"PHYTOCHEMICAL PROFILE OF THE SEA GRAPE CAULERPA

LENTILLIFERA (J. AGARDH) FROM TWO SELECTED FARMS IN CEBU,

PHILIPPINES”

An Undergraduate Thesis
Presented to the
Faculty of College of Education
Cebu Technological University-Main Campus
MJ Cuenco Ave, Cor R. Palma St., 6000 Cebu City, Philippines

In Partial Fulfillment on the subject

Undergraduate Thesis/ Research Paper/ Research Project in Science Education
SCI M 4120

Alquizalas, Jezelle Mae Marie P.

Tanuco, Isabel Marie D.
Ceniza, Glein G.
Java, Jill

February 2024
 ii

APPROVAL SHEET

The research entitled “PHYTOCHEMICAL PROFILE OF THE SEA

GRAPE CAULERPA LENTILLIFERA (J. AGARDH) FROM TWO SELECTED
FARMS IN CEBU, PHILIPPINES” prepared and submitted by JEZELLE MAE
MARIE ALQUIZALAS, GLEIN CENIZA, JILL JAVA and ISABEL MARIE
TANUCO in partial fulfillment of the requirements for the subject SCI-M 4120, has
been examined and is recommended for acceptance and acceptance for Oral
Examination.

RESEARCH ADVISORY COMMITTEE

MARIA SALUD DELOS SANTOS, Ph.D.

Chairman

JONATHAN O. ETCUBAN, LPT. PhD-TM, PhD-E ALITA S. LABIAGA, MAT-S

Member Member

JABIN J. DEGUMA, DEV. ED. D., RGC IRENE O. MAMITES, Dev. Ed.
Member Member

REGINA E. SITOY, LPT, Dev. Ed. D.

Member
________________________________________________________________

Approved by the Committee on Oral Defense with a grade __________.

PANEL OF EXAMINERS

MARIA SALUD DELOS SANTOS, Ph.D.

Chairman

JONATHAN O. ETCUBAN, LPT. PhD-TM, PhD-E ALITA S. LABIAGA, MAT-S

Member Member

JABIN J. DEGUMA, DEV. ED. D., RGC IRENE O. MAMITES, Dev. Ed.
Member Member

REGINA E. SITOY, LPT, Dev. Ed. D.

Member
 iii

ACCEPTED and APPROVED in partial fulfillment of the requirements of SCI-M

4120 and for the degree of Bachelor of Secondary Education Major in
Science.

Comprehensive Examination:
Date of Oral Examination: February 12, 2024

REYLAN G. CAPUNO, Ph.D., Dev.Ed.D.

Dean, College of Education
 iv

ABSTRACT

Caulerpa lentillifera, a type of green seaweed prevalent in Southeast Asian

countries, including the Philippines, is known for its potential bioactive compounds
with various biological activities. However, these remain largely unexplored. The
main objective of this study was to determine the secondary metabolites of C.
lentillifera ethanolic extracts taken from two selected farms in Cebu, Philippines.
Ethanol was used as the solvent for preparing the extracts of C. lentillifera. The
ethanolic extracts of C. lentillifera were subjected to qualitative phytochemical
screening using standard tests and procedures. These extracts were tested for
various phytoconstituents, including alkaloids, carbohydrates, flavonoids,
glycosides, phenols, proteins, saponins, steroids, sterols, tannins, terpenoids, and
coumarin. The measured physico-chemical parameters of seawater were limited
only to salinity, temperature, and pH using a handheld refractometer. The results
of the phytochemical tests on extracts from Kalawisan, Lapu-Lapu and Lamintak
Norte, Medellin, Cebu, Philippines, showed a moderate to high presence of most
bioactive compounds, except for sterols, which were only slightly present in both.
The study noticed that the physico-chemical parameters of seawater have little
effect on the phytochemical content of the same seaweed in different places. Both
ethanolic extracts of C. lentillifera yield a higher number of phytoconstituents. The
present phytochemical screening study revealed the richness of C. lentillifera in
bioactive compounds with potential biomedical and pharmacological applications
but necessitated more investigation. Further screening of the recorded
phytoconstituents involving quantitative detection and bioactivity assessment was
recommended for future studies involving similar seaweed.

Keywords: green seaweed; ethanolic extract; phytoconstituents; bioactive

compounds; physico-chemical parameters
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ACKNOWLEDGEMENT

Imbued with immense gratitude and appreciation, the researchers wish to

express their heartfelt gratitude to all the people who gave them great support and

assistance in bringing this research into reality.

First and foremost, the researchers would like to express their profound

gratitude to God Almighty for the life strength, zeal, and determination to

accomplish this research endeavor. Without Him and His blessings, this would not

have been possible.

The researchers would also like to thank their research adviser, Prof. Alita

S. Labiaga, for her continuous help and support in shouldering all the research

expenses, her invaluable insights, her patience, and constructive criticism and

encouragement throughout this research work. Her door was always open

whenever the researchers ran into a trouble spot or had a question about research

or writing.

The researchers would like to extend their deepest gratitude to their

research instructor, Dr. Maria Salud M. Delos Santos, who constantly encourages

the researchers and imparts her research knowledge.

The researchers would like to express their sincerest gratitude to the Dean

of the College of Education, Dr. Reylan G. Capuno, for allowing them to conduct

their research.

It is with great pleasure and privilege that we acknowledge their deepest

regards and gratitude to the owners of C. lentillifera farms, Mr. Vicente Ybañez
 vi

and Mr. Eduardo Florita, for letting us collect the samples of C. lentillifera in their

farms and accommodating us throughout their stay.

The researchers would like to thank Mr. Joel B. Clapano, a fisheries

technologist at the Bureau of Fisheries and Aquatic Resources (BFAR-7), for

identifying the seaweed used in their research work.

The researchers thank Dr. Rosalyn Alburo, Center Director of Biodiversity,

Environment, and Nature Resource Center (BENRC) of Cebu Technological

University- Argao Campus, for sharing her knowledge and expertise and allowing

them to use the rotary evaporator.

The researchers would like to thank the research assistant, Mr. Gabriel

Fortuna, who provided additional assistance in operating the rotary evaporator

properly.

Lastly, the researchers would like to thank their parents for their prayers,

guidance, courage, and financial support in pursuing this study. They were the

primary source of inspiration and their ultimate role models who were always there

every step of the way.

Our thanks and appreciation also go to our colleagues and people who have

helped us with their abilities.

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DEDICATION

This study is lovingly dedicated to our parents. They inspire us, give us

strength during tough times, and continually support us emotionally, spiritually, and

financially throughout this research journey.

To our brothers, sisters, relatives, mentor, friends, and classmates who

gave words of encouragement which have been instrumental in the completion

and enhancement of our study.

And lastly, we dedicated this research to the Almighty God, thank you for

the guidance, strength, wisdom, protection and unwavering will, enabling us to

have a positive mindset and successfully conclude this research.

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TABLE OF CONTENTS

Page

TITLE PAGE-----------------------------------------------------------------------------------------i

APPROVAL SHEET ------------------------------------------------------------------------------ii

ABSTRACT -----------------------------------------------------------------------------------------iii

ACKNOWLEDGEMENT--------------------------------------------------------------------------v

DEDICATION -------------------------------------------------------------------------------------vii

TABLE OF CONTENTS------------------------------------------------------------------------viii

LIST OF TABLES----------------------------------------------------------------------------------xi

LIST OF FIGURES-------------------------------------------------------------------------------xii

CHAPTER I: INTRODUCTION-----------------------------------------------------------------1

Background of the Study ---------------------------------------------------------------1

Theoretical Background-----------------------------------------------------------------5

Research Framework -------------------------------------------------------------------7

Objectives ----------------------------------------------------------------------------------8

Hypothesis----------------------------------------------------------------------------------8

Significance of the Study----------------------------------------------------------------9

Scope and Limitation-------------------------------------------------------------------11

 ix

CHAPTER II: REVIEW OF RELATED LITERATURE ---------------------------------12

Related Legal Basis---------------------------------------------------------------------12

Phytochemicals--------------------------------------------------------------------------13

Caulerpa Species Structure-----------------------------------------------------------15

Caulerpa lentillifera----------------------------------------------------------------------16

Methods of Extraction------------------------------------------------------------------19

Factors Affecting the Seaweed’s Phytoconstituents----------------------------20

Synthesis of Related Literature and Studies-------------------------------------22

