Preliminary Identification of Secondary Metabolites through Phytochemical

Screening of Hylocereus polyrhizus (Dragon Fruit) Peels and

Comparative Evaluation Against Other Studies

By:

GEMMALYN H. BAUTISTA
 Abstract

Green chemistry calls for further ventures of identifying compounds in the environment
which can be useful to humans and do not leave harmful footprints that can harm the people and
the environment. Thus, this investigatory project on preliminary identification of secondary
metabolites through phytochemical screening of Hylocereus polyrhizus (dragon fruit) peels and
comparative evaluation against other studies was conducted. It used a descriptive-comparative
research design to qualitatively identify the presence of secondary metabolites and compare the
results to other studies. The phytochemical screening was conducted at the Center for Natural
Sciences at Saint Mary’s University following the protocols of Guevara (2005) using Thin Layer
Chromatography. The peels of H. polyrhizus was found to contain essential oils, steroids, fatty
acids, sugars, anthaquinones, coumarins, phenols, alkaloids and flavonoids. Also, the results of
this study has major differences with other studies but the studies consistently identified
flavonoids to be present in two varieties of dragon fruits (H. polyrhizus and H. undatus). It is
then recommended to explore more on the properties of dragon fruits such as its antioxidant,
antibacterial or cytotoxic properties.
Keywords: flavonoids, green chemistry, plant compounds, Thin Layer Chromatography

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 Table of Contents

Title Page............................................................................................................................. 1

Abstract................................................................................................................................ 2

CHAPTER 1 THE PROBLEM AND ITS BACKGROUND................................. 4

Rationale........................................................................................... 4

Statement of the Objectives............................................................... 6

Conceptual Framework..................................................................... 6

Significance of the Study.................................................................. 8

Scope and Delimitation..................................................................... 8

Definition of Terms........................................................................... 9

CHAPTER 2 REVIEW OF RELATED LITERATURE AND STUDIES............. 10

Green Chemistry............................................................................... 10

Dragon Fruit...................................................................................... 13

Plant Secondary Metabolites and Phytochemical Screening............ 14

Related Studies.................................................................................. 16

Synthesis............................................................................................ 19

CHAPTER 3 RESEARCH METHODOLOGY...................................................... 20

Research Design................................................................................ 20

Research Environment...................................................................... 20

Research Instruments........................................................................ 21

Data Gathering Procedures............................................................... 23

Data Analysis.................................................................................... 24

CHAPTER 4 RESULTS AND DISCUSSION....................................................... 25

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 Section 1: Phytochemical Screening................................................. 25

Section 2: Comparative Evaluation................................................... 27

CHAPTER 5 SUMMARY, CONCLUSION AND RECOMMENDATION......... 29

Summary............................................................................................ 29

Summary of Findings........................................................................ 29

Conclusion......................................................................................... 30

Recommendations.............................................................................. 30

References............................................................................................................................. 32

4
 Chapter 1

THE PROBLEM AND ITS BACKGROUND

Rationale

The world has been experiencing the hazardous effects of synthetic compounds that

humans produce, and these compounds contaminate the food we consume and the products we

use. For this reason, chemists started to venture on Green Chemistry to design chemical products

and processes which can reduce or eliminate the utilization and generation of hazardous

substances. As part of pollution prevention, chemists nowadays also try to reduce such sources of

pollutions from synthetic materials. One of the principles of green chemistry is that chemical

products and processes must be carefully designed such that at the end of their functions, they

will not persist in the environment and eventually break down into harmless degradation

products (Kirchhoff, 2013). These things are potentially found in the secondary metabolites of

plants which are organic molecules that can be used as food additives, flavor, pharmaceuticals,

and other industrial materials.

With the emergence of green chemistry, there has been an increasing interest in searching

for more environment-friendly materials to be used in laboratories and for healthier sources of

compounds necessary for better human growth and development. This search includes the

identification of polyphenolic compounds which are excellent antioxidant and bioactive free

radical scavengers which play significant roles in human health (Cheah, Eid, Aziz, Ariffin,

Elmahjoubi & Elmarzugi, 2016). Antioxidants are natural or man-made substances that can

protect human cell from being damaged, and these substances can be found in many foods

including fruits and vegetables. There has been many evidences that fruits are rich sources of

5
antioxidants (Sekar, et al, 2016). According to Nurul and Asmah (2014), fruits and vegetables

contains in them a large number of antioxidants that protect the human body from chronic

diseases. These compounds are found to be abundant in tropical fruits. These phenolic and

flavonoid compounds in plants which include antioxidants, antibacterial, and hepatoprotectors

are produced as defense mechanism against stressful environment conditions. One plant which is

a potential source of these compounds is the dragon fruit. Dragon fruit is considered rich in

vitamins, fiber, phosphorus, calcium, magnesium, phytochemical and antioxidants (Romero,

Waing & Valentino, 2017).

