trends happening in Asia remain multifactorial. Several studies

have suggested that diet change may have been the prime cause of increased Western-style diet

consumption in the region, resulting in an elevated increase in incidence and mortality among

Asians. Others have also proposed that the increasing incidence and mortality may have been

caused by comprehensive systematic PSA screening, although little conclusive evidence is

present (Chen et al.,2012).

B. Pathophysiology

A normal prostate requires androgens—testosterone and dihydrotestosterone (DHT) to

function and develop optimally. The growth and expansion of the prostate glandular

component of the prostate depend on DHT, which is primarily formed from testosterone under

the influence of 5-alpha-reductase by attaching and actuating the androgen receptor (AR)

cytoplasm of human prostate epithelial cells. Recent studies have established that androgens’

activation of this specific receptor promotes prostatic growth, resulting in benign prostate

hyperplasia (BPH)—a normal condition for males approaching advanced age. However, this

pathway causes the AR to be activated without androgen stimulation—initiating carcinogenesis

among the prostate epithelial cells, eventually resulting in PCa (Alukal & Lepor, 2016; Kim et

7 of 26
al., 2018; Chhabra et al., 2018; Nevedomskaya et al., 2018). It has been proposed that blockage

of DHT production by 5-alpha-reductase inhibitors may significantly reduce PCa incidence

among men but may pose a higher risk of high-grade prostate cancer; thus, it does not

significantly promote survival.

Current Treatment Options and Side Effects

A. Surgery and Radiation Therapy

The primary type of prostate cancer surgery is radical prostatectomy, wherein a surgical

incision is made, and the entire prostate and seminal vesicles (or a part of it) are surgically

removed. This procedure may be accomplished with either of two methods, the retropubic or

suprapubic incision (lower abdomen) or a perineum incision (through the skin between the

scrotum and the rectum) (Johns Hopkins Medicine, n.d.). Other methods include robotic

prostatectomy, bilateral orchiectomy, and many others (Cancer.net, 2019). Surgery is generally

effective in eradicating prostate cancer tumors (usually by removing the whole prostate gland).

However, this usually leads to unwanted side effects. A study describes patients as “feeling

like a changed man with a lost sex life” as surgery may lead to erectile dysfunction (Hedestig

et al., 2005). Patients who undergo radical prostatectomy also have a significantly higher

chance of experiencing urinary incontinence than those who undergo other treatment options

(Resnick et al., 2013). A study by Donovan et al. states that prostatectomy poses the worst

effects on sexual function and urinary incontinence. They conducted a trial involving three

groups, a group for radical prostatectomy, one for radiation therapy, and another assigned to

active monitoring. Radical prostatectomy had the most significant adverse effect on urinary

continence for six months, although some recovery was observed.

On the other hand, radiation therapy pertains to the use of high-energy rays to eliminate

cancer cells (Cancer.net, 2019). It is used to slow the growth of or destroy tumors in early-

stage cancers confined to the prostate gland and treat people with advanced/recurrent prostate

8 of 26
cancer by slowing cancer growth and reduce pain. Mainly, there are two types of radiation

therapy used for prostate cancer: external beam radiation (EBM) and brachytherapy (internal

radiation). Both require different mechanisms, but they cause similar side effects and

complications. Although advances in radiation therapy have considerably improved the

prognosis for prostate cancer patients, the aftereffects it causes are quite harmful. While

patients who undergo surgery are more likely to be diagnosed with erectile and urinary

problems, radiation therapy patients generally have a higher chance of experiencing bowel

dysfunction (Shrader-Bogen et al., 1997).

B. Complications of the Current Treatment Strategies

Since androgens are the primary fuel of PCa tumors, androgen deprivation therapy (ADT)

has been an initial treatment for PCa. Current treatment strategies for prostate cancer also

encompass surgery, proto-beam therapy, radiotherapy, or chemotherapy basing on the clinical

state, histological grade, and serum levels of PSA (Mokbel et al., 2019; Chen & Zhao, 2013).

However, prostate cancer is highly molecular heterogeneous, comprised of a phenotypically

diverse population of cancerous cells, and its carcinogenesis is involved with several steps

through which cancer cells are accessing and bypassing surrogate survival pathways that may

result in an elevated increase of drug resistance and presents a challenge for novel therapeutic

strategies (Shoag & Barbieri, 2016; Chen & Zhao, 2013). While significant steps have been

made in attaining prostate cancer treatments, the therapeutic strategies available for

metastasized prostate cancer are still comprehensively deficient, and the prognosis remains

unclear (Mokbel et al., 2019).