Gaps Bridged-----------------------------------------------------------------------------23

CHAPTER III: MATERIALS AND METHODS--------------------------------------------24

Research Design -----------------------------------------------------------------------24

Sampling Site ----------------------------------------------------------------------------25

Research Sample -----------------------------------------------------------------------25

Experimental Procedures -------------------------------------------------------------26

Collection and Identification of Seaweed --------------------------------26

Ethanolic Extraction of C. lentillifera --------------------------------------27

Phytochemical Screening Process ----------------------------------------27

Water Quality Parameter Measurements -----------------------------------------28

 x

Ethical Considerations -----------------------------------------------------------------29

Data Privacy------------------------------------------------------------------------------29

Statistical Treatment -------------------------------------------------------------------30

Definition of Terms ---------------------------------------------------------------------30

CHAPTER IV: RESULTS AND DISCUSSION ------------------------------------------33

CHAPTER V: CONCLUSION AND RECOMMENDATION---------------------------42

REFERENCES -----------------------------------------------------------------------------------44

APPENDICES-------------------------------------------------------------------------------------56

A Approval Sheet (Proposal)---------------------------------------------------56

B Ethics Certificate----------------------------------------------------------------57

C Workplan--------------------------------------------------------------------------59

D GAD Score-----------------------------------------------------------------------60

E Transmittal Letters-------------------------------------------------------------64

F Certificate of Authentication--------------------------------------------------79

G Accession Records-------------------------------------------------------------81

H Phytochemical Screening Results------------------------------------------83

I Computations--------------------------------------------------------------------95

CURRICULUM VITAE--------------------------------------------------------------------------96
 xi

LIST OF TABLES

Table Page

1 Qualitative Phytochemical Screening of metabolites 28

(Guevarra, 2005)

2 Results of the Phytochemical Screening from the ethanolic 34

extract of Caulerpa lentillifera

3 Some physico-chemical parameters of water in Kalawisan 36

C. lentillifera farm

4 Some physico-chemical parameters of water in Medellin C. 37

lentillifera farm

5 T-test results between physico-chemical parameters of two 37

localities
 xii

LIST OF FIGURES

Figure Page

1 Research Framework of the Study 7

2 Close up image of C. lentillifera 16

3 Freshly collected sample of c. lentillifera 26

4 Morphology of Caulerpa lentillifera J. Agardh. 1837 33

5 Test for Alkaloids (Medellin) 83

6 Test for Carbohydrates (Medellin) 83

7 Test for Cardiac glycosides (Medellin) 84

8 Test for Flavonoids (Medellin) 84

9 Test for Phenols (Medellin) 85

10 Test for Proteins (Medellin) 85

11 Test for Saponin (Medellin) 86

12 Test for Sterols (Medellin) 86

13 Test for Steroids (Medellin) 87

14 Test for Tannins (Medellin) 87

15 Test for Terpenoids (Medellin) 88

16 Test for Coumarin (Medellin) 88

17 Test for Alkaloids (Kalawisan) 89

18 Test for Carbohydrates (Kalawisan) 89

19 Test for Cardiac glycosides (Kalawisan) 90

20 Test for Flavonoids (Kalawisan) 90

21 Test for Phenols (Kalawisan) 91

 xiii

22 Test for Proteins (Kalawisan) 91

23 Test for Saponin (Kalawisan) 92

24 Test for Sterols (Kalawisan) 92

25 Test for Steroids (Kalawisan) 93

26 Test for Tannins (Kalawisan) 93

27 Test for Terpenoids (Kalawisan) 94

28 Test for Coumarin (Kalawisan) 94

 1

INTRODUCTION

This chapter presented the problem and issues related to the study. This

includes the background of the study, theoretical background, research

framework, objectives, hypothesis, significance of the study, and finally, the study's

scope and limitations.

Background of the Study

The Philippines is renowned for its rich diversity of macroalgae, including

approximately 1200 recorded taxa, of which 306 are green seaweed species (Ang

et al. 2013). In the Central Visayas region, particularly in Bohol and Cebu Islands,

a variety of green seaweed species can be found, such as the locally known sea

grapes called "lato" (Delan et al. 2015). Sea grapes belong to the phylum

Chlorophyta, specifically the Caulerpaceae family in the order Bryopsidales, with

ninety-seven recognized taxa (Guiry and Guiry, 2019). Caulerpa lentillifera J.

Agardh and Caulerpa racemosa (Forsskal) J. Agardh are well-known among

various sea grapes. They can be found in the wild or cultivated in ponds, open

lagoons, and even concrete water tanks in the Visayan region, particularly in Cebu

province, including Mactan and Camotes Islands, which are best harvested during

dry (peak) seasons (Trono, 1969; Pereira, 2016; Dumilag, 2019; Estrada et al.

2020).

C. lentillifera is highly regarded for its potential health benefits, such as

being rich in vitamins, minerals, polyunsaturated fatty acids (PUFAs) from omega

3 group (DHA, EPA, ALA), and dietary fibers, making it preferred for raw
 2

consumption or as an ingredient in salads along with vegetables (García-Vaquero

et al. 2017; Chen et al. 2019; Zubia et al. 2020). Its distinctive appearance is

characterized by its long, tubular branches resembling a bunch of grapes. This

seaweed typically thrives in marine environments like coastal areas, reefs, and

shallow waters in tropical and subtropical regions of the Indian and Pacific Oceans.

Its morphology is favorable for marketing because of its higher antioxidant value

than other seaweed species (Wichachucherd et al. 2019).

Being a marine resource, C. lentillifera is renowned for its abundant and

diverse array of chemical constituents, including polyphenols, sterols, vitamins,

high mineral content such as zinc and iron, and trace elements including cobalt,

selenium, and valium that meet daily body requirements (Peña Rodriguez et al.

2011). It is also a valuable source of commercially essential polysaccharides such

as alginate, agar, and carrageenan (Nguyen et al. 2011). Research on seaweed

polysaccharides is essential as they have shown promising potential in providing

various health benefits, including antioxidant, anti-inflammatory, and antitumor

activities (Zhang et al. 2021). Despite its abundance and promising nutritional

value, limited information is still available about the phytoconstituents of C.

lentillifera, particularly about how their composition might vary in different locations.

The influence of geographic locations has been explored by researchers, which is

necessary for a comprehensive understanding of its chemical composition for

potential application and, above all, consumer safety. Potential variations in the

abundance, diversity, and concentrations of phytoconstituents across different

locations will be identified through this analysis.

 3

The geographical location and seasonal variations along with

environmental conditions (e.g. temperature, light intensity, salinity, nutrition, pH)

significantly contribute to the chemical diversity observed in seaweeds (Lorenzo et

al. 2017; de Melo et al. 2021), making it a noteworthy aspect for research. These

factors play a noteworthy role in shaping the interplay of compounds and

metabolites in seaweeds, suggesting that their biological activities stem from a

combination of metabolites rather than individual compounds (Wirawan et al. 2022;

Palaniyappan et al. 2023).

Although highly adaptable to various environments, C. lentillifera is

considered a stenohaline macroalgae. It is sensitive to lower salinities caused by

rainfall during the monsoon season, affecting its growth and mortality (Rabia,

2016). C. lentillifera can tolerate a salinity range of 20 to 50 and can develop at 30

to 40 parts per thousand salinities. The seaweed can withstand water

temperatures of about 25°C and 30°C. Pond depth should be about 0.5 m and

areas of about 0.5 ha are usual (Trono and Ganzon-Fortes, 1988). A study by

Delan et al. (2015) investigated the impact of habitat on the quality traits of C.

lentillifera collected from Bohol and Cebu islands. The study revealed significant

variations in heavy metal concentrations (Hg and Pb) among the seaweed

sources, with higher levels found in Cebu samples. Wild Cebu samples had higher

lethal values, lower bacterial counts, and different proximate composition than

Bohol samples. E. coli levels showed no significant variation across all samples,

and direct pollutant indicators (PO4, NH3, and NO3) were detected at low levels

in both island samples. These findings highlighted the influence of habitat on the
 4

quality characteristics of sea grapes and their potential as indicators of

environmental pollutants. It has been confirmed that, aside from the limited studies

on its bioactive phytochemicals, no studies have been reported to evaluate the

influence of varying geographical locations on the phytoconstituents of this

seaweed species. Further investigations are necessary to address this knowledge

gap and understand the seaweed's phytochemical composition.