The dragon fruit from the Cactacae family comes in three types or varieties: Hylocereus

undatus which has white flesh with pink skin; Hylocereus polyrhizus which has red flesh with

pink/red skin; and Selenicereus megalanthus which has white flesh with yellow skin. Based on

observation, it is the red variety which is very much available in the Philippines. Dragon fruit is a

perennial climbing cactus with a triangular fleshy stems with thorns. Their phytochemical

properties have been explored by several studies through laboratory procedures however,

according to Chaiyasut, et al (2016) in their study exploring phytochemical properties of widely

used fruits, some phytochemical compounds are influenced by geographical location and climate

conditions, and even cultivation techniques, including, according to Nurul and Asmah (2014),

water availability, and transport, availability and storage. In the Philippines, phytochemical

properties of the dragon have not been explored yet. This is important to understand further

similarities or differences of the chemical constituents of dragon fruit in the Philippines

compared to other countries.

Hence, this study sought to evaluate the red pitaya or commonly known as dragon fruit,

available in the province on its phytochemical properties, and eventually compare the results of

6
this study to the results of previously conducted studies to identify similarities and differences,

not only on laboratory procedures used, but also on the identified phytochemicals. This study

used the peels because according to Romero, Waing and Valentino (2017), it is the part of the

plant which is underused, not given attention and rarely explored.

Statement of the Objectives

This study primarily sought to characterize using phytochemical screening Hylocereus

polyrhizus (red dragon fruit).

Specifically, it aimed to give light to the following objectives:

1. To identify the phytochemical compounds present in the peels of Hylocereus polyrhizus

(red dragon fruit); and

2. To compare and evaluate similarities and/or differences from the phytochemical

compounds found in this study and in other varieties of dragon fruit in other countries.

Conceptual Framework

Plants have been a source of many organic materials in our households – medicines, food

additives, coloring, ornaments and others. The values of these plants lie on their physical and

chemical properties. In chemistry, plants have been sources of many compounds that help

develop organic products in pharmaceutical studies and in laboratory procedures. Their active

chemical substances such as phenolic compounds are synthesized as secondary metabolites to

respond in stress conditions, wounding and UV radiations. They also contain flavonoids,

stilbenes, hydrolysable, and tannins, lignins and lignans (Anokwuru, Anyasor, Ajibaye, Fakoya

& Okebugwu, 2011).

7
 Flavonoids are the colored compounds in plants. These are soluble in water and alcohol,

and can be extracted by chopping and macerating plant parts. There are also other organic

compounds in plant parts that give colors such as flavonols, acylated flavonoids, anthocyanins,

glucosylated acylated anthocyanin, quinines, imines, polymethines, napthaquinones,

anthraquinonoids, indigoids and dihydropyrans diarylmethanes carotene (Thote, 2015). The

framework below summarizes the concepts and variables involved in the study:

INPUT PROCESS OUTPUT

- Crude Peel
Hylocereus Extraction
- Phytochemical
polyrhizus (red - Phytochemical
Properties
dragon fruit) Screening
- Comparative Evaluation
peels - Document
Analysis

Figure 1. Research Paradigm

Based on Figure 1, the study tried to explore on the phytochemical properties of crude

peel extracts of Hylocereus polyrhizus (red dragon fruit) through phytochemical screening. There

will also be a comparative evaluation of the results of this study to the results of other

phytochemical screenings of dragon fruit from other studies through analyzing published

researches. Hence, in this research, the framework seemed to be supported by the concept of the

properties of plants on their chemical compositions.

8
Significance of the Study

This study is deemed to be beneficial to the following:

Chemists. The results of this study could open new avenues for exploring the

phytochemical properties of Hylocereus polyrhizus (dragon fruit) peel extracts. Also, the product

of this study will enable them to advocate further use of Green Chemistry in their respective

laboratories based on the compounds identified.

Science Teachers. They can use the results of the study as an instructional material on

demonstrating benefits of local fruits and vegetables. This could also serve as a direction in

studying other compositions of dragon fruit through other laboratory tests.

Students. This study could serve as their basis on further understanding phytochemical

compounds in plants or organic materials.

Future Researchers. This study can open other opportunities in exploring other sources

of organic compounds that can be used in other areas such as acid-base indicators.

Scope and Delimitation

This study is mainly focused on the analysis of the contents of Hylocereus polyrhizus

(dragon fruit) by identifying the active compounds of the extracts obtained from the peels

through phytochemical screening The study also includes evaluation of the similarities and

differences on the results of this study to the results of other previously conducted studies.