As the years go by, these current therapeutic strategies for PCa are gradually becoming

invasive and aggressive, coupled with elevating toxicity, resistance, and side effects. Moreover,

these treatments are also posing a considerable burden in the health care system as the early

detection tools that diagnose patients at the outset stages of cancer before it becomes life-

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threatening results in vast diagnosis and therapy costs. Thus, dietary compound agents found

in natural food samples are being researched in hopes of a non-invasive and convenient

treatment for PCa for potency in blocking the process of carcinogenesis as preliminary data

suggest that these agents may have chemopreventive properties. Characterized by its long

latency period, high incidence, molecular heterogeneity, and clinical ambiguity, several studies

have noted that prostate cancer is an ideal candidate for potential chemopreventive

interventions (Van Poppel & Tombal, 2011; Chhabra et al., 2018; Shoag & Barbieri, 2016;

Mokbel et al., 2019). In its long pre-clinical phase, chemopreventive interventions could attain

a considerable diminution of PCa prevalence as it may play a considerable influence in PCa

progression in many stages.

Studied Natural Chemopreventive Agents against Prostate Cancer

The term chemoprevention was first used in the late 1970s and referred to cancer

prevention by selective use of phytochemicals or analogs. Chemoprevention refers to the

prevention of cancer through the selective use of phytochemicals. The idea of using naturally

derived chemicals as potential chemopreventive agents has advanced the field dramatically.

An extensive number of chemopreventive agents present in natural products have been

evaluated using various experimental models throughout the years. It has been recognized that

single chemopreventive agents may not always be enough to provide chemopreventive

efficacy. Therefore, the new concept of combination by multiple agents or consuming “whole

foods” has become an increasingly attractive area of study for natural chemoprevention (Mehta

et al., 2010).

A. Tea Polyphenols

Tea polyphenols are the primary active compounds found in teas. This is the general

term for polyphenol compounds in tea and has been shown to have sound effects on

antioxidant, anti-inflammatory, cancer prevention, and regulation of lipid metabolism (Yan et

10 of 26
al., 2020). Epidemiologic observations and laboratory studies have indicated that tea

polyphenolic compounds may reduce the risk of various illnesses, including cancer. Research

investigating polyphenols, particularly those found in green and black tea, and their role in

preventing and progressing certain diseases and cancers is a growing field (Chhabra et al.,

2018). Green tea catechins (GTCs) proved to inhibit cancer growth in several experimental

models effectively (Bettuzzi et al., 2006).

B. Vitamins & Minerals

Other natural chemopreventive agents against prostate cancer are vitamins and

minerals. Several studies have evaluated vitamins as potential chemopreventive agents against

PCa. Some well-studied vitamins and minerals with the potential for prostate cancer

chemoprevention are vitamins E and D and selenium (Brawley, 2002; Bemis et al., 2006;

Özten-Kandas & Bosland, 2011; Mokbel et al., 2019). Although remarkably divergent in

nature, selenium and vitamin E share an antioxidant activity that may affect the prostate

epithelium’s protective effects. Selenium (in the organic form) has been reported to inhibit cell

growth of multiple prostate cancer cell lines, potentially through its effects on the expression

of critical cell-cycle genes and the AR protein that drives hormone-dependent prostate cancer

cell growth. The E vitamins are fat-soluble compounds that act as antioxidants in cell

membranes and inhibit lipid peroxidation. The main form of vitamin E found in the human

body is α-tocopherol. In vitro studies [68,71,72] of both α-tocopherol and α-tocopherol

succinate (VES) have indicated that these compounds act as anti-prostate cancer agents through

protection against DNA damage, induction of apoptosis, and inhibition of cell growth and AR

signaling (Bemis et al., 2006).

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Colocasia esculenta

A. Overview of the Plant

Colocasia esculenta, or taro, is a plant grown for its edible corms, roots, and vegetables,

originating from Southeast Asia. (Rashmi et al., 2018) It is also known as taro and is widely

planted in places with high rainfall conditions, usually by small farmers. It is native to India

and the Malay peninsula. Cultivation throughout tropical and subtropical Asia, the Caribbean,

Pacific Islands (including northern Australia), and tropical Africa (from East to West) is

possible. It allows for a wide range of sunlight conditions, from full sun to shaded conditions

with coconut, coffee, or cocoa trees. Taro tubers are essential sources of carbohydrates and are

considered staple foods in tropical and subtropical countries. Besides its nutritional value, taro

is traditionally utilized as a medicinal plant and provides bioactive compounds with important

biological properties (Rashmi et al., 2018).

B. Nutritional Content of the Corm

Taro corm is a good source of minerals and the small granule size of its starch helps

increase the bioavailability of its nutrients due to efficiency of digestion and absorption. Taros

have a high amount of β-carotene in the corm and will impart vitamin A and antioxidant

property in the body. In terms of protein content of the corm, it is relatively higher towards the

corm’s periphery than towards its center (Temesgen et al., 2015). Taro root has much more

fiber than potatoes, improving digestive function and alleviating issues such as diarrhea,

stomach ulcers, acid reflux, and constipation. Taro tubers are rich in starch and contain

anthocyanins, cyanidin 3-glucoside. Anthocyanins are known to improve blood circulation by

decreasing capillary fragility, enhance eyesight, act as potent antioxidants, and act as anti-

inflammatory agents to inhibit human cancer cell growth (Rashmi et al., 2018). These

carbohydrates are proven to stabilize blood sugar, help with weight management, and reduce

the risk of diabetes. Taro root also has high potassium levels, a mineral that helps control high

12 of 26
blood pressure by breaking down excess salt. Potassium reduces stress on the cardiovascular

system and enables the development of chronic heart problems.