The present study performed qualitative phytochemical screening on C.

lentillifera ethanolic extracts to identify unexplored secondary metabolite

components with potential therapeutic importance. It was sought to compare the

detected phytoconstituents of C. lentillifera samples collected from two selected

farms in Cebu, Philippines, to determine the geographic location that exhibits a

more significant number of recorded phytoconstituents, which can be valuable

input for industries such as food, cosmetics, and pharmaceuticals. A novel

dimension to existing knowledge was provided by this comparison by considering

the influence of geographical location on the total number of phytoconstituents in

seaweed. To the best of the researcher's knowledge, the impact of geographical

location on the biochemical compositions of C. lentillifera was evaluated for the

first time in the current study. The data collected will also support local

communities, particularly fisherfolk and farmers, in implementing sustainable

phycoculture practices and responsible wild harvesting of seaweed, aligning with

the United Nations' Sustainable Development Goals, explicitly promoting

economic growth, good health, and well-being.

 5

Theoretical Background

Ecological niche theory can be applied to understand the growth and

distribution patterns of C. lentillifera in different environmental conditions. The

ecological niche theory introduced by Joseph Grinnell (1917) focused on the

habitat requirements of a species for it to survive and reproduce, while Hutchinson

(1959) expanded on this definition by considering the multiple resources that a

species requires, such as the physical space it occupies, temperature, moisture,

food requirements, and interactions with other species. Seaweed species like C.

lentillifera provide ecosystem services such as environmental benefits and the

provision of food and have a substantial impact on the achievement of the UN's

Sustainable Development Goals (SDGs). It is correlated to Republic Act No.

10654, which supported the significance of seaweed species as an integral marine

resource (Raffo et al. 2022).

Ecological niche models are valuable tools for predicting habitat distribution

(Mota-Vargas et al. 2013; Peterson, Ball & Cohoon, 2002) and rely on

environmental factors and data on where the species (Morrison et al. 2012) inhabit.

It offered valuable insights into the preferences of individual species by examining

factors such as temperature, light availability, nutrient availability, and interactions

with other organisms (Parmesan and Yohe, 2003; Poloczanska et al. 2013).

Temperature, in particular, profoundly influenced seaweeds' survival, recruitment,

growth, and reproduction, and seaweed distributions are correlated with sea

surface temperature (SST) isotherms (Lüning et al. 1990).

 6

Factors such as temperature, salinity, light, water movement, tidal and

current patterns, dissolved oxygen, and nutrient availability influenced seaweed

dispersal, recruitment, survival, growth, and distribution. These factors can vary

spatially and temporally, affecting the overall dynamics of seaweed populations.

Some environmental habitats of seaweed have different salinity and temperature

that affect their growth and bioactive compounds, and C. lentillifera tends to grow

better under natural light and longer lighting duration (Fakhrulddin, I. M, et. al.

2021). Stress generated by salinity can trigger plants and algae to produce higher

secondary metabolites (Gengmao et al. 2015). This condition might affect the

growth of C. lentillifera, resulting in the production of several secondary

metabolites. The diverse tolerance of C. lentillifera to the natural environment

adaptation makes it an inversive species in some places (Z. Muta Harah, 2014).

When understood, the ecological niche of seaweed species like C. lentillifera can

lead to information on their growth and distribution patterns and their potential for

providing ecosystem services and non-food products. The present study

determined significant variations in the phytochemical constituents found in

selected locations, and light was shed on how the environmental factors where

seaweed grows influence the overall number of recorded phytoconstituents.

 7

Research Framework

INPUT PROCESS OUTPUT

- Experimental plant: - Collection and Identification of - Phytochemical
Seaweed Seaweed profile of sea
(Caulerpa - Seaweed Ethanolic Extraction grape
Lentillifera, J Agardh, - Phytochemical Screening Process Caulerpa
1837) • Alkaloids lentillifera from
• Carbohydrates two selected
- Chemicals and • Cardiac glycosides farms in Cebu,
reagents • Flavonoids Philippines
• Phenols
• Proteins
• Saponins
• Sterols
• Steroids
• Tannins
• Terpenoids
• Coumarin

- Measurements of the Physico-

chemical Parameters of the
Seawater

- Data Analysis

Figure 1. Research Framework of the Study

The research framework of the study was represented in a flow chart (Fig.

1) illustrating how the study was conducted. The researchers presented the

variables of the study using the Input-Process-Output framework. This study

focused on the determination of the C. lentillifera bioactive compounds. The C.

lentillifera as the experimental plant, chemicals, and reagents for phytochemical

screening serve as the research input. The research process includes collecting

the sample, extraction, phytochemical screening, and water quality parameters

 8

measurement. Lastly, the research output was to identify if the study proved the

presence of secondary metabolites in the C. lentillifera extracts.

Objectives

The main objective of this study was to determine the phytochemicals

present in the ethanolic extract of C. lentillifera. Specifically, this study sought to

achieve the following objectives:

1. evaluate the influence of geographical locations on the biochemical

composition of ethanolic extract of C.lentillifera;

2. describe the influence of some physico-chemical parameters of

seawater on seaweed’s bioactive compounds in terms of

● salinity

● temperature

● pH

Hypothesis

Based on the objectives provided, the researchers formulated the null

hypothesis:

H0: There is no significant relationship between the detected

phytoconstituents of C. lentillifera samples collected and physico-chemical

parameters of seawater from two selected farms in Cebu, Philippines

 9

Significance of the Study

C. lentillifera is found in tropical and subtropical coastal waters and is widely

used in traditional medicine and cuisine in Southeast Asia, such as the Philippines.

Some studies suggest that it has significant potential as a source of natural

remedies and functional food ingredients. Its phytochemical constituents provide

several health benefits that can aid in preventing and treating various diseases.

The generalization of this study had redounded to the benefit of society due

to its significant contribution to the vast knowledge of phytochemicals. This study

intended to analyze the presence or absence of different phytoconstituents in C.

lentillifera used to prevent and treat various diseases. It provided alternative

substances that could be beneficial in identifying new substances that offered

potential health benefits. This research could have led to the discovery of

alternative substances that promoted a greater understanding of traditional

medicines and practices, leading to a greater understanding of their efficacy and

potential uses. The findings of this study could have been highly significant and

beneficial, particularly to the following:

Economy. The Philippines' coastline is the second longest in Asia and the fourth

longest in the world. However, the country's abundant marine resources remain

relatively unexplored and underdeveloped, particularly its often-remarkable

seaweed resources (Marquez et al. 2014). The potential of seaweed resources in

the country may improve people's social, economic, and health conditions because

methods to cultivate, extract, and characterize the chemical constituents of

seaweeds will encourage more investors to focus on marine-derived drugs and

 10

products significantly contributing to SDG 8 (Decent Work and Economic Growth)

and would likely provide increased job opportunities and increased income for

individuals and communities.

Industry. The findings of this study can be applied in the food and pharmaceutical

industries, implying that C. lentillifera is a viable source of bioactive substances

that can be used to create a product for public consumption. The potential health

benefits of C. lentillifera as a natural remedy and functional food ingredient can aid

in achieving the SDG on good health and well-being.

Healthcare. Previous studies have reported that seaweeds like C. lentillifera have

been traditionally used for medicinal purposes; however, the researchers believe

there is still limited information on its total phytoconstituents. Identifying bioactive

compounds in this seaweed can help validate its use as an alternative medicine

and potentially lead to the development of new drugs with therapeutic applications.

Community. Sea grape (C. lentillifera) thrives abundantly and highly consumed

seaweed species in numerous coastal areas, particularly Cebu, Philippines. The

findings of this study hold immense potential to educate and raise awareness

within the community about the diverse health benefits of seaweed consumption.

These findings can be explored further for developing novel food products using

C. lentillifera as a critical ingredient, offering a sustainable livelihood opportunity

and serving as an alternative food source among the local population.

 11

Scope and Limitation

This study solely focused on the phytochemical analysis of bioactive

compounds in the ethanolic extract of C. lentillifera, following the procedure

outlined by Guevarra et al. (2005). The algal specimens were freshly collected from

two selected farms across the province of Cebu, Philippines, where C. lentillifera

was harvested from the wild and cultivated through phycoculture practices (Trono,

1997). The collected samples from these selected locations were characterized

and compared based on the presence of bioactive compounds.