The study did not explore on antimicrobial, antioxidant or any medicinal properties using

true and controlled experiments with living organisms, and other laboratory tests not within the

researcher’s capability. Furthermore, the selection of the fruit was based on a single variety, red

peel and reddish-purplish flesh, obtained from one farm only.

9
Definition of Terms

The following terms in this study were conceptually and operationally defined for better

understanding:

Comparative Evaluation refers to the technique of comparing a set of data against a set

of criteria of descriptions. In this study, it was described as done by comparing the results of this

study against the results of previously conducted studies on red dragon fruit.

Dragon Fruit refers to a fruit under the family of cactus plants with soft peel and soft

flesh usually cultivated in tropical regions of the world. In this study, the red dragon fruit

(Hylocereus polyrhizus) was characterized.

Phenolic Compounds, also called phenolics or polyphenolics, refer to more than 8,000

ocmpounds found in plants which possess at least one aromatic ring with one or more hydroxyl-

substituents. These include esters, methyl ethers and glycosides (Bhuyan & Basu (2017). In this

study, only the phenolic compounds which can be identified the set of tests were included.

Phytochemical Screening refers to the technique used to determine the active chemical

constituents of the plant material (Matias, 2010). The technique will determine the secondary

plant metabolites to characterized the obtained samples from the peels of red dragon fruit.

10
 Chapter 2

REVIEW OF RELATED LITERATURE AND STUDIES

This chapter presents a discussion of literature of the concepts and/or variables involved

in the study to enlighten the readers. Connections of the present study to related studies are also

provided to establish any gap that supports this research to be feasible.

Green Chemistry

Green chemistry, also known as sustainable chemistry, is the designing of chemical

products and processes that help eliminate or reduce the utilization and generation of hazardous

chemical substances. It is applied across the life cycle of chemical products which includes the

design, manufacture, use, and ultimate disposal. This philosophy prevents pollution, by any

means, at the molecular level by applying innovations on scientific solutions to the real-world

environmental problems we are facing. This approach also reduces negative impacts of the

utilization and generation of chemical products and processes on human health and the

environment (United States Environmental Protection Agency, 2017).

Green chemistry has 12 principles established by the American Chemical Society which

have been serving as major guiding principles for scientific researches to design synthesis routes

toward a particular product or process. The 12 principles can be summarized into reduction of

certain factors such as hazard, waste, energy, risk, environmental impact and cost (Torres &

Promentilla, 2015). The principles are as follows:

1. Prevent waste – designing chemical syntheses to prevent waste, and leave no waste to

treat or clean up.

11
2. Maximize atom economy – designing syntheses by which the final product will contain

the maximum proportion of the raw (starting) materials; wasting few or no atoms at all.

3. Design less hazardous chemical syntheses – designing synthesis that will use and

generate chemical substances with little or no toxicity to humans and the environment.

4. Design safer chemicals and products – designing chemical substances that are fully

effective yet have little or no toxicity at all.

5. Use safer solvents and reactions conditions – avoiding the use of solvents, separation

agents and other auxiliary chemicals which are harmful and pose threats to humans and

the environment.

6. Increase energy efficiency – running chemical reactions at room temperature and

atmospheric pressure as much as and whenever possible.

7. Use renewable feedstocks – utilizing starting materials (also known as feedstocks) that

are renewable (often found in agriculture products or wastes of other processes) rather

than depletable (often found in fossil fuels or mining operations).

8. Avoid chemical derivatives – avoiding the use of blocking or protecting groups, or any

temporary modifications, if possible. Derivatives use additional reagents and generate

chemical wastes.

9. Use catalysts, not stoichiometric reagents – minimizing wastes by using catalytic

reactions (catalysts are effective even in small amounts and can carry out a single

reaction many times).

10. Design chemicals and products to degrade after use – designing chemical products that

will break down to harmless substances after use to prevent accumulation in the

environment.

12
 11. Analyze in real time to prevent pollution – real-time monitoring and control (including

in-process) during chemical syntheses minimizes or eliminates formation of harmful by-

products.

12. Minimize the potential for accidents – designing chemicals and their physical states

(solid, liquid or gas) to minimize the potential for chemical accidents in laboratories

including explosions, fires and releases to the environment.

Generally speaking, green chemistry is achieved by designing efficient, effective and

environmentally benign chemical substances and processes. According to Paul Anastas (n.d.),

the Director of the Green Chemistry Institute at the American Chemical Society, it is a rapidly

expanding field in sciences to answer problems on environmental threats which can offer

limitless possibilities to continuously improve chemicals and chemical processes. In 2005, Ryoji

Noyori, a Nobel Prize awardee in chemistry for 2001, had identified three key developments in

green chemistry: a) use of supercritical carbon dioxide as a green solvent, b) aqueous hydrogen

peroxide for clean oxidations, and c) the use of hydrogen in asymmetric synthesis. The Nobel

Prize in 2005 was awarded based on several advances in green chemistry. Synthesis methods

were developed that are more efficient, simpler to use and environmentally friendlier.