C. Nutritional Content of the Stem and Leaves

The stem extract of C. esculenta from a study of Asaduddin et al. (2021) positively

contained Flavonoid, Terpenoid, Saponin, and Steroid based on TLC assays. Based on

nutritional minerals test, C. esculenta contained vitamin C, vitamin E, Potassium, and %

Calcium. Flavonoids, Terpenoid, Saponin, and steroid had been proved as potent treatment for

oxidative stress. The leaves of taro have been reported to be rich in minerals like Ca, P, Fe, and

vitamins. The high level of dietary fibre found in the taro leaf are also advantageous for their

active role in the regulation intestinal transit, increasing dietary bulk and feces consistency due

to their ability to absorb water (Rashmi et al., 2018). The protein fraction is rich in essential

amino acids of threonine, leucine, arginine, valine, and phenylalanine. Among the essential

amino acids, methionine, lysine, cystine, phenylalanine and leucine are relatively abundant in

the leaf than the corm (Temesgen et al., 2015).

D. Known Medical Use and Anti-Metastatic Activity

Taro corms are a natural pedigree of bioactive proteins, which behave as

immunostimulators on total bone marrow and spleen cells from different mice strains (Rashmi

et al., 2018). Taro roots and their edible leaves contain antioxidants. Quercetin, which comes

from the vegetable purple pigment, is an antioxidant that protects the body from free radicals

or molecules built in the body due to aging and lifestyle and results in cell damage or cancer.

Taro root also plays an integral part as an antioxidant in our body. The elevated levels of

vitamin A, C, and other resin antioxidants boost our immune system and help dispose of

dangerous free radicals from our system. Cryptoxanthin, located in the taro root, is directly

connected to a lowered chance of developing lung and oral cancers. Iron and copper’s dual

presence makes it a critical food to prevent anemia and boost circulation throughout the body.

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Kundu et al. (2012) have shown that a water-soluble extract of taro (TE) potently inhibits lung

colonizing ability as well as spontaneous metastasis from mammary gland-implanted tumors,

in a murine model of highly metastatic ER, PR and Her-2/neu negative breast cancer. TE

modestly inhibits proliferation of some, but not all, breast, and prostate cancer cell lines. They

also had considerably good finds on TE treatment blocking tumor cell migration and inhibiting

prostaglandin E2 (PGE2) synthesis and downregulated cyclooxygenase (COX) 1 and 2 mRNA

expression.

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 CHAPTER 3

METHODOLOGY

Plant Material

Fresh stem, leaves, and corm of Colocasia esculenta will be acquired from one of the

researchers’ backyard in Albuquerque, Bohol, and will be brought to a laboratory within Cebu

City plastic bags. The stem, leaves, and corm will be washed with tap water containing

detergent to eliminate all soil debris, lingering organisms, and adhering dirt particles. The

samples will be cut into small pieces, washed with distilled water, and then dried at room

temperature for 72 hours until constant weights are achieved. Dried samples will be kept in the

fridge at four celsius before the extraction process.

Extraction of the Crude Extracts from Stem, Leaves, and Corm of C.esculenta

Dried stem, leaves, and corm will be milled to fine powders using a local grinder. Each

of the 250g samples from different Colocasia esculenta will be macerated in 1000 mL in

methanol (99.9%; absolute) for 72 hours at room temperature and then filtered with Whatman

filter paper no. 1. The residue from the extraction will be soaked again with the same solvent

mixture for 72 hours until the solvent layer will become colorless. The solvents will be mixed

and evaporated under reduced pressure at 40°C using a rotary evaporator. 10 mL of each stem,

leaf, and corm extract will be measured out from the concentrate for phytochemical screening,

and the remaining would be tested for cytotoxic assays. The concentrated crude extracts will

be stored at −20°C for further testing and use.

Phytochemical Screening

Each of the methanolic stem, leaves, and corm extracts will be screened for the presence

or absence of alkaloids, saponins, tannins, anthraquinones, cardiac glycosides, steroids,

carbohydrates, flavonoids, and phlobatannins according to the methods described by Evans

15 of 26
(1996) and Ajayi et al. (2011) with some modifications. This study will use only pure and

analytical-grade chemicals and solvents.

Detection of alkaloids

On a steam bath, each extract (1.0 ml) was stirred with 5 ml diluted hydrochloric acid,

filtered, and 1.0 ml of each Dragendoff’s/Mayer’s/reagent Wagner’s was added to 1.0 ml

separate portions of filtrate. The presence of alkaloids is indicated by a cloudy orange-

red/slightly yellow/turbid brown color in respective reagents.

Detection of saponins

Frothing Test: 0.2 ml of each extract will be mixed with 5.0 ml distilled water, gently

shaken for 20 min. The persistence of foams indicates the presence of saponins.

Detection of tannins

Each extract (1.0 ml) will be stirred with 1.0 ml ferric chloride. A greenish-black

precipitate will indicate the presence of tannins; 1.0 ml extract will also be mixed with 1.0 ml

bromine water. A reddish-brown turbid color will reveal the presence of tannins.