The results and conclusions were thus accurate only for the presence or

absence of phytochemical constituents in ethanolic C. lentillifera bioactive

compounds. However, the results could have been used as a basis for similar

studies that would be conducted in the future. The study's results were collected

and analyzed through observation by the researchers using the appropriate

apparatus provided right after the experimentation. The study was conducted

during the academic year 2023-2024.

 12

REVIEW OF RELATED LITERATURE

The review of literature for this study focused on the chemical composition,

properties, and potential applications of C. lentillifera. The information gathered

from this review might contribute to a better understanding of the potential health

benefits of consuming this green seaweed and provide insights for future research.

Towards the end of the chapter, a synthesis of the related literature and studies

and the gap discovered will be presented. Also, related legal bases are included.

Related Legal Basis

The conduct of research involving the collection, identification, and

analysis of natural resources, including seaweeds, is subject to various laws and

regulations in different countries, including the Philippines. The following are some

of the legal bases for conducting a study on the phytochemical analysis of green

seaweed Caulerpa lentillifera:

1. Republic Act No. 10654, amending the Philippine Fisheries Code of

1998, governs the management, conservation, and protection of the

country's fisheries and aquatic resources, including seaweed. It

directs the Department of Agriculture, through the Bureau of

Fisheries and Aquatic Resources, to develop and implement policies

and programs for the sustainable development of fisheries and

aquatic resources.

2. Republic Act No. 8423 establishes the Philippine Institute of

Traditional and Alternative Health Care (PITAHC) to promote and

 13

accelerate the development of traditional and alternative healthcare

practices in the Philippines. This law also establishes a traditional

and alternative healthcare development fund and encompasses

various aspects related to research and development.

3. Department of Agriculture Administrative Order No. 242 or the

National Plan of Action for the Conservation and Management of

Sharks - This administrative order provides for the conservation and

management of sharks and their habitats, which includes coral reefs

where seaweeds grow. It mandates integrating conservation and

management measures for sharks and their habitats in developing

policies and programs related to fisheries and aquatic resources.

Therefore, to conduct a study on the phytochemical analysis of green

seaweed C. lentillifera in the Philippines, researchers must comply with the

relevant laws and regulations, such as obtaining permits or licenses for collecting

and utilizing seaweed resources and ensuring the ethical conduct of research. By

following these legal guidelines, our research maintains its integrity, upholds

environmental sustainability, and supports the development of traditional and

alternative healthcare practices in the Philippines.

Phytochemicals

Phytochemicals are compounds found in plants that have been extensively

studied as potential medicinal agents in recent years. Phytochemicals exhibit

diverse bioactivities, such as antioxidant, antimicrobial, antivirus, and anticancer.

Scientists are actively investigating the antioxidant properties of plants to

 14

determine their ability to delay or reduce the harmful effects of free radicals

(Antolovich et al. 2002).

Natural products can be classified into three main groups: primary

metabolites, secondary metabolites, and high molecular weight polymeric

materials (Hanson J.R. et al. 2003). Primary metabolites, such as nucleic acids,

amino acids, and sugars, are present in all cells and play a central role in cellular

metabolism and reproduction. High molecular weight polymeric materials, such as

cellulose, lignins, and proteins, contribute to cellular structure. Natural product term

refers to any naturally occurring compounds but usually means secondary

metabolites.

Phytochemical analysis is the study of the chemical components and

characteristics of plants. This procedure entails detecting and measuring various

plant-based substances, such as alkaloids, flavonoids, and terpenoids, all of which

have significant therapeutic and dietary benefits (Alam et al. 2020). These

compounds are responsible for the distinctive colors, flavors, and smells of fruits,

vegetables, and other plant-based foods.

In a more recent study, Ghasemi Pirbalouti et al. (2017) reviewed the

phytochemical composition of various medicinal plants and highlighted their

potential pharmacological activities, including antioxidant, anti-inflammatory, and

antimicrobial effects. The authors emphasize the importance of continued

research to identify new bioactive compounds with therapeutic potential. Overall,

phytochemical analysis is a critical area of research that has the potential to

contribute to the development of new drugs and functional food ingredients.

 15

Caulerpa species Structure

Caulerpa spp. is a genus of green seaweed widely distributed in tropical

and subtropical regions such as the Philippines (Estrada & Dionisio, 2020). It is

distinguished by its distinct morphology, consisting of a single cell that can grow

several meters long, with branches and leaves that resemble those of higher plants

(Huisman et al. 2020). Caulerpa has a distinct structure that makes it resistant to

predators and environmental stresses such as high salinity and temperature

fluctuations (Chen et al. 2019).

Caulerpa species have three parts: rhizoids, stolons, and fronds. Rhizoids

are root-like structures that anchor the plant to the substrate, whereas stolons are

horizontal stems that connect the rhizoids and fronds. Fronds are leaf-like

structures responsible for photosynthesis and reproduction (Skelton et al. 2016).

Caulerpa lentillifera

Caulerpa lentillifera is a type of green seaweed in the Caulerpaceae family.

It has a distinct appearance, with long, slender, cylindrical branches resembling

green spaghetti and small, spherical air bladders known as "lentil balls" (Trono,

1997). It is most commonly found in tropical and subtropical regions, especially the

Indian and Pacific Oceans. It can attach to rigid substrates such as rocks, coral

reefs, and mangrove roots and grows in shallow waters, usually at depths of 1-2

meters (Ganzon-Fortes, 2015). It is a siphonous macroalgae, a giant single cell

with multiple nuclei, and can grow to 30 cm long. Instead of leaves, the algae have

bubbles that burst in the mouth, releasing an umami taste.

 16

The algae's upright twigs are light green, simple, and branched. They carry

transparent spherical twigs that measure around 1.5 mm in diameter. The main

body of the algae, called the thallus, consists of a horizontal branched stolon and

erect branches. The erect branches are densely covered with numerous short

branchlets along their length. Each small branchlet comprises a short stalk and a

round tip, measuring 1 and 3 mm in diameter. A noticeable feature of this species

is the distinct narrowing between the base of the round tips and the stem end. The

upright branches can grow up to 15 cm in height.

Figure 2. Close up image of C. lentillifera

C. lentillifera was traditionally harvested from the wild. However, the

accidental introduction of C. lentillifera to fish ponds on the island of Mactan,

province of Cebu, Central Visayas, Philippines, in the 1950s led to the first

commercial cultivation of seaweed. Approximately 400 hectares of ponds in Cebu

produce 12 to 15 tons of fresh C. lentillifera per year. The seaweed is typically

 17

harvested two months after initial planting and every two weeks after that based

on growth rates (Trono, 1987).

A study by Saito et al. (2010) described that C. lentillifera is high in

polyunsaturated fatty acids, including omega-3 fatty acids. Furthermore, this

seaweed contains high levels of minerals such as zinc, iron, calcium, potassium,

magnesium, sodium, and copper, all necessary for meeting the body's daily

nutritional requirements (Pea-Rodriguez et al. 2011). Seaweeds may have strong

antioxidant systems to protect themselves from oxidative damage because they

are constantly exposed to light and oxygen, which promote the production of free

radicals and oxidizing agents (Nguyen et al. 2011).

Caulerpa species are rich in vitamins and minerals (37.2%-46.2% dry

weight) and carbohydrates/dietary fiber (25.1%-55.4% dry weight). They have a

low lipid content (0.29%-1.64% dry weight) and a variable protein content. They

are also essential sources of commercially valuable polysaccharides like alginate,

agar, and carrageenan (Nguyen et al., 2011). Seaweed-derived bioactive

compounds have multiple potential applications in various industries, including

food, cosmetics, and pharmaceuticals. (Gao et al. 2021) Studying seaweed

polysaccharides is crucial because they have shown promising potential for

various health benefits, such as antioxidant, anti-inflammatory, and antitumor

activities. (Zhang et al. 2021). It is typically fresh or salted (Mary et al., 2009). C.

lentillifera has a high carbohydrate content. The -1,3-xylooligosaccharides

produced by C. lentillifera have antioxidant, anti-clotting, and anticancer properties

(induction of apoptosis in MCF-7 human breast cells) activities (Sun et al. 2017).
 18

In addition to food applications, C. lentillifera is used to treat wastewater

accumulated in shrimp farms (Apiratikul et al. 2011).