Nowadays, many students are profoundly interested in the sustainability of the Earth’s

resources and the planet itself. They have grown public concern on global warming and

greenhouse gases which resulted to their wanting to understand how human actions could affect

their health and the health of the environment. To answer such wanting, students have already

ventured into green chemistry as a mainstream practice supported by the academe, industry and

government (Hjeresen, Schutt & Boese, 2000).

13
Dragon Fruit

Gunasena, Pushpakumara and Kariyawasam (2007) comprehensively described and

studied dragon fruit. The content of this part of the literature largely came for their descriptions.

Dragon fruit is a climbing vine cactus species which has received worldwide recognition, first, as

an ornamental plant and then as a fruit crop. Its fruit is considered as the most beautiful in the

Cactacae family usually with a bright red skin studded with green scales, and white or red flesh

with tiny black seeds. Its flower was named “Noble Woman” or “Queen of the Night” for it

possesses beauty that stands among others. This crop is well established in China, Australia,

Malaysia, Israel, Taiwan, Nicaragua, Vietnam and the Philippines. The table below summarizes

the common or vernacular names of dragon fruit:

Table 1
Common and Vernacular Names of Dragon Fruit in Other Countries or Languages
Country/Language Common/Vernacular Name
Chinese Zunlongguo
Colombian Pitahaya Roja/Blanca, Flor de Calis, Pitajaya
English Strawberry Pear, Dragon Fruit, Rep Pitaya, Red Pitahaya,
Night Blooming Cereus, Belle of the Night, Conderella
Plant, Queen of the Night
Hawaii Paniniokapunahou, Papipi Pua, Panani O Ka
Indonesian Buah Naga
Israel Pitaya
Mexico Junco, Flor de Calis, Pitajava, Pithaya Roja, Tasajo
Portuguese Cato-barse, Cardo-ananas
Vietnam Dragon Fruit, Thanh Long
Spanish Chaca, Chak-wob, Flor de Caliz, Junco Tapatio, Pitahaya,
Pithaya Orejona, Tuna, Nopal, Pitjaya, Reina de la Noche,
Zacamb
Sri Lanka Dragon Fruit

This fruit or plant is nutritious with a variety of uses, although the plant parts have not

been extensively studied yet, particularly on the chemical components. The fruit is the most

14
edible part of the plant and its fruit pulp constitutes about 70-80% of the ripe fruit. The flavor of

the fruit can be likened to the taste of the kiwi fruit. The fruit can be processed or included into

other products such as juice, jam, ice cream, jelly and even candies.

The fruit also possesses medicinal properties with its richness in antioxidants to prevent

certain types of cancer and even diabetes. It also neutralizes toxic substances such as heavy

metals in the body, reduces cholesterol and lowers high blood pressure. In diabetes, it controls

blood sugar levels and promotes better dental health (Romero, Waing & Valentino, 2017).

Plant Secondary Metabolites and Phytochemical Screening

Phytochemical are the chemical that are naturally present in plants. These phytochemicals

are beneficial to humans as they become more useful in medicine with vital roles in combating

diseases or illnesses such as asthma, arthritis, cancer and diabetes (Banu & Cathrine, 2015).

Phytochemicals are also called plant secondary metabolites. Scientific sources described these

compounds whose major role is not for normal growth, development and reproduction, but for

defense purposes to protect the plant from external harm. These compounds are by-products of

reactions among the primary metabolites. Secondary metabolites may often be created by

modified synthetic pathways from primary metabolite, or share substrates of primary metabolite

origin. According to Tiwari and Rana (2015), unlike primary metabolites, absence of secondary

metabolites does not directly result to the death of the plant, but rather, it can cause long-term

impairment of an important function for the organism’s survival.

In human life, these compounds are used as medicines, flavorings, or relaxing drugs,

especially the essential oils which can be obtained from it. It is stated that these secondary

metabolites are classified into three major groups: terpenoids, alkaloids and phenolics.

15
Terpenoids include steroids, carotenoids and gibberelic acid. Alkaloids contains the nitrogen

atom and some of its members are atropine, codeine, morphine, nicotine, solanine and tomatine.

For phenolics, these include tannins, glycosides, saponins and flavonoids (Kabera, Semana,

Mussa & He, 2014).

