Preliminary Phytochemical Analysis of Selected

Seagrasses in Guiuan, E. Samar

A Research Proposal

Presented to the Panel of Critiques

Guiuan National High School

Guiuan, E. Samar

In Partial Fulfillment of

the Requirements for the subject

Research II

by

Glenn Dioshua O. Altar

Regine C. Betorio

Kirk Raffy L. Abud

February 2020
 Chapter 1

INTRODUCTION

Background of the Study

Seagrass is an angiosperm that lives in marine environment (Richmond 1997).

Seagrasses have adjusted to develop in beach front marine situations in both tropical

and calm areas (Coles et al. 2002). In general seagrasses inhabit the tidal and subtidal

zones of shallow and sheltered localities of seas, gulfs, bays, backwaters, lagoons

and estuaries. They typically lean toward sloppy, sandy, clayey and coral rubble

substrate; however, they likewise develop on rocks and in fissure (Jagtap et al. 2003).

They are known to be highly productive and serves as nursery grounds, food source

and shelter for many sea organisms (Richmond 1997). They also reduce coastal

erosions and provide habitat to both attached and free-living organisms (Hemminga &

Duarte 2003). At least 12 seagrass species have been distinguished in the Philippines

out of the 50 species around the world (Fortes 1990), while 9 species can be found in

Guiuan, Eastern Samar (Cabacaba et al. 2018).

In addition, the significance of seagrasses has been archived in many coastal

communities around the world, including India (Kannan et al. 2010), Africa (Pillay et

al. 2010), Canada (Wyllie-Echeverria & Cox 1999; Kuhnlein & Turner 1992), Mexico

(Felger & Moser 1973; Felger & Moser 1986), and Sweden (Rönnbäck et al. 2007).

Some of these studies describe the role of seagrasses in ecosystem function

(Rönnbäck et al. 2007), while others highlight their economic and traditional value,

such as the research of Wyllie-Echeverria and Cox (Wyllie-Echeverria & Cox 1999)

concerning wild and commercial gathering of seagrass Zostera marina by North

American fishing communities during the early to mid-1900s for use as green manure
and insulating products. Seagrasses have likewise been read for their potential use in

present day drug (Kannan et al. 2010; Alam et al. 1994; Arumugam et al. 2010;

Anantharaman et al. 2010). Recent inquiries on seagrass phytochemistry has

demonstrated its significance as wellspring of antioxidants (Kannan et al. 2010),

antibacterial agents (Arumugam et al. 2010), minerals (Anantharaman et al. 2010) and

potential anticancer compounds (Folmer et al. 2010).

Meanwhile, plant products have been part of phytomedicines for a very long

time (Cragg & Newman 2001). Plants produce phytochemicals to ensure themselves

against ecological dangers like predator bugs, contamination and diseases (Oliveira

2018). This can be derived from barks, stems, roots, flowers, fruit, and seeds (Cragg

& Newman 2001). On the other hand, most of the developing countries in the world

still rely on herbal preparations for the treatment of some diseases despite having

conventional medicines (Nwabuisi 2002). Some activities of phytochemicals are; anti-

microbial which are present in phenols, flavonoids, tannins, coumarins, terpenoids,

and alkaloids; anti-diarrheal which are present in phenols, flavonoids, terpenoids,

glycosides, and saponins (Tiwari et al. 2011).

Phytochemicals from seagrasses have been found to have significant functions

such as the tannin cells being specialized to produce phenolic compounds, which play

defensive roles against microorganisms and herbivores (Tempel 1982; Kuo &

McComb 1989; Cariello & Zanetti 1979). Chemical constituents of several seagrasses

have been described including the antibiotic flavone glycoside from T. testudium

(Jensen et al. 1998), one sugar derivative from Ruppia maritime (Aquino 2004),

phenolic compounds from P. oceanica (Todd et al. 1993), diterpens from R. maritima

(Dellagreca 2000) and steroids and fatty acids from Zostera japonica (Kuo & McComb

1989; Gillan et al. 1984; Sanina et al. 2004).

 Apart from medicinal purposes, seagrasses also have other uses like the

Enhalus acoroides which is used as fishing nets in Moluccas and New Guinea. It is

also used as mats, ropes and paper making. In the Philippines and Indonesia, the

seeds of E. acoroides are sometimes eaten (https://uses.plantnet-

project.org/en/Enhalus_acoroides).

To date, little information exists about the phytochemical components of these

species of seagrasses. Hence, the goals of this study are to determine if flavonoids,

tannins, saponins, phenols, alkaloids, carbohydrates, proteins and amino acids, and

coumarins are present in Enhalus acoroides, Thalassia hemprichii, and Halodule

uninervis.