Dried C. lentillifera contains approximately 10% protein, 16.76%

polyunsaturated fatty acids, 1% lipids, 38.6% carbohydrates, and 37% ash by

weight. According to previous studies, it is also rich in essential minerals, omega-

3 fatty acids, dietary fibers, and vitamins A and C (Matanjun et al. 2009; Sharma

and Rhyu 2014). C. lentillifera extracts have been traditionally used to treat high

blood pressure, rheumatism, and diabetes, as well as bacterial and fungal

infections (Sharma et al. 2015). Studies suggest that C. lentillifera extracts have

potential as a drug or functional material to treat cancer and diabetes mellitus

(Nguyen et al. 2011; Sharma and Rhyu 2014).

Commonly found in C. lentillifera are phenolic antioxidants that may be used

to prevent liver disease (Vargas-Mendoza et al. 2014). Phenolic antioxidants work

by chelating metal ions, preventing radical formation, and improving the antioxidant

endogenous system (Khairy & El-Sheikh, 2015). C. lentillifera antioxidants are

essential in preventing free radical-induced diseases (Yamaguchi et al. 1998).

Several studies have recently revealed that C. lentillifera has the potential for

pharmaceutical and agricultural applications (Khairy & El-Sheikh, 2015).

Antioxidants derived from edible C. lentillifera protect against cancer cells.

These cause multiple damages to cancer cells by affecting their cytoskeletal

framework, cellular metabolism, cell cycle, endoplasmic reticulum function,

mitochondrial respiration, and enzymatic machinery. They are also known to

impact the immune system and play a role in diabetes (Mehra et al. 2019).
 19

Methods of Extraction

In the pharmaceutical context, extraction involves separating biologically

active components from plant or animal tissues while leaving inactive or inert

constituents behind. It is achieved through the use of selective solvents in standard

extraction procedures. Various preparations are employed for this purpose,

including decoctions, infusions, fluid extracts, tinctures, pilular extracts (semisolid),

and powdered extracts.

Maceration is a method where the whole or coarsely powdered crude drug

is placed in a stoppered container along with the solvent and left to stand at room

temperature for a minimum of three days. The mixture is then strained, pressed,

and filtered to obtain the desired extract.

Infusion involves the maceration of the crude drug for a short duration using

either cold or boiling water as the solvent. This process allows for the extraction of

active constituents.

Digestion is a form of maceration where gentle heat is applied during

extraction. This method aids in the release of active compounds from the crude

drug.

Decoction, on the other hand, involves boiling the crude drug in a specified

volume of water for a defined period. Afterward, the mixture is cooled, strained,

and filtered. A decoction is particularly suitable for extracting water-soluble

constituents that are heat-stable.

 20

It is important to note that the products obtained from plant sources through

these extraction methods are typically impure liquids, semisolids, or powders and

are intended for oral or external use only (Handa et al., 2008).

Factors Affecting the seaweed’s seaweed’s phytoconstituents

Numerous studies indicated that the composition of compounds in marine

algae can vary due to factors such as geographic location, environmental

conditions (e.g., temperature, light intensity, salinity, nutrition, pH), biotic factors

(e.g., pathogens, predators), and genetic structure (Wirawan et al. 2022;

Palaniyappan et al. 2023). The phytoconstituents present in macroalgae, such as

polysaccharides, phenolic compounds, pigments, and fatty acids, are known to

possess a wide range of bioactive properties responsible for protecting them

quickly from any changes that may occur in marine environments, including the

abnormal increase in temperature and radiation. These factors could stimulate or

inhibit the production of macroalgal bioactive constituents (Mæhre et al. 2014).

Understanding the factors that influence the phytoconstituents in macroalgae is

crucial for optimizing their production and harnessing their potential benefits.

Environmental factors play a significant role in shaping the phytochemical

composition of macroalgae. Light intensity and quality have been shown to

influence the synthesis and accumulation of specific phytoconstituents in

macroalgae. For instance, red and blue light wavelengths have been reported to

enhance the production of phycobiliproteins and pigments in red and green

macroalgae, respectively (Gao et al. 2020). Temperature variations also have a

notable impact on the phytoconstituent profiles of macroalgae. Higher

 21

temperatures have been found to increase the production of secondary

metabolites, including phenolic compounds and antioxidants, in certain macroalgal

species (Boo et al. 2019).

Nutrient availability, particularly nitrogen and phosphorus, plays a crucial

role in the growth and metabolism of macroalgae and can significantly affect their

phytoconstituent profiles. Nitrogen deficiency, for example, can stimulate the

synthesis of phenolic compounds, while phosphorus limitation may enhance the

accumulation of lipids in macroalgae (Kumar et al. 2018). Moreover, seasonal

changes and geographic factors contribute to the variations in phytoconstituent

composition observed in macroalgae. Fluctuations in light availability, temperature,

and nutrient levels throughout the year can result in seasonal variations in the

concentration of bioactive compounds, such as pigments and polysaccharides

(Lorenzo et al. 2017; de Melo et al. 2021). Furthermore, variations in water

temperature, salinity, nutrient availability, and light conditions across different

regions can lead to distinct phytochemical compositions in macroalgae from

different locations (Wang et al. 2021).

Biotic interactions also play a significant role in shaping the

phytoconstituents in macroalgae. Grazing pressure from herbivorous marine

organisms, such as sea urchins and fish, can induce defense mechanisms in

macroalgae, leading to the synthesis of secondary metabolites, including

phenolics and terpenoids, which serve as feeding deterrents (Liu et al. 2022).

Additionally, epiphytic organisms, such as bacteria and fungi, can colonize the

surfaces of macroalgae and influence their phytoconstituent profiles. Some

 22

epiphytes can produce bioactive compounds that interact with the host macroalga,

leading to changes in its secondary metabolite production (Tang et al. 2021).

Synthesis of the Related Literature and Studies

The researchers had been motivated to explore the potential of seagrape

(C. lentillifera) as a rich source of bioactive compounds. Seaweed was renowned

for its diverse bioactive compounds, including antioxidants, antimicrobials, and

anti-inflammatory agents, which held immense potential in the food,

pharmaceutical, and cosmetic industries (Gao et al. 2021). In the coastal waters

of Central Visayas, C. lentillifera, locally known as “lato,” was traditionally

consumed for its potential health benefits and served as a vital source of income

for local communities. While there had been studies investigating its nutritional

value and some bioactive compounds, the researchers firmly believed there were

still unexplored compounds waiting to be discovered. Furthermore, certain factors,

such as geographical locations, which could potentially influence the

phytoconstituents of seaweed, still need to be understood and have received

limited attention. Hence, the researchers set out to conduct a comprehensive

phytochemical analysis of sea grapes (C. lentillifera) to understand their bioactive

composition better. They also sought to contribute to the existing body of

knowledge by investigating the impact of geographical location on the diversity and

abundance of phytoconstituents in C. lentillifera. Through this study, they aspired

to shed light on the untapped potential of C. lentillifera and provide valuable

insights into the influence of geographical factors on its phytoconstituents for

potential application across various industries.

 23

Gaps Bridged

The researchers conducted a review of related literature and studies. They

discovered that the studies at that time focused on the chemical constituents,

properties, and potential health benefits of C. lentillifera. Despite the potential

health benefits and industrial applications of C. lentillifera, little was known about

its bioactive composition and quantity. The impact of extrinsic factors such as

geographical location on its compound composition has yet to be explored.

Therefore, the study of the phytochemical analysis of green seaweed C. lentillifera

aimed to bridge this research gap by comprehensively analyzing its bioactive

composition and potential applications. Additionally, by investigating the influence

of geographical factors on the compound composition, the study intended to

contribute to a better understanding of how such factors could shape the

phytoconstituents of the seaweed.

 24

MATERIALS AND METHODS

The study’s research techniques are detailed in this chapter. It includes a

review of the research design. It focused on the processes involved in identifying

the phytochemical constituents of C. lentillifera. Additionally, it elucidates the

sampling site, the research sample used, the sources of data, and the statistical

treatment employed to ensure accurate interpretation of the collected data.

Research Design

A descriptive-comparative research design was used to determine the

bioactive compounds in the ethanolic extract of C. lentillifera between samples

collected from two selected farms within Cebu, Philippines. This research design

uses statistical analysis to prove or disprove a hypothesis (Shuttleworth, 2018).