The secondary metabolites or phytochemical are obtained from the plants through

phytochemical screening. It refers to the extraction, screening and identification of bioactive

chemical compounds found in plants. This procedure starts with collection of specimen and the

drying stage. Extraction methods follow which is scientifically done to separate the active

portions of the plant from the inactive portions. During extraction, different solvents can be used

such as distilled water, ethanol or methanol. These standardized procedures aims to

therapeutically obtain the desired portions and eliminate unwanted material to carefully and

correctly identify the needed phytochemicals. The basic parameters that can affect the quality of

extraction are the quality of the plant part, the solvent for extraction, and lastly, the extraction

procedure. In phytochemical screening, water, acetone, alcohol, chloroform and ether are

commonly used as solvents. The table below summarizes what solvents to use to purposively

identify active components of a plant material:

Table 2
Potentially Identified Secondary Metabolites in Different Extraction Solvents
Water Ethanol Methanol Chloroform Ether Acetone
Anthocyanins Anthocyanins
Starches
Tannins Tannins Tannins
Saponins Saponins
Terpenoids Terpenoids Terpenoids Terpenoids Terpenoids
Polypeptides
Lectins
Polyphenols Polyphenols
Polyacetylenes
Flavonols Flavonols
Sterols

16
 Water Ethanol Methanol Chloroform Ether Acetone
Alkaloids Alkaloids
Xanthoxylines
Totarol
Quassinoids
Lactones
Flavones
Phenones
Flavonoids
Coumarins
Fatty Acids
Phenols

Phytochemical screening can involve different tests based on standard methods:

Table 3
Different Test Procedures to Identify Secondary Metabolites
Secondary Metabolites Test Procedure
ALKALOIDS Mayer’s Test
Wagner’s Test
Dragendorff’s Test
Hager’s Test
CARBOHYDRATES Molisch’s Test
Benedict’s Test
Fehling’s Test
GLYCOSIDES Modified Borntrager’s Test
Legal’s Test
SAPONINS Froth Test
Foam Test
PHYTOSTEROLS Salkowski’s Test
Libermann Burchard’ Test
PHENOLS Ferric Chloride Test
TANNINS Gelatin Test
FLAVONOIDS Alkaline Reagent Test
Lead Acetate Test
PROTEINS and AMINO AIDS Xanthoproteic Test
Ninhydrin Test
DITERPENES Copper Acetate Test

Related Studies

17
 Kanchana, Devi, Iatha and Spurthi (2018) conducted a phytochemical evaluation of

Hylocereus undatus using the ethanolic extracts carried out using standard methods. The

screening revealed the presence of carbohydrates, saponins, flavonoids, tannins and phenolic

compounds, glycosides, oils, alkaloids, proteins and amino acids, terpenoids, and steroids. These

results were considered as significant anti-Parkinson’s and laxative activity of the fruit.

In 2017, Romero, Waing and Valentino conducted a study on the phytochemical

screening and bioassay of the antibacterial activity of Hylocereus undatus and Hylocereus

polyrhicus fruit peel and identified that the fruit peels contain terpenoids and cardiac glycosides,

including saponins and steroids in the ethanol extracts. More phytochemicals were found when

alcohol is used as solvent.

Manihuruk, Suryati and Arief (2017) also conducted a study on the effectiveness of red

dragon fruit peel extract as a colorant, antimicrobial and antioxidant in beef sausage. Their

phytochemical screening using distilled water revealed the presence of phenol hydroquinone,

flavonoids, triterpenoids, steroids, saponin and tannins.

Fidrianny, Ilham and Hartati (2017) also revealed in their study about the super red

dragon fruit (Hylocereus costaricensis) from West Java, Indonesia that this fruit contains gallic

acid, flavonoids, tannins and phenolic acids. Also, it has betalain and anthocyanin. These

compounds were attributed to the antioxidant profile of the fruit where phenolic compounds

found in the flesh of super red dragon fruit were the major contributors to its antioxidant

activities.

Cheah, Eid, Aziz, Ariffin, Elmahjoubi and Elmarzugi (2016) revealed in the study of the

phytochemical properties and health benefits of Hylocereus undatus. They used content analysis

18
of previous researches to gather the necessary data and they reported that this dragon fruit

contains betalains, gallic acid and betacyanins. Ascorbic acid was also found to be present in the

dragon fruit, including dehydroascorbic acid and its intermediate monodehydroascorbic acid

which are all essential in the human immune system to fight against diseases.

Chaiyasut, et al (2016) explored on the anthocyanin profiles and antioxidant activities of

fruits, vegetables and flowers in Thailand. Anthocyanins are considered as water-soluble

pigments most commonly present in fruits, vegetables and flowers. They have antioxidant, anti-

inflammatory and antibacterial properties. The study then found out that ma-kiang extract

showed high cyaniding 3-glucoside content and antioxidant property. Further, the tests showed

that anthocyanin content vary at different climates and geographical locations.