Detection of anthraquinones

Each extract (2.0g) in 10 ml methanol, steamed for 5 min and filtered, 2.0 ml filtrate

will be added to 2.0 ml chloroform, shaken thoroughly, chloroform layer will be taken off. 5.0

ml distilled water will be added, which will be shaken with 5.0 ml dilute ammonia solution.

The red color in the upper ammonia phase indicates the presence of anthraquinones.

Detection of cardiac glycosides

Legal’s test: 1.0 ml of each extract will be dissolved in 5.0 ml pyridine with two drops

of 2% Sodium Nitroprusside and two drops of 20% NaOH. The presence of cardenolide is

indicated by a deep red color that fades to brown.

16 of 26
Detection of steroids

Liebermann-Burchard’s test: 0.5 g extract will be dissolved in 2.0 ml acetic anhydride,

cooled in ice, and carefully added to 1.0 ml concentrated H2SO4. The presence of steroids is

indicated by a blue-green ring.

Detection of carbohydrates

Fehling’s Test: 1.0 mL of each extract will be added to 2.0 ml of Fehling’s solution,

and the mixture will be boiled for 5 minutes. The presence of reducing sugars is indicated by a

red precipitate.

Molisch Test: 1.0 ml of each extract will be carefully added to 1.0 ml Molisch’s reagent,

followed by 1.0 ml concentrated H2SO4. The presence of carbohydrates is indicated by a

reddish ring.

Detection of flavonoids

Alkaline Reagent Test: 0.2 ml diluted NaOH will be added to 0.2 ml of each extract,

gently shaken. A dirty yellowish-brown precipitate indicates the presence of flavonoids.

Lead acetate Test: In 0.2 ml 10% lead acetate, 0.2 ml of each extract will be added

acetate and gently shaken. The presence of flavonoids will be indicated by the existence of a

dark brown precipitate.

Detection of phlobatannins.

Each extract (1.0 mL) will be boiled in 2.0 mL aqueous HCl at a concentration of 1%.

The presence of phlobatannins is indicated by a red precipitate.

Analytical studies

Analytical studies will be carried out to isolate and identify compounds in stem, corm,

leaf extracts. The following methods were previously described by Chakraborty et al. (2015).

17 of 26
Thin Layer Chromatography

The taro stem, corm, and leaf methanol extract compounds will be separated using thin-

layer chromatography (TLC). As a mobile phase, a mixture of solvents (hexane: acetone,

60:40) will be used to separate the compounds according to their mobility. Separated spots will

be visualized under UV light, and the Rf factor will be calculated using the equation below.

Distance traveled by the solute front

Rf factor =
Distance traveled by the solvent front

Gas Chromatography-Mass Spectrometry

The methanol extract of taro corm, leaves, and stem will be analyzed using GC-MS.

The instrument will be a gas chromatographic instrument with a mass spectrometer detector

and a column. Helium will be utilized as the carrier gas, with a flow rate of 1 ml/min. The

temperature program below will be used in the following manner. For a total run time of 30

minutes, the oven temperature will be held at 60°C for 2 minutes before ramping up to 300°C

at a rate of 10°C/min with a 4-minute hold time. The injector will be kept at a temperature of

300°C0. The ion trap will run at 70 eV and have a scan range of 50 to 600 m/z. In split mode,

a 1 l sample will be taken (10:1). The intermediate and final products will be identified using

mass spectral data from the Wiley registry.

Cell line and Cell Plating

Human prostate epithelial adenocarcinoma cell lines (PC3 and DU145), directly

acquired from willing hospital centers in Cebu City, will be used throughout the study. Cell

lines will be cultured in RPMI-1640M supplemented with 10% FBS, L-glutamine, 100 units/ml

penicillin, and 100 µg/ml streptomycin 25cm2 tissue culture flasks in a humidified atmosphere

of 5% CO2 at 37°C. Maintenance cultures will be passaged weekly, and the culture medium

will be changed bi-weekly. Once the cells have reached >80% confluency, they will be washed

in sterile phosphate buffer saline (PBS), and 1 mL trypsin ethylenediaminetetraacetic acid

(EDTA) will be added to the flask, which will then be kept in a CO2 incubator at 37°C for 5–

18 of 26
10 minutes to permit the cells to will be detached from the flask. The cell will be centrifuged,

the supernatant will be discarded, and the cell pellet will be resuspended in a culture medium

to make a single-cell suspension. Trypan blue exclusion will be used to count viable cell density

in a hemocytometer and then diluted with a medium containing 5% FBS to produce an optimal

plating density of 1 × 105 cells/ml for PC3.