Statement of the Problem

This study answered the question, “What are the phytochemicals present in the

selected seagrasses found in Guiuan, E. Samar?”

Specifically, the following question was answered:

1.) Do E. acoroides, T. hemprichii, and H. uninervis contain flavonoids, tannins,

saponins, phenols, alkaloids, carbohydrates, proteins and amino acids, and

coumarins?

Hypothesis
The following is the hypothesis of the study:

1.) E. acoroides, T. hemprichii, and H. uninervis does not contain flavonoids,

tannins, saponins, phenols, alkaloids, carbohydrates, proteins and amino acids, and

coumarins.

Conceptual Framework

Phytochemical
Seagrasses
Consituents

Scope and Delimitation

This study focused only on determining the presence or absence of the

phytochemicals; alkaloids, tannins, phenols, flavonoids, saponins, carbohydrates,

coumarins, and proteins and amino acids of E. acoroides, T. hemprichii and H.

uninervis. This was accomplished by treating chemical reagents to the extracts. For

the flavonoids test, sodium hydroxide and hydrochloric acid was used. For the tannins

and phenols test, ferric chloride was used. For the alkaloids test, potassium iodide was

used. For the carbohydrates test, iodine solution was used. For the proteins and amino

acids test, concentrated nitric acid was used. For the coumarins, sodium hydroxide

was used.

This study was conducted on the month of August till the month of September.

The phytochemical screening was done in the chemistry laboratory in Eastern Visayas

State University (EVSU).

Significance of the Study

Determining the phytochemical constituents of these seagrasses will open

many applications for the three seagrasses such as pharmaceutical, and industrial

applications. Thus, the results of this study will benefit:

Department of Health: This study will give this department an idea on the different

medicinal applications of these seagrasses according to their phytochemical

constituents.

Department of Environment and Natural Resources: This study will give this

department an idea on both the medicinal and industrial applications of these

seagrasses. They can make different organic products from these seagrasses

according to their phytochemical constituents.

Community: This study will give the community an idea on the uses of these

seagrasses and also be their source of income.

Definition of Terms

Phytochemical: Phytochemicals are plant-generated chemicals by primary or

secondary metabolism (Molyneix 2007). In this study, phytochemical is the one being

determined from the three seagrasses.

Extraction: Extraction means the target material can be isolated when combined with

other substances. The mixture contacts a solvent in which the material of interest is

soluble, but the other present substances are insoluble

(www.chemicool.com/definition/extraction.html). In this study, this refers to the

process wherein the dried leaves were grinded, then mixed with water and sealed,

then stored in a cabinet to homogenize.

Maceration: Maceration is a technique of extraction which is carried out at room

temperature. The plant is dipped in a liquid within an airtight container, based on the

plant and liquid used for a variable time

(https://albrigiinherba.com/contacts/extraction/maceration/). In this study, this refers to

the process of extraction that was used.

Homogenization: Homogenization is the mechanism by which two immiscible liquids

are mixed into an emulsion (McClements 2008). In this study, this process was used

in obtaining the extract.

Reagents: A reagent is a substance added to a system that causes a chemical reaction

or to test if there is a reaction (McNaught &Wilkinson 2000). In this study, this refers

to the chemicals that was treated to the extracts.

 Chapter 2

Review of Related Literature

Seagrasses are false grasses, however they do take after them and have

numerous likenesses with them in an amazing science cycle. Seagrasses normally

structure thick knolls that look like more well-known prairies or fields. Seagrasses are

vascular plants. The vascular framework in the seagrasses comprises of an inner

system of cylinders that is constant from the roots to the leaf tips, shipping water,

supplements, and gases for tissue development. Due to this framework, seagrass

developing in sand or mud can utilize the exceptionally focused supplements of the

base, supplements that might be hundreds or thousands of times more gathered than

those in the overlying water section. This source, the base residue, is inaccessible to

all other marine plants; these others must depend for development just on

supplements broke up in the water. Seagrasses can ingest supplements from water

through their leaves like green growth, yet are most certainly not restricted to that

source (Meñez Ernani et al. 1983).

Seagrasses, at that point, are a component for returning supplements to other

marine life that would somehow or another be lost in the silt. They are the main plants

in the ocean that can do this. The noteworthiness of this double supplement source is

reflected in development rates that are high or then again higher than those of some

other plant network in the ocean. The roots and rhizomes not just assimilate

supplements from the residue, yet in addition forestall the development of base silt

(Meñez Ernani et al. 1983).