Sekar, et al (2016) compare the properties of methanolic extracts of red and white

dragoin fruits. Both fruits contain the following phytochemicals: carbohydrates, proteins and

amino acids, steroids and sterols, glycosides, flavonoids, and fixed oils. The results further

showed that the methanolic extracts of the red dragon fruits showed significantly higher and

better antioxidant activity than its white counterpart. Thus, the red dragon fruit is recommended

for further investigation and characterization.

The variability in nutritional composition including the phytochemical properties of red

pitaya (dragon fruit) from Australia and Malaysia were studied by Nurul and Asmah (2014). The

work determined optimum ethanol concentration to extract the phenolic, flavonoids and

betacyanin contents of the dragon fruit. Carbohydrate was found to be the predominant

macronutrient while potassium for the major mineral and vitamin A for the vitamin content. It

was also found out that the phytochemical contents of the dragon fruit from Malaysia and

19
Australia significantly differs. This is suggested by environmental factors like soil and climate

that could affect chemical compositions of organic matters.

Susanti, Utomo, Syukin and Redjeki (2012) conducted a phytochemical screening on the

white dragon fruit using the crude extract. The screening showed the presence of triterpenoid,

alkaloid, flavonoid, saponin, and showed the absence of sterol and tannin.

Synthesis

In a nutshell, several studies have been conducted already to characterize the red dragon

fruit. Other studies explored on their chemical compounds and visual appearance through several

laboratory techniques such as UV/visible spectroscopy and Fourier transform infrared

spectroscopy. However, very few studies have tried to verify the phytochemical compounds

responsible for the capabilities of these extracts to be of greater use in medicine or any field.

Thus, this study adopts some of the ideas of the previous studies conducted by using the

peel of Hylocereus polyrhizus (dragon fruit) and the basic experimental procedures of

determining the set up using the extract prepared. The extract will also go through phytochemical

screening to characterize the plant compounds responsible for its potentials.

20
 Chapter 3

RESEARCH METHODOLOGY

Research Design

This investigatory project used a descriptive-comparative research design. This design

works when the research considered two variables and describe them side-by-side, whether

qualitatively or quantitatively. This study qualitatively described the phytochemical compounds

found in the peel extracts and compared the identified plant secondary metabolites to the results

of previously conducted studies.

Research Environment

The Hylocereus polyrhizus (red dragon fruit) was collected from a dragon fruit stand in

Diadi, Nueva Vizcaya whose supply of dragon fruits come from one farm only. Dragon fruit

farming in Nueva Vizcaya is becoming a trend due to the province’s geographical location and

climatic conditions.

Also, the phytochemical screening will be done at Center for Natural Sciences, Saint

Mary’s University, Bayombong, Nueva Vizcaya. The center provides services to student

researchers inside and outside the university with an affordable and reasonable cost. It has

produced results which were part of published researches in local, national and international

publication platforms. The center follows careful and standard laboratory procedures for the

accuracy of results.

21
Research Instruments

The following laboratory equipment, apparatus and chemicals and materials were used

for gathering the necessary data for the study:

Plant Extraction:

1. Dragon fruit peel

2. Laboratory oven

3. Laboratory blender

4. Extraction solvent

5. Cotton-lined Buechener funnel

6. Beakers, test tubes and graduated cylinders

Phytochemical Screening

1. Analytical grade dichloromethane, methanol, ethyl acetate and ethanol

2. Silica gel F254 aluminum-backed pre-coated TLC sheets

3. UV light sets

4. Different spray reagents:

Spray Reagents Compounds tested

Preliminary Test Essential Oils

Vanillin sulfuric acid
Napthol-sulfuric acid
Metanolic potassium hydroxide
(KOH-MetOH)
Potassium ferricyanide-ferric
chloride
Dragendorff’s reagent
Antimony (III) chloride
Higher alcohols, steroids, triterpenes,
essential oils, phenols, fatty acids
Sugars
Anthraquinones, coumarins, anthrones
Tannins, flavonoids, phenols
Alkaloids
Flavonoids, steroids

22
 Ninhydrin Amino acid

Data Gathering Procedures

A. Plant Collection and Extraction

The fruits of Hylocereus polyrhizus (dragon fruit) were harvested from a single

fruit stand in Diadi, Nueva Vizcaya. The peels of the fruits were thoroughly washed in

running water; air dried at room temperature (or dried using an oven), and powdered

using a laboratory blender. The plant material was soaked in an 80% ethyl alcohol for 48

hours. The mixture was filtered using a cotton-lined Buechner funnel. The solvent was

removed by evaporation in a water bath set at 40° C.

B. Phytochemical Screening

The phytochemical screening was based from Guevara (2005). Thin Layer

Chromatography (TLC) was used in the determination of the extract’s secondary

metabolites. Ethanolic crude extract of the peels was spotted to TLC silica gel, 60 F254.