In Vitro Cell Viability Assay

To determine cell viability, the ability of the cells to cleave the tetrazolium salt MTT

[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide by the mitochondrial enzyme

succinate dehydrogenase, as described previously by Kumar et al. (2018) and Chakraborty et

al. (2015) with some modifications, will be used. Prior to being stimulated by each methanolic

extract, 150μL of cell suspension will be seeded into 96-well cell culture plates and incubated

for 24 hours at 37°C and 5% CO2 to permit the cells to attach to the wells. After incubation

and attachment, each well will have 20μL of supernatant pipetted out. The cells will then be

treated with stem, corm, and leaf extracts (at concentrations of 200, 100, 50, 10, and 7.5 g/ml,

respectively) as a sample test. The extracts will be dissolved in 10% dimethylsulfoxide

(DMSO) along with an aliquot of the sample solution, which will be serially diluted in RPMI-

1640 medium to twice the required final maximum test concentration (DMSO 1 percent v/v).

A series of four serial dilutions will be performed to generate five sample concentrations. The

required final sample concentrations will be achieved by adding aliquots of 100 μL of each of

these different sample dilutions to the appropriate wells containing 100 μL of growth medium.

All extract concentrations will be maintained in triplicates. As controls, wells with only

200 μL of medium will be used.

MTT Assay

After 48 hours, 20 μl of MTT (5 mg/ml stock in PBS) will be added to each well

containing 200 μl medium and incubated for 4 hours at 37°C. Excess MTT will be discarded,

19 of 26
and the formed formazan crystals will be solubilized with 100 μl of DMSO before being

measured at 550 nm using a microplate reader for absorbance values of the formazan as a

measure of viable cells. In three separate experiments, each extract and control will be tested

in triplicates. Cells that have been treated will be compared to cells that have not been treated.

Tetrazolium salts are cleaved to formazan dye only in viable cells by cellular enzymes found

only in metabolically active cells (viable cells). As a result, the amount of formazan dye

produced is proportional to the number of viable cells in the culture and is measured as

absorbance using spectrophotometry. Toxin-exposed cells are expected to have reduced

activity. In comparison to the control, the percentage of cell viability was calculated as follows.

mean absorbance value of treated cells (test)

% of cell viability = ( ) × 100
mean absorbance of value untreated cells (control)

The percentage of growth inhibition will be calculated using the following formula:

absorbance value of sample

% of cell inhibition = 100 − ( ) × 100
absorbance value of control

Statistical Analysis

Experimental results will be expressed as mean ± SEM. One-way analysis of variance

(ANOVA) will be employed to detect differences between the groups of treated and control

and one-tailed unpaired 𝑡-test using GraphPad Prism software (GraphPad Prism 5.0, GraphPad

Software, Inc., CA, USA). Values showing p ≤ 0.05 will be considered statistically significant.

The IC50 values will be calculated from nonlinear regression analysis, plotted between the

percentage of cell inhibition and Log concentration using GraphPad Prism software.

20 of 26
 Literature Cited

Alukal, J. P., & Lepor, H. (2016). Testosterone Deficiency and the Prostate. Urologic Clinics
of North America, 43(2), 203–208. doi:10.1016/j.ucl.2016.01.013

Asaduddin, A. H., Maulani, U. N., Sari, A. Y., Hawari, K., & Ayusari, A. A. (2021, April).
Taro Ice Cream: Addition of Colocasia esculenta Stem to Improve Antioxidant Activity
in Ice Cream. In IOP Conference Series: Materials Science and Engineering (Vol. 1143,
No. 1, p. 012038). IOP Publishing.

Bemis, D., Katz, A., & Buttyan, R. (2006). Clinical trials of natural products as
chemopreventive agents for prostate cancer. Expert Opinion on Investigational Drugs,
15(10), 1191-1200. https://doi.org/10.1517/13543784.15.10.1191

Bettuzzi, S., Brausi, M., Rizzi, F., Castagnetti, G., Peracchia, G., & Corti, A. (2006).
Chemoprevention of Human Prostate Cancer by Oral Administration of Green Tea
Catechins in Volunteers with High-Grade Prostate Intraepithelial Neoplasia: A
Preliminary Report from a One-Year Proof-of-Principle Study. Cancer Research, 66(2),
1234-1240. https://doi.org/10.1158/0008-5472.can-05-1145

Brawley O. W. (2002). The potential for prostate cancer chemoprevention. Reviews in urology,
4 Suppl 5(Suppl 5), S11–S17.

Cancer.Net (2019, November). Prostate Cancer - Types of Treatment. Cancer.Net.

https://www.cancer.net/cancer-types/prostate-cancer/types-treatment.