Seagrasses are very important in the ecosystem. They comprise a shelter and

asylum for creatures in the ocean. They are particularly significant as nurseries for
fish, shrimps, lobsters, scallops, also, other important nourishment creatures. The

insurance that these plant frameworks manage the cost of reaches out past their

related creature life to the coast itself. Albeit a large number of the creature relationship

with seagrasses are backhanded and subtle, there are striking special cases:

dugongs, ocean turtles, a few fish, and ocean urchins and different spineless creatures

feed straightforwardly on these plants (Meñez Ernani et al. 1983).

In addition, some portion of the significance of seagrasses is their high

profitability. Seagrass beds have been appeared to be among the most gainful

territories of the earth, including those under escalated horticulture. Seagrasses have

a supplement of connected green growth, called epiphytes, which add to the high

efficiency of the seagrass biological system. This essential efficiency is the main

connection in the nourishment chains of these biological systems, and by and large

backings a high efficiency in the related creature network (Meñez Ernani et al. 1983).

Meanwhile, medicinal plants have been of age long remedies for human and

animal diseases because they contain components of therapeutic value (Nostro et al.

2000). Some of them are also used for prophylactic purposes. An increasing interest

in herbal remedies have been incorporated into orthodox medicinal plant include

malaria, epilepsy, infantile convulsion, diarrhea, dysentery, fungal and bacterial

infections (Sofowora, 1996). Medicinal herb is considered to be a chemical factory as

is contains multitude of chemical sompounds like alkaloids, glycosides, saponins,

resins, oleoresins, sesquiterpene, lactones and oils (Singh, 2005). India has one of

the oldest, richest and most diverse cultural tradition.

Phytochemical screening involves the identification of various bioactive

compounds such as tannin, saponin, phenolic compounds, glycosides etc. Screening

is a series of continuous processes involving selection of promising plants, collection

and authentication of plant materials, proper method of processing and packaging,

extraction and identification of phytochemicals using standard method (Tiwari et al.

2011).

Carica papaya is an herbaceous plant with prominent leaves (20-60 cm long)

and is a member of the Caricaceae family, indigenous to the tropical region of Mexico,

Central America and Northern South America. All parts of the papaya plant can be

used as medicine; the fruit flesh, flowers, seeds and the flowers. Many scientific

investigations have been conducted to evaluate the biological activities of various part

of C. papaya including their fruits, shoots, leaves, seeds, roots or latex (Kovendan et

al. 2012).

Likewise, Prasad et al. (2012) carried out a phytochemical evaluation of leaves

of five different species of A. heterophyltus using four solvents viz. acetone, hexane,

water and petroleum ether. The result revealed that all the five species of Jackfruit

contains phytochemicals such as alkaloids, cardiac glycosides and carbohydrates. On

the other hand saponin, phenol and flavanoid are absent in petroleum ether, acetone

and hexane extract but present in water extract terpenoid is absent in water, hexane

and acetone extract. Tannin was also not detected in acetone extract.

For the phytochemical analysis of seagrasses, the study of Girija et al. (2013)

determined the phytochemical constituents of Halodule pinifolia, Halophila ovalis, and

Syrongodium isoetifolium. H. pinifolia showed the presence of carbohydrate, cardiac

glycosides, coumarins, flavonoides and sugar. H. pinifolia showed the presence of

alkaloids, phytosteroides, protein and amino acids while H. ovalis showed the

presence of carbohydrate, coumarins, flavonoides and sugar.

 Consequently, the methanol extract of Syringodium isoetifolium showed the

presence of protein, resin, glycosides, reducing sugar, saponins, acidic compounds,

Phenols, cardiac glycosides and alkaloids. The phytochemical constituents such as

carbohydrates and phenols were moderately present. Steroid, tannins sterols,

terpenoids, catachols and flavonoids were absent (Mani 2012).

 Chapter 3

Research Design

The researcher used Experimental Research Design. The design of this study

is experimental design because the researcher wants to determine the phytochemical

constituents in the selected seagrasses by observing for color changes when chemical

reagents was treated to their extracts. According to QuestonPro.com, “The simplest

example of an experimental research is conducting a laboratory test. As long as

research is being conducted under scientifically acceptable conditions – it qualifies as

an experimental research. A true experimental research is considered to be

successful only when the researcher confirms that a change in the dependent variable

is solely due to the manipulation of the independent variable.”

(https://www.questionpro.com/blog/experimental-research/)

Methodology

Collection of seagrasses

E. acoroides, T. hemprichii, and H. uninervis were collected in Brgy.

Pagnamitan in the month of September. Two hundred grams of each seagrass species

was collected with the assistance of a marine biologist from DENR-CENRO Guiuan

sub-office for the identification. The collected seagrass was placed in a cloth bag and

brought to the laboratory. Samples of each specimen was sent to Visayas State

University for species verification.