The TLC plates was developed in 5:1 ratio of CHCl3:MeOH solvent system. UV light

was used to visualize the UV active spots corresponding to a certain metabolite. Spray

reagents was used on the TLC plates to identify the different classes of compounds

present in the extract. For the presence of higher alcohols, steroids, triterpenes, essential

oils, phenols, fatty acids, vanillin-was used. For the detection of sugar, alpha-naphthol-

sulfuric acid was used. KOH-MeOH was used to detect the presence of anthrones,

coumarins and antraquinones. Potassium ferricyanide-ferric chloride reagent was used to

detect the presence of flavonids, phenols and tannins. For the detection of alkaloids,

23
 Dragendorff’s reagent was used. Antimony (III) chloride was used in the detection of

flavonoids and steroids and ninhydrin was used in the detection of amino acids.

Data Analysis

The data from the study were provided by the Center for Natural Sciences of Saint

Mary’s University. It was qualitatively reported and described with respect to the potentials of

the peels of dragon fruit based on related literature and studies. Furthermore, the evaluation was

done by summarizing and discussing results from previous studies available in published

research journals. The study generally used laboratory reports and content analysis.

24
 Chapter 4

RESULTS AND DISCUSSION

Section 1: Phytochemical Screening

Using thin layer chromatography (TLC), the plant extract was spotted on marked and

labeled, and exposed under UV light and hot plate to check the separation of the different

compounds. For typical visualization of the secondary metabolites different reagents were

utilized. The table below summarizes the different secondary metabolites found in the dragon

fruit peel extracts:

Table 4
Secondary Metabolites found in Red Dragon Fruit Peel Extracts
Spray Reagents Secondary Metabolites Presence/Absence
Preliminary Tests Essential Oils +
Vanillin sulfuric acid Higher Alcohols -
Steroids +
Triterpenes -
Fatty Acids +
Napthol-sulfuric acid Sugars +
Metanolic potassium Anthraquinones +
hydroxide (KOH-MetOH) Coumarins +
Anthrones -
Potassium ferricyanide-ferric Tannins -
chloride Phenols +
Dragendorff’s reagent Alkaloids +
Antimony (III) chloride Flavonoids +
Ninhydrin Amino acid -
+ Present, – Absent

25
 Based on Table 4, the secondary metabolites found in the extracts of Hylocereus

polyrhizus were essential oils, steroids, fatty acids, anthraquinones, coumarins, phenols, alkaloids

and flavonoids. These results give light to the potentials of the peel extracts of red dragon fruit.

The presence of coumarin, a phenolic compound, helps in the plant’s defense mechanism against

herbivores and fungi, and poses an antimicrobial activity against both fungi and bacteria (Mazid,

Khan & Mohammad, 2011). The red dragon fruit then is a potential source of pesticidal defense

compounds. The presence of flavonoids gives the red dragon fruit the capacity for pigmentation

and defense which protect cells from UV-B radiation as they accumulate in the epidermal layers

of the fruit and absorb light strongly in the UV-B region (Lake, Field, Davey, Beerling &

Lomax, 2009). Alkaloids on the other hand, which are usually synthesized from amino acids, act

as defensive elements against microbial infection (Mazid, Khan & Mohammad, 2011). Although

the screening conducted did not resulted to the presence of amino acids, the presence of alkaloids

can be attributed to the presence of these acids which can be considered as non-protein amino

acids which is of the same family with alkaloids, nitrogen-containing secondary metabolites.

For anthraquinones, these naturally occurring secondary metabolites found in lichens,

fungi and higher medicinal plants have given the red dragon fruit the potential to become a

source of laxative compounds which are used to treat constipation. Reports also showed that

anthraquinones contain cathartic, anti-inflammatory, anticancer, antimicrobial, diuretic, DNA-

binding, and vasorelaxant activities (Chien, Wu, Chen & Yang, 2014).

On the presence of phenols or phenolic compounds, this fulfills a broad range of

physiological roles in plants. It helps plants to cope with constantly changing environmental

challenges over evolutionary time. They serve as defense compounds against high light, low

temperatures, pathogen infection and nutrient deficiency (Lattanzio, 2013). According to Sultan

26
and Rauf (2015), the steroids found in plants play important roles in the physiology and

biochemistry, and they can be extensively used as anti-hormones, contraceptives, anti-cancer,

cardiovascular agents, osteoporosis drugs, antibiotics, anesthetics, anti-inflammatories, and anti-

asthmas. These things make the peels of red dragon fruit to be potentially useful in medicine.