Sampling Site

The algal specimens for this study were collected from two carefully

selected locations within Cebu, Philippines, guided by approval from local

authorities and the owners of seagrape ponds (see Appendix E). The first location

is a commercial seawater culture pond in Kalawisan, Lapu-Lapu, Mactan Island,

Cebu (10.284093° N, 123.934694° E). The second location is also a plantation in

Dawis, Lamintak Norte, Medellin, Cebu (11.108388° N, 123.960691° E). The

collection of samples took place last October 2023, a season of commercial

production. The collected samples were authenticated at the Bureau of Fisheries

and Aquatic Resources (BFAR) VII located along Arellano Boulevard, Pier Area,

Cebu City (refer to Appendix F). The extraction and testing process was conducted
 25

at the Biology and Chemistry Laboratory in Cebu Technological University- Argao

Campus, located at VHJX+VG2, Ed, Isidro Kintanar St., Argao, 6021 Cebu City

and Cebu Technological University-Main Campus located alongside MJ Cuenco

Ave, Cor R. Palma St., 6000 Cebu City, Philippines respectively.

Research Sample

In this experimental study, the researchers focused on utilizing Caulerpa

lentillifera, locally known as “lato,” as the primary subject of investigation (see

Figure 3). A total of three (3) kilograms of fresh biological samples of C. lentillifera

were collected and analyzed from each site. Caulerpa lentillifera, commonly known

as sea grapes, is a type of edible green algae that thrives in the Asia-Pacific’s

coastal regions, particularly in the Philippines. This unique species belongs to the

ulvophyte group of green algae and is characterized by its siphonous structure. It

is a single, giant cell with multiple nuclei, capable of growing up to 30 cm in length.

The taxonomical classification of C. lentillifera, as listed in Algaebase - a

comprehensive global database for seaweed taxonomy, is as follows: Plantae

(Kingdom), Chlorophyta (Phylum), Ulvophyceae (Class), Bryopsidales (Order),

Caulerpaceae (Family), Caulerpa (Genus), and C. lentillifera (Species).

 26

Figure 3. Freshly collected sample of C. lentillifera

Experimental Procedures

Collection and Identification of Seaweed

The seaweed samples were carefully cleaned on-site with seawater to

remove sand and other particles, then placed in a polyethylene bag with seawater

and stored in a secure, airtight container without direct exposure to ice or intense

light during transport. The experts from the Bureau of Fisheries and Aquatic

Resources (BFAR Region VII) authenticated the collected samples in Cebu City.

Following procedures by Tsuda and Abott (1985), as cited by Geraldino et al.

(2005) with slight adjustments, the specimens were preserved in plastic jars with

a 7% formalin solution in seawater and placed at the herbarium with accession

number 2024.6.1-4 and 2024.7.2-4 (see Appendix F).

 27

Ethanolic Extraction of C. lentillifera

The preparation of the extract followed the protocol described by Guevarra

et al. (2005) with minor modifications. Approximately three (3) kilograms of fresh

C. lentillifera were harvested from each site and initially rinsed on-site with

seawater to eliminate epiphytes, sand, calcareous particles, and other adhering

debris. The samples were carefully detached from their substratum by hand,

placed in a polyethylene bag, and transported to one of the researcher’s houses.

Upon arrival, the samples underwent a second thorough washing with seawater,

rinsing with prepared sterile water, and air-dried for seven days, as Deyab (2016)

recommended. The air-dried C. lentillifera samples were weighed, cut into small

pieces, and ground using a household blender. The dried powdered samples were

submerged and macerated in 95% ethanol for at least 72 hours at 1:4 w/v

concentrations. The extract was gently filtered using a filter cloth followed by

Whatman filter paper No. 1. This process was repeated until all the compounds

had been extracted. The filtrate from each sample was concentrated in a rotary

evaporator (RE 100-Pro) at a temperature of 40˚C and a speed of 100-110 rpm to

remove the alcohol. The residue was collected and weighed to obtain the

percentage yield. Finally, the crude C. lentillifera ethanolic extracts were stored in

a refrigerator set at 4°C until the phytochemical screening process.

Phytochemical Screening Process

The prepared ethanolic extracts of C. lentillifera were brought to the

Chemistry Laboratory at Cebu Technological University-Main Campus for

evaluation of the presence of phytoconstituents using different qualitative chemical

 28

tests described by Guevarra (2005), as cited by Labiaga et al. (2021). The extracts

were tested for phytoconstituents such as alkaloids, carbohydrates, flavonoids,

glycosides, saponins, steroids, and tannins. (Table 1).

Table 1. Qualitative Phytochemical Screening of metabolites (Guevarra,

2005)

PHYTOCONSTITUENTS TESTS

Alkaloids Wagner’s Reagent

Carbohydrates Molisch’s test

Cardiac glycosides Keller Kelliani’s test

Flavonoids Alkaline reagent test

Phenols Ferric Chloride test

Proteins 1% ninhydrin solution in acetone

Saponin Foam test

Sterols Liebermann-Burchard test

Steroids

Tannins Braymer’s test

Terpenoids Salkowki’s test

Coumarins NaOH test

Measurements of the Physico-chemical Parameters of the Seawater

The researchers measured the physico-chemical parameters of the

seawater during the seaweed collection. Salinity, temperature, pH, and dissolved
 29

oxygen were measured on-site with a handheld refractometer (YY-1010) and

recorded every three seconds.

Ethical Considerations

The researchers secured an approved biosafety/ biorisk clearance from the

Institutional Biorisk Management Committee (IBMC) of Cebu Technological

University with a protocol number of 2023-MA-054 (refer to Appendix B). The

researchers are committed to upholding the highest standards of integrity and

professionalism throughout the study. The researchers presented their findings

entirely and truthfully. The information gathered was not falsified or

misrepresented. Detailed records of all research activities, including data

collection, were kept. The work was scrutinized thoroughly and critically before

conclusions and recommendations were made.

Data Privacy

The researchers adhered rigorously to the highest ethical standards and

complied with data privacy laws to ensure integrity and confidentiality. A robust

and comprehensive data management system was implemented, employing

stringent security measures for storing, encoding, coding, accessing, and sharing

data. The project leader kept raw data. These raw data were accessible only to

researchers. The researchers did the encoding and coding. Access to the

completed research files was restricted to the institution and the target publisher

for monitoring and validation purposes. If other researchers would like to make a
 30

secondary data analysis for a valid reason, they can make a request to the office

of research with the researchers' consent.

Statistical Treatment

The data gathered was subjected to statistical analysis. An independent

sample T-test was carried out using MS Excel to determine whether there is a

significant relationship between the physico-chemical parameters of seawater and

seaweed bioactive compounds from two different locations. The data analysis was

set at a 95% confidence level, with a statistical significance of P ≤ 0.05. All

quantitative and qualitative data were analyzed, and the results were presented in

tables.

Definition of Terms

To fully understand some of the technical terms used in the study, the terms

are conceptually and operationally defined:

Alkaloids. These are a group of naturally occurring compounds that contain at

least one nitrogen atom in a heterocyclic ring and have pharmacological effects on

living organisms.

Bioactivity. The ability of a compound to interact with biological systems, including

therapeutic and dietary benefits.

Carotenoids. A class of pigments that give plants their yellow, orange, and red

colors. They have antioxidant properties and have been associated with the

prevention of chronic diseases.

 31

Caulerpa lentillifera. It is a green seaweed known as "sea grapes" or "green

caviar". It is found in tropical and subtropical regions and has small, round,

translucent green leaves resembling caviar beads. It was used as a source of

extract in the study and a test medium/subject to demonstrate the presence of

phytochemicals.

Flavonoids. A class of polyphenolic compounds abundant in fruits, vegetables,

and seaweeds. They have antioxidant and anti-inflammatory properties and have

been linked to a reduced risk of chronic diseases.

Phycobiliproteins. Water-soluble pigments are found in cyanobacteria and some

algae, including C. lentillifera. They have antioxidant and anti-inflammatory

properties and have been used as natural colorants and nutraceuticals.

Phytochemical analysis. Identifying and quantifying the chemical components in

plants or plant-based materials, including their structure and function. It involves

various techniques such as extraction, isolation, and characterization of bioactive

compounds, which can help determine their potential health benefits and

applications. This study focuses on determining the presence of phytochemical

constituents in C. lentillifera.

Phytochemicals. Secondary metabolites with significant bioactivity are found in

plants, including antioxidant, antimicrobial, antivirus, anticancer, and other

properties.
 32

Polyphenols. A class of phytochemicals that are widely distributed in plants,

including seaweeds. They have antioxidant and anti-inflammatory properties and

have been associated with the prevention of chronic diseases.