Chan, J. M., Stampfer, M. J., & Giovannucci, E. L. (1998). What causes prostate cancer? A
brief summary of the epidemiology. Seminars in Cancer Biology, 8(4), 263–
273. doi:10.1006/scbi.1998.0075

Chen, C., Naidoo, N., Yang Q., Hartman, M., Verkooijen, H.M. Loy, E.Y., Bouchardy, C.
Chia, K.S., Chia, S.E. (2012) A comparative population-based study of prostate cancer
incidence and mortality rates in Singapore, Sweden and Geneva, Switzerland from
1973 to 2006. BMC Cancer 12(222). doi:10.1186/1471-2407-12-222

Chen, F. Z., & Zhao, X. K. (2013). Prostate cancer: current treatment and prevention
strategies. Iranian Red Crescent medical journal, 15(4), 279–284.
https://doi.org/10.5812/ircmj.6499

Chhabra, G., Singh, C. K., Ndiaye, M. A., Fedorowicz, S., Molot, A., & Ahmad, N. (2018).
Prostate cancer chemoprevention by natural agents: Clinical evidence and potential
implications. Cancer letters, 422, 9–18. https://doi.org/10.1016/j.canlet.2018.02.025

Culp, M. B., Soerjomataram, I., Efstathiou, J. A., Bray, F., & Jemal, A. (2019). Recent Global
Patterns in Prostate Cancer Incidence and Mortality Rates. European
Urology. doi:10.1016/j.eururo.2019.08.005

Donovan, J. L., Hamdy, F. C., Lane, J. A., Mason, M., Metcalfe, C., Walsh, E., … Neal, D. E.
(2016). Patient-reported outcomes after monitoring, surgery, or radiotherapy for

21 of 26
 prostate cancer. New England Journal of Medicine, 375(15), 1425–1437.
https://doi.org/10.1056/nejmoa1606221

Ha Chung, B., Horie, S., & Chiong, E. (2019). The incidence, mortality, and risk factors of
prostate cancer in Asian men. Prostate international, 7(1), 1–8.
https://doi.org/10.1016/j.prnil.2018.11.001.

Hedestig, O., Sandman, P., Tomic, R., & Widmark, A. (2005). Living after radical
prostatectomy for localized prostate cancer. A qualitative analysis of patient narratives.
Acta Oncologica, 44(7), 679-686. https://doi.org/10.1

Hercberg, S., Preziosi, P., Briançon, S., Galan, P., Triol, I., Malvy, D., Roussel, A. M., &
Favier, A. (1998). A primary prevention trial using nutritional doses of antioxidant
vitamins and minerals in cardiovascular diseases and cancers in a general population:
the SU.VI.MAX study--design, methods, and participant characteristics.
SUpplementation en VItamines et Minéraux AntioXydants. Controlled clinical
trials, 19(4), 336–351. https://doi.org/10.1016/s0197-2456(98)00015-4

Ito, K. (2014). Prostate cancer in Asian men. Nature Reviews Urology, 11(4), 197–
212. doi:10.1038/nrurol.2014.42

Kim, E. H., Brockman, J. A., & Andriole, G. L. (2018). The use of 5-alpha reductase inhibitors
in the treatment of benign prostatic hyperplasia. Asian journal of urology, 5(1), 28–32.
https://doi.org/10.1016/j.ajur.2017.11.005

Kimura T. (2012). East meets West: ethnic differences in prostate cancer epidemiology
between East Asians and Caucasians. Chinese journal of cancer, 31(9), 421–429.
https://doi.org/10.5732/cjc.011.10324

Kundu, N., Campbell, P., Hampton, B., Lin, C.-Y., Ma, X., Ambulos, N., … Fulton, A. M.
(2012). Antimetastatic activity isolated from Colocasia esculenta (taro). Anti-Cancer
Drugs, 23(2), 200–211. https://doi.org/10.1097/cad.0b013e32834b85e8

Lippman, S. M., Goodman, P. J., Klein, E. A., Parnes, H. L., Thompson, I. M., Jr, Kristal, A.
R., Santella, R. M., Probstfield, J. L., Moinpour, C. M., Albanes, D., Taylor, P. R.,
Minasian, L. M., Hoque, A., Thomas, S. M., Crowley, J. J., Gaziano, J. M., Stanford,
J. L., Cook, E. D., Fleshner, N. E., Lieber, M. M., … Coltman, C. A., Jr (2005).
Designing the Selenium and Vitamin E Cancer Prevention Trial (SELECT). Journal of
the National Cancer Institute, 97(2), 94–102. https://doi.org/10.1093/jnci/dji009

Lippman, S. M., Klein, E. A., Goodman, P. J., Lucia, M. S., Thompson, I. M., Ford, L. G.,
Parnes, H. L., Minasian, L. M., Gaziano, J. M., Hartline, J. A., Parsons, J. K., Bearden,
J. D., 3rd, Crawford, E. D., Goodman, G. E., Claudio, J., Winquist, E., Cook, E. D.,
Karp, D. D., Walther, P., Lieber, M. M., … Coltman, C. A., Jr (2009). Effect of
selenium and vitamin E on risk of prostate cancer and other cancers: The Selenium and
Vitamin E Cancer Prevention Trial (SELECT). JAMA, 301(1), 39–51.
https://doi.org/10.1001/jama.2008.864

22 of 26
Mehta, R., Murillo, G., Naithani, R., & Peng, X. (2010). Cancer Chemoprevention by Natural
Products: How Far Have We Come? Pharmaceutical Research, 27(6), 950-961.
https://doi.org/10.1007/s11095-010-0085-y

Mokbel, K., Wazir, U., & Mokbel, K. (2019). Chemoprevention of Prostate Cancer by Natural
Agents: Evidence from Molecular and Epidemiological Studies. Anticancer research,
39(10), 5231–5259. https://doi.org/10.21873/anticanres.13720