Preparation of Seagrass
 The seagrasses were washed with running tap water along with stroking the

leaves to remove the algae. This process was repeated twice for each species. Then

it was rinsed with distilled water. The seagrasses were air dried using a net. It took 7

days for the seagrasses to completely dry.

Extraction of seagrasses

The leaves of every species were grinded using a blender. For the flavonoids,

alkaloids, carbohydrates, proteins and amino acids, and coumarins tests, the

extraction of maceration method was used (Tiwari et al. 2011). Ten g of the grinded

dried leaves of E. acoroides were mixed with a 100mL distilled water in an Erlenmeyer

flask (Luay et al. 2018). The process was repeated for the T. hemprichii and H.

uninervis. The flasks were sealed with aluminum foil and stored in a cabinet to

homogenize for 24 hours. After 24 hours, it was filtered using Whatman (no.1) filter

paper. The filtrate was placed in petri dishes and left to evaporate through an

improvised water bath in absence of a rotary evaporator at around 40°C until all the

solvent was totally removed (Tiwari et al. 2011). Same process was done to the T.

hemprichii and H. uninervis. For the tannins, saponins, and phenols tests, 0.5g of the

grinded dried leaves of E. acoroides was mixed with 20 ml distilled water and heated

until boiled. After boiling, it was filtered with Whatman no.1 filter paper (Tiwari et al.

2011). The same process was done to the T. hemprichii and H. uninervis.

Phytochemical Screening

Detection of Flavonoids: Alkaline Reagent Test was used. Crude extract was

dissolved in 20mL of distilled water. Three mL of the aqueous extract was placed in
each of the 4 test tubes and labeled trial 0, 1, 2, and 3. Trial 1-3 were treated with 4

drops of 2% sodium hydroxide solution. Formation of intense yellow color, which

becomes colorless on addition of acetic acid, indicates the presence of flavonoids

(Tiwari et al. 2011).

Detection of Tannins: Ferric chloride Test was used. Three mL of the aqueous

extract was placed in each of the 4 test tubes and labeled trial 0, 1, 2, and 3. Trial 1-3

were treated with 4 drops of ferric chloride. Formation of bluish black and brownish

green coloration indicates the presence of tannins (Tiwari et al. 2011).

Detection of Phenols: Ferric chloride Test was used. Three mL of the aqueous

extract was placed in each of the 4 test tubes and labeled trial 0, 1, 2, and 3. Trial 1-3

were treated with 4 drops of ferric chloride. Formation of bluish black coloration

indicates the presence of phenols (Tiwari et al. 2011).

Detection of Saponins: Foam test was used. Three mL of the aqueous extract was

placed in each of the 4 test tubes and labeled trial 0, 1, 2, and 3. 2mL of distilled water

was added to trial 1-3. The mixture was shaken vigorously. Formation of foam

indicates the presence of saponins (Tiwari et al. 2011).

Detection of Alkaloids: Wagner’s test was used. Extracts was dissolved in dilute

Hydrochloric acid and filtered. Three mL of the aqueous extract was placed in each of

the 4 test tubes and labeled trial 0, 1, 2, and 3. Trial 1-3 were treated with 4 drops of

Wagner’s reagent (Iodine in Potassium Iodide). Formation of brown/reddish precipitate

indicates the presence of alkaloids (Tiwari et al. 2011).

Detection of Carbohydrates: Crude extract was mixed with 20mL distilled water.

Three mL of the aqueous extract was placed in each of the 4 test tubes and labeled

trial 0, 1, 2, and 3. Trial 1-3 were treated with 4 drops of iodine solution. A dark blue
or purple coloration indicates the presence of the carbohydrate (Yadav & Agarwala

2011).

Detection of Proteins and Amino Acids: Xanthoproteic Test was used, crude extract

was mixed with 20mL distilled water. Three mL of the aqueous extract was placed in

each of the 4 test tubes and labeled trial 0, 1, 2, and 3. Trial 1-3 were treated with 4

few drops of conc. Nitric acid. Formation of yellow color indicates the presence of

proteins (Tiwari et al. 2011).

Detection of Coumarins: The method of Rizk, A.M., was used. Crude extract was

mixed with 20mL distilled water. Two mL of the aqueous extract was placed in each

of the 4 test tubes and labeled trial 0, 1, 2, and 3. A 3 ml of 10% NaOH was added to

2 ml of aqueous extract. Formation of yellow color indicates the presence of coumarins

(Savithramma et al. 2011).

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