Saponins. A hygroscopic amorphous saponin combination is utilized as a

foaming, emulsifying, and detergent agent.

Tannins. Polyphenolic compounds bind to proteins, amino acids, and alkaloids

and precipitate.
 33

RESULTS AND DISCUSSION

In this chapter, the data gathered in this study, along with its analysis and

interpretation, are presented. It revealed the presence of bioactive compounds in

the ethanolic extracts of C. lentillifera and the significant relationship between the

measured physio-chemical parameters of seawater.

Seaweed identification

The results of the collected seaweed samples have led to the identification

of Caulerpa lentillifera (J. Agardh, 1837), as depicted in Figure 3 below. The

species identification was examined based on the morphological and anatomical

features of the seaweed (Trono et al. 1997). These characteristics include a thallus

composed of elongated horizontal stolons with a few rhizoidal branches beneath

and numerous upright, grape-like branches above.

Figure 4. Morphology of Caulerpa lentillifera J. Agardh. 1837

 34

Qualitative phytochemical analysis

The extraction yields of C. lentillifera were 1.17% for Kalawisan and 5.67%

for Medellin (see Appendix I); therefore, samples from the latter hold the most

significant yield. This is because the majority of the content of fresh C. lentillifera

is water. Salt crystals were produced upon drying and removed by washing the

product with sterile water and air-drying it again for three days.

Using standard tests and procedures, the qualitative phytochemical

screening was performed on the C. lentillifera extracts. The results confirmed that

biologically active compounds were present in the C. lentillifera extract. These

findings are expressed as (+) for the presence and (-) for the absence of the

phytoconstituents, as summarized in the table below.

Table 2. Results of the Phytochemical Screening from the ethanolic extract

of Caulerpa lentillifera

Phytochemicals Chemical Test CEE.K CEE.M

Alkaloids Wagner’s reagent +++ ++

Carbohydrates Molisch test +++ +++

Cardiac Keller-Killiani’s test +++ +++

glycosides

Flavonoids Alkaline Reagent test ++ +++

Phenols Ferric Chloride test +++ +

Proteins 1% ninhydrin solution +++ +++

in ethanol
 35

Saponins Foam test +++ +++

Sterols Liebermann-Burchard + +

test

Steroids +++ +

Tannins Braymer’s test +++ +

Terpenoids Salkowki’s test +++ +++

Coumarin NaOH test +++ +++

Key:
CEE.K: C. lentillifera Ethanolic Extract Kalawisan
CEE.M: C. lentillifera Ethanolic Extract Medellin
-: Negative (absent)
+: Positive (slightly present)
++: Positive (moderately present)
+++: Positive (highly present)

The ethanolic extracts of C. lentillifera collected from two selected farms in

Cebu, Philippines, have been analyzed for their phytochemical composition. The

results revealed the presence of several secondary metabolites, including

alkaloids, carbohydrates, cardiac glycosides, flavonoids, phenols, proteins,

saponins, sterols, steroids, tannins, terpenoids, and coumarin. Table 2 shows that

both samples tested highly positive for carbohydrates, cardiac glycosides,

proteins, saponins, terpenoids, and coumarins but had only traces of sterols. It

contrasts with the findings of Shevchenko et al. (2009), who claimed that C.

lentillifera is abundant in sterols, with clionasterol being the most prevalent

steroidal element in the sterol fraction. The weak presence of sterols could be due
 36

to the poor solubility of the said phytochemical in ethanol (Hanani, 2021) and

relatively its growth condition is affected by various factors such as salinity and

nutrient levels (Guo et al. 2015). The results also indicated a slight variation in the

recorded phytoconstituents between the two sampling sites. The C. lentillifera

sample taken from Kalawisan, Lapu-Lapu City (CEE.K), was abundant in alkaloids,

phenols, steroids, and tannins. In contrast, the C. lentillifera sample from Lamintak

Norte, Medellin (CEE.M) demonstrated a moderate presence of alkaloids but was

deficient in phenols, sterols, steroids, and tannins. These differences could be

attributed to the effect of extraction method, choice of solvent, geographical

location, environmental condition, and seasons of collection (Ekpo et al. 2009;

Gosch et al. 2012; Estrada et al. 2021; Nurkolis et al. 2022).

Table 3. Some physico-chemical parameters of water in Kalawisan C.

lentillifera farm
 37

Table 4. Some physico-chemical parameters of water in Medellin C.

lentillifera farm

The physico-chemical parameters of the two locations, Medellin and

Kalawisan, were tested using the refractometer (YY-1010). Based on the above

data, the results showed a difference in the parameters recorded from the two

localities. The salinity values were relatively higher at Medellin (Table 4) than at

Kalawisan (Table 3), which measured 2. 07, 2.36, and 2.46, respectively. For

temperature, the values range from 33.9 to 36 °C. The temperature increases from

the first data set to the second set. It could indicate a rise in temperature over time

due to evaporation or a temperature difference between two different periods.

Lastly, the pH values are relatively close, indicating that the water's acidity levels

are fairly consistent across the different areas and data sets.
 38

Table 5. T-test results between physico-chemical parameters of two

localities

Table 5 demonstrates that the p-value for salinity is 0.0241824. Since the

value is less than 0.05, it was found that the effect of salinity on the bioactive

compound of seaweed is statistically significant. However, for temperature and pH,

the p-values calculated are higher than 0.05; therefore, parameter changes might

not significantly impact the bioactive compounds of the seaweed.

Discussion

Phytochemicals are bioactive compounds present in plants that, while not

contributing to nutrition, play other significant roles (Kumar et al. 2009). Seaweeds

are particularly rich in these phytochemicals, which include a wide array of

compounds such as carotenoids, phycobilins, fatty acids, polysaccharides,

vitamins, sterols, tocopherol, and phycocyannins, among others (Bhat et al. 2005;

Hanani, 2021). These diverse phytochemicals have been found to exhibit a vast

array of biological activities—the present study aimed to identify the unexplored

secondary metabolites in green algae C. lentillifera.

Polar solvents such as ethanol have been found to yield a moderate to high

presence of flavonoids and phenols (Table 2), suggesting that this seaweed

species could serve as a potential medicinal resource (Mehmood et al. 2022).

Numerous research studies demonstrate that flavonoids possess a broad

spectrum of pharmacological benefits, including anti-inflammatory, antimicrobial,

and antitumor, and their potential for preventing clotting and fighting cancer (Sun
 39

et al. 2017). However, among these properties, flavonoids are best described as

powerful antioxidants alongside polyphenols and phenols, which constitute a

diverse group of substances responsible for safeguarding biological systems

against harmful effects of reactive oxygen species (ROS) on macromolecules

(Atmani et al. 2009; Silva et al. 2017; Yap et al. 2019).

The presence of alkaloids in C. lentillifera in the current study supports the

findings of Lunagariya et al. (2019) which identified the bisindolic alkaloid caulerpin

in various Caulerpa species. In various studies, Caulerpin has shown significant

pharmacological potential, including anti-inflammatory and pain-relieving

(antinociceptive), anticancer, antibacterial, antidiabetic, and antiplasmodial

activities (De Souza et al. 2009; Darmawan et al. 2020). However, despite these

promising findings, researchers stress that isolating the specific type of flavonoids

and phenols present in C. lentillifera, along with alkaloid caulerpin needs to be

further optimized and explored for future studies to fully understand its

mechanisms of action and potential side effects (Tong et al. 2022).

Both extracts of C. lentillifera were found to contain phytochemicals such as

steroids and terpenoids (refer to Table 2). These findings align with the research

conducted by Tapotubun et al. (2019) and Asmara, D. A. (2015) on Caulerpa sp.

from the Kei Islands and Tual Coastal Waters in Indonesia. According to Angka

and Suhartono (2000), steroid/triterpenoids, lipid-soluble compounds, can

penetrate the cell walls of gram-positive and gram-negative bacteria. They can

inhibit bacterial growth by interfering with protein synthesis and altering the cell

structure. It is corroborated by the fact that triterpenes, which make up a significant

 40

portion of seaweed's lipid substances, have been isolated in large numbers (over

4000 triterpenoids), as stated by Seigler, D.S. (1998). Saponins, which are a type

of terpenoids, have been found to exhibit several beneficial properties. These

include reducing cholesterol levels, exhibiting cytotoxic effects, and possessing

antibacterial and antiviral activities (Bailly & Vergoten, 2020). Tannins show an

anticancer property perceptible from their inhibitory activity towards growth (Mazni

et al. 2016). Green algae, known to contain significant amounts of coumarins,

could have potential applications in treating fungal infections, tumors, and asthma

and may also help reduce capillary permeability (Liu, 2011). To the researchers'

knowledge, no reported studies have explicitly mentioned the specific presence of

saponins, tannins, and coumarins.