Mukhtar, H., & Ahmad, N. (2000). Tea polyphenols: prevention of cancer and optimizing
health. The American Journal of Clinical Nutrition, 71(6).
https://doi.org/10.1093/ajcn/71.6.1698s

Nevedomskaya, E., Baumgart, S. J., & Haendler, B. (2018). Recent Advances in Prostate
Cancer Treatment and Drug Discovery. International journal of molecular
sciences, 19(5), 1359. https://doi.org/10.3390/ijms19051359

Ozten-Kandaş, N., & Bosland, M. C. (2011). Chemoprevention of prostate cancer: Natural

compounds, antiandrogens, and antioxidants - In vivo evidence. Journal of
carcinogenesis, 10, 27. https://doi.org/10.4103/1477-3163.90438

Radical Prostatectomy. Johns Hopkins Medicine. (n. d.).

https://www.hopkinsmedicine.org/health/treatment-tests-and-therapies/radical-
prostatectomy.

Rashmi D.R., Raghu N, Gopenath T.S, Pradeep P., Pugazhandhi B., Murugesan K., Ashok G.,
Ranjith M.S., Chandrashekrappa G.K., Kanthesh M.B. (2018). Taro (Colocasia
esculenta): An overview. Journal of Medicinal Plants Study, 6(4), 56-161

Resnick, M., Koyama, T., Fan, K., Albertsen, P., Goodman, M., & Hamilton, A. et al. (2013).
Long-Term Functional Outcomes after Treatment for Localized Prostate Cancer. New
England Journal of Medicine, 368(5), 436-445.
https://doi.org/10.1056/nejmoa1209978

Shoag, J., & Barbieri, C. E. (2016). Clinical variability and molecular heterogeneity in prostate
cancer. Asian journal of andrology, 18(4), 543–548. https://doi.org/10.4103/1008-
682X.178852

Shrader-Bogen, C., Kjellberg, J., McPherson, C., & Murray, C. (1997). Quality of life and
treatment outcomes. Cancer, 79(10), 1977-1986. https://doi.org/10.1002/(sici)1097-
0142(19970515)79:103.0.co;2-r

Skvortsov, S., Skvortsova, I. I., Tang, D. G., & Dubrovska, A. (2018). Concise Review:
Prostate Cancer Stem Cells: Current Understanding. Stem cells (Dayton, Ohio), 36(10),
1457–1474. https://doi.org/10.1002/stem.2859

Sung, H., Ferlay, J., Siegel, R. L., Laversanne, M., Soerjomataram, I., Jemal, A., & Bray, F.
(2021). Global cancer statistics 2020: GLOBOCAN estimates of incidence and
mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for
Clinicians. https://doi.org/10.3322/caac.21660\

23 of 26
Syed, D. N., Khan, N., Afaq, F., & Mukhtar, H. (2007). Chemoprevention of Prostate Cancer
through Dietary Agents: Progress and Promise. Cancer Epidemiology Biomarkers &
Prevention, 16(11), 2193–2203. doi: 10.1158/1055-9965.epi-06-0942

Temesgen, M., & Retta, N. (2015). Nutritional potential, health and food security benefits of
taro Colocasia esculenta (L.): A review. Food Science and Quality Management, 36,
23-30.

The alpha-tocopherol, beta-carotene lung cancer prevention study: design, methods, participant
characteristics, and compliance. The ATBC Cancer Prevention Study Group.
(1994). Annals of epidemiology, 4(1), 1–10. https://doi.org/10.1016/1047-
2797(94)90036-1

Van Poppel, H., & Tombal, B. (2011). Chemoprevention of prostate cancer with nutrients and
supplements. Cancer management and research, 3, 91–100.
https://doi.org/10.2147/CMR.S18503

Yan, Z., Zhong, Y., Duan, Y., Chen, Q., & Li, F. (2020, January 23). Antioxidant
mechanism of tea polyphenols and its impact on health benefits. Animal Nutrition.
https://www.sciencedirect.com/science/article/pii/S2405654520300032.

Chhabra, G., Singh, C. K., Ndiaye, M. A., Fedorowicz, S., Molot, A., & Ahmad, N. (2018).
Prostate cancer chemoprevention by natural agents: Clinical evidence and potential
implications. Cancer letters, 422, 9–18. https://doi.org/10.1016/j.canlet.2018.02.025

Katnoria J.K., Savita, Kaur A., Bakshi A., Nagpal A.K. (2020) Cancer Chemoprevention by
Natural Plant Products and Their Derivatives: Clinical Trials. In: Kumar M., Sharma
A., Kumar P. (eds) Pharmacotherapeutic Botanicals for Cancer Chemoprevention.
Springer, Singapore. https://doi.org/10.1007/978-981-15-5999-0_13

Kundu, N., Campbell, P., Hampton, B., Lin, C. Y., Ma, X., Ambulos, N., Zhao, X. F.,
Goloubeva, O., Holt, D., & Fulton, A. M. (2012). Antimetastatic activity isolated from
Colocasia esculenta (taro). Anti-cancer drugs, 23(2), 200–211.
https://doi.org/10.1097/CAD.0b013e32834b85e8