Section 2: Comparative Evaluation

Table 5
Phytochemical Screening Results of Dragon Fruits
Kanchana,
Romero,
Nurul & Devi, Pushpa Manihuruk,
Cheah, et al. Waing & Sekar, et al.
Asmah Latha & Suryati &
Results of this (2016) Valentino (2016)
(2014) Spurthi Arief (2016)
Study (2017)
(2018)
(H. polyrhizus)
H. undatus H. undatus
H. undatus H. polyrhizus H. undatus H. polyrhizus and H. and H.
polyrhizus polyrhizus
Essential Oils Essential Oils Essential Oils Essential Oils
Steroids Steroids Steroids Steroids Steroids
Phenols Phenols Phenols Phenols
Fatty Acids Fatty Acids Fatty Acids
Sugar
Anthraquinones
Coumarines
Alkaloids Alkaloids
Flavonoids Flavonoids Flavonoids Flavonoids Flavonoids Flavonoids
Betalains
Gallic Acid
Betacyanins Betacyanin
Ascorbic Ascorbic
Acid Acid
Carbohydrate Carbohydrate
s s
Saponins Saponins Saponins
Tannins Tannins
Glycosides Glycosides Glycosides
Amino Acids Amino Acids
Terpenoids Terpenoids
Hydroquinone
Triterpenoids

Based on the table above, four studies on H. polyrhizus were compared to the present

study. Some of the similarities of the results are the presence of phenols, steroids, fatty acids, and

27
flavonoids. For the studies on H. undatus when compared to the present study, the similarities

are the presence of essential oils, fatty acids, phenols, and flavonoids. This result implies that

phytochemical screening on different studies is different and does not use the same set of

reagents. However, these results confirm the presence of the secondary metabolites which can be

bases for further investigation of the medicinal use of the dragon fruit. For almost all studies

presented, except one, flavonoids are present. These are important polyphenolic compounds with

a wide range of biological applications such as anti-inflammatory, antibacterial, antiviral, anti-

allergy, and cytotoxic antitumor. They serve as antioxidants which are capable of preventing and

even counteracting the damage caused in human tissues by the normal effect of physiological

oxidation (Susanti, Utomo, Syukri, & Redjeki, 2012). This confirms that dragon fruits are

essentially healthy for human beings to consume as it is a rich source of antioxidants.

On the other hand, the studies presented in Table 5 confirms the presence of other

secondary metabolites which the present study was not able to identify like saponins, tannins and

ascorbic acid. Also, the evaluation produced the presence of glycosides and terpenoids in H.

undatus but not in H. polyrhizus but this needs further investigation.

28
 Chapter 5

SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

Summary

This investigatory project primarily studied the secondary metabolites present in the peel

of Hylosereus polyrhizus (red dragon fruit) through phytochemical screening, and compared the

results to the results of previously conducted studies. It used a descriptive-comparative research

design to qualitatively analyze the presence of secondary metabolites in this study and other

studies. The phytochemical screening was conducted at the Center for Natural Sciences at Saint

Mary’s University following the protocols of Guevara (2005) using Thin Layer Chromatography.

Summary of Findings

1. The secondary metabolites found in the extracts of Hylocereus polyrhizus peels are

essential oils, steroids, fatty acids, sugars, anthaquinones, coumarins, phenols, alkaloids

and flavonoids.

2. Varieties of dragon fruit present different phytochemicals based on the comparative

evaluation of different studies. Based on reports, glycosides and terpenoids are found in

H. undatus (white variety) but not in H. polyrhizus (red variety).

29
 3. The presence of phenols, steroids, fatty acids and flavonoids were found to be present in

all phytochemical screening of H. polyrhizus, while essential oils, phenols, fatty acids and

flavonoids for H. undatus.

Conclusion

Based on the findings of the study, the following conclusions are forwarded:

1. The peels of Hylocereus polyrhizus (red dragon fruit) contains biologically active

chemical compounds which can be used as defense mechanism against pathogens; thus,

making it a potential source of antibacterial compounds which can be used for medicinal

purposes.

2. The phytochemical screening from different studies varies based on the procedures

conducted and does not follow the same standard procedures based on the differences of

results.

Recommendations

The following recommendations are forwarded for the improvement of the study and for

the conduct of further similar studies:

1. Chemists, and even biologists, should explore on other secondary metabolites that can be

found in dragon fruits to maximize the utilization and potentials of the fruit, especially on

the other parts of the plant.

30
2. Science teachers should encourage science-inclined students to explore on other forms of

characterizing dragon fruits such as identifying its antioxidant, antibacterial or cytotoxic

properties.

3. For future researchers, it is recommended to:

a. utilize other reagents to identify other secondary metabolites;

b. include more researches on phytochemical screening of dragon fruits and other

plants in the Cactacae family;

c. identify total phenolic content (TPC) and total flavonoid content (TFC) of the

different varieties of dragon fruit; and

d. explore on the other potential uses of dragon fruit extracts such as acid-base

indicators.

31
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