Phenolic compounds, recognized for their antioxidant capabilities, are

believed to aid algae such as C. lentillifera to defend against abiotic stressors like

salinity and UV radiation (de la Coba et al. 2009; Nurkolis et al. 2023). Antioxidants

are known to counteract or decelerate cellular damage instigated by free radicals

- unstable molecules produced by the body in response to environmental stressors

(Yap et al. 2019). In Medellin, a marginal presence of phenols was detected in the

ethanolic extract of C. lentillifera compared to Kalawisan (Table 2). It has been

shown that the concentration of phenolic compounds in algae can fluctuate based

on habitat, seasonal changes, and local environmental factors such as salinity,

light, and nutrient availability (Jormalainem and Honkanen, 2008). A study by

Monsour et al. (2018) revealed that variations in salinity levels between the Red
 41

Sea and the Mediterranean Sea did not significantly affect the total phenol content

of Codium tomentosum.

In both locations where the seaweed is found, the water quality is brackish,

resulting in relatively lower salinity levels ranging from 0.96% to 2.48%. It differs

from previous research findings, which suggest that C. lentillifera can withstand

salinity levels of 30-40% (Guo et al. 2015; Wichachucherd et al. 2019). In the

current study, the seaweed was grown in open ponds without controlling light

exposure, subjecting it to broad daylight and high temperatures. Optimal conditions

for C. lentillifera culture were 27.5 ºC with a light intensity of 40 µmol photons m-

2s-2. In comparison, stress conditions were observed at 30 ºC with a light intensity

of 100 µmol photons m-2s-2. These findings align with previous studies (Guo et al.

2014). As seaweeds grow at low temperatures, there are changes in their bioactive

compounds, such as carbohydrates, protein, amino acids, lipids, fatty acids, ash,

and moisture (Palaniveloo K. et al. 2021). In specific geographical locations,

natural selection factors, geographical distance, and environmental factors, such

as temperature and salinity, influence seaweed's growth and total

phytoconstituents (Sun and Lin, 2003). The results obtained in the present study

suggested the identified phytochemical compounds may be the bioactive

constituents and this green alga proved to be an increasingly valuable reservoir of

bioactive compounds of substantial medicinal merit.

 42

CONCLUSION AND RECOMMENDATION

In this chapter, the conclusion and recommendations of the study are

presented. The conclusions were based on scientific interpretation, observation,

and explanation. The recommendations of the study were indicated to ease the

work during the process.

Conclusion

The qualitative phytochemical screening confirmed the presence of diverse

secondary metabolites in both extracts. Six (6) out of twelve (12) compounds

tested highly positive on the tests: carbohydrates, cardiac glycosides, proteins,

saponins, terpenoids, and coumarins. However, traces of sterol were found in both

extracts. Both extracts exhibited moderate to high amounts of alkaloids, flavonoids,

phenols, steroids, and tannins. These compounds possess potential bioactivities,

including anti-inflammatory, antimicrobial, and antioxidant properties, hinting at

their potential for developing functional foods, medicinal treatments, and

nutraceutical products. Also, it was revealed that there is a significant relationship

between the physicochemical parameters of seawater, such as salinity, and the

seaweed’s phytoconstituents.

Recommendation

Additional investigation into the isolation and characterization of

phytoconstituent from the sea grape C. lentillifera might be performed to determine

the various biological activities of the compound. The study was only based on

qualitative analysis and screening and was limited to using ethanol as the solvent.
 43

The researchers recommended further screening involving quantitative detection,

such as HPLC and bioactivity assessment of the various phytochemicals, to

understand the specific metabolites in similar seaweed.

 44

References

Alam, M. N., Bristi, N. J., and Rafiquzzaman, M. (2020). “Review on in vivo and in

vitro methods evaluation of antioxidant activity”. Saudi Pharmaceutical

Journal, 28(11), 1425-1434. https://doi.org/10.1016/j.jsps.2020.10.014

Angka S.L. Suhartono M.G. Marine product biotechnology. Pusat Kajian

Sumberdaya Pesisir dan Lautan. Institut Pertanian Bogor. Bogor. 2000,149

[in Indonesian].

Antolovich, M., Prenzler, P., Robards, K., & Ryan, D. (2002). "Phenolic compounds

and antioxidant activity of Olea europaea L. leaves and stems." Food

chemistry, 79(3), 353 358. DOI: 10.1016/S0308-8146(02)00133-9

Ang, Jr. P. O., Leung, S. M., Choi, M. M. (2013). A verification of reports of marine

algal species from the Philippines. Philipp J Sci 142:5–49

Asmara D.A. (2015). Kandungan Senyawa Fenol Anggur Laut (Caulerpa sp) Segar

dan Rebus dari Perairan Tual, Maluku. Skripsi. Departemen Teknologi Hasil

Perairan Fakultas Perikanan dan Ilmu Kelautan Institut Pertanian Bogor.

Skripsi. p.26

Bailly, C., & Vergoten, G. (2020). Esculentosides: Insights into the potential health

benefits, mechanism of action, and molecular target. Phytomedicine. 79

Bhat, S.V., B.A. Nagasampagi, and M. Sivakumar. (2005). Chemistry of Natural

Products. Narosa Publishing House, India

 45

Caulerpa lentillifera J.Agardh, 1837. In World Register of Marine Species.

Retrieved February 5, 2024.

https://www.marinespecies.org/aphia.php?p=taxdetails&id=211475

Chen, X., Sun, Y., Liu, H., Liu, S., Qin, Y., & Li, P. (2019). Advances in cultivation,

wastewater treatment application, bioactive components of Caulerpa

lentillifera and their biotechnological applications. PeerJ, 7, e6118. doi:

10.7717/peerj.6118

Darmawan, M., Fajarningsih, N.D., Sihono, Irianto, H.E. (2020). Caulerpa:

Ecology, Nutraceutical and Pharmaceutical Potential. In: Nathani, N.M.,

Mootapally, C., Gadhvi, I.R., Maitreya, B., Joshi, C.G. (eds) Marine Niche:

Applications in Pharmaceutical Sciences Springer, Singapore.

https://doi.org/10.1007/978-981-15-5017-1_17

Delan, G., Legados, J., Pepito, A., et al. (2015). The Influence of Habitat on the

Quality Characteristics of the Green Macro Alga Caulerpa lentillifera Agardh

(Caulerpaceae, Chlorophyta). Tropical Technology Journal, 19(10).

https://doi.org/10.7603/s40934-015-0010-4

de la Coba, F. Aguilera, J. Fogueroa, F. L., de Galvez, M. V. and Herrera, E.

(2009). Antioxidant activity of mycosporine-like amino acids isolated from

three red macroalgae and one marine lichen, J. Appl. Phycol., 21: 161-169

De Souza, É.T.; Pereira de Lira, D.; Cavalcanti de Queiroz, A.; Costa da Silva,

D.J.; Bezerra de Aquino, A.; Campessato Mella, E.A.; Prates Lorenzo, V.;

De Miranda, G.E.C.; De Araújo-Júnior, J.X.; De Oliveira Chaves, M.C.; et

 46

al. The Antinociceptive and Anti-Inflammatory Activities of Caulerpin, a

Bisindole Alkaloid Isolated from Seaweeds of the Genus Caulerpa. Mar.

Drugs 2009, 7, 689-704. https://doi.org/10.3390/md7040689

Deyab, M., Elkatony, T., & Ward, F. (2016). Qualitative and quantitative analysis

of phytochemical studies on brown seaweed, Dictyota dichotoma.

International Journal of Engineering Development and Research, 4(2), 674-

678. Algal extracts. Proceedings of the National Academy of Sciences, India

Section B: Biological Sciences, 85(2), 643-651

Drewnowski, A., & Gomez-Carneros, C. (2000). Bitter taste, phytonutrients, and

the consumer: a review. The American Journal of