Mukherjee, P. K. (2019). Bioassay-Guided Isolation and Evaluation of Herbal Drugs. Quality

Control and Evaluation of Herbal Drugs, 515–537. https://doi.org/10.1016/b978-0-12-
813374-3.00013-2

Ramawat, K., & Goyal, S. (2008). Natural Products in Cancer Chemoprevention and
Chemotherapy [Abstract]. Herbal Drugs: Ethnomedicine to Modern Medicine, 153-
171. https://doi.org/10.1007/978-3-540-79116-4_10

Karthikeyan, Murugesan & Gnanasekaran, Ashok & Dr, Rashmi & Ts, Gopenath &
Palanisamy, Pradeep & Gk, Chandrashekrappa & Basalingappa, Kanthesh & Bm,
Kanthesh. (2018). Taro (Colocasia esculenta): an overview. Journal of Medicinal Plants
Studies, 6(4), 156-161.

Rawla P. (2019). Epidemiology of Prostate Cancer. World journal of oncology, 10(2), 63– 89.
https://doi.org/10.14740/wjon1191

24 of 26
Stan, S.D., Kar, S., Stoner, G.D. and Singh, S.V. (2008), Bioactive food components and cancer
risk reduction. J. Cell. Biochem., 104, 339-356. https://doi.org/10.1002/jcb.21623

Tsao, A. S., Kim, E. S., & Hong, W. K. (2004). Chemoprevention of cancer. CA: a cancer
journal for clinicians, 54(3), 150-180

Chakraborty, P., Deb, P., Chakraborty, S., Chatterjee , B., & Abraham, J. (2012). Cytotoxicity
and antimicrobial activity of Colocasia esculenta. Journal of Chemical and
Pharmaceutical Research, 2015. https://www.jocpr.com/articles/cytotoxicity-and-
antimicrobial-activity-of-colocasia-esculenta.pdf.

Ajayi, G. O., Olagunju, J., Ademuyiwa, O., & Olubukola, C. M. (2011, May). Gas
chromatography-mass spectrometry analysis and phytochemical screening of ethanolic
root extract of Plumbago zeylanica, LinnGas chromatography-mass spectrometry
analysis and phytochemical screening of ethanolic root extract of Plumbago zeylanica,
Linn. Gas chromatography-mass spectrometry analysis and phytochemical screening of
ethanolic root extract of Plumbago zeylanica, Linn.
https://www.researchgate.net/publication/277009746_Gas_chromatography-
mass_spectrometry_analysis_and_phytochemical_screening_of_ethanolic_root_extract
_of_Plumbago_zeylanica_Linn.

Evans W C (1996). Trease and Evans Pharmacognosy, 14th Edition, Bailiere Tindal W.B.
Sauders company ltd; London, pp. 224-228, 293-309, 542-575

Kumar P, Nagarajan A, Uchil PD. Analysis of Cell Viability by the MTT Assay. Cold Spring
Harb Protoc. 2018 Jun 1;2018(6). doi: 10.1101/pdb.prot095505. PMID: 29858338.

Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R.L., Torre, L.A. and Jemal, A. (2018), Global
cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide
for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians, 68: 394-424.
https://doi.org/10.3322/caac.21492

Ribeiro Pereira, P., Bertozzi de Aquino Mattos, É., Nitzsche Teixeira Fernandes Corrêa, A.
C., Afonso Vericimo, M., & Margaret Flosi Paschoalin, V. (2020). Anticancer and
Immunomodulatory Benefits of Taro (Colocasia esculenta) Corms, an Underexploited
Tuber Crop. International Journal of Molecular Sciences, 22(1), 265.
https://doi.org/10.3390/ijms22010265

Pawar, H. A., Choudhar, P.D., & Kama, S. R. (2018). (PDF) Anticancer and
Immunomodulatory Benefits of Taro ... Medicinal & Aromatic Plants.
https://www.researchgate.net/publication/347989714_Anticancer_and_Immunomodulat
ory_Benefits_of_Taro_Colocasia_esculenta_Corms_an_Underexploited_Tuber_Crop.

B, P., & Armis, R. (2020, June 2). Update in prostate cancer: overview and
diagnosis. Medicina & Laboratorio. https://www.medigraphic.com/cgi-
bin/new/resumenI.cgi?IDARTICULO=93549.

25 of 26
Simsek, S., & Nehir El, S. (2015). In vitro starch digestibility, estimated glycemic index and
antioxidant potential of taro (Colocasia esculenta L. Schott) corm. Food chemistry, 168,
257–261. https://doi.org/10.1016/j.foodchem.2014.07.052.

Foster-Powell, K., Holt, S. H., & Brand-Miller, J. C. (2002). International table of glycemic
index and glycemic load values: 2002. The American journal of clinical nutrition,
76(1), 5–56. https://doi.org/10.1093/ajcn/76.1.5

26 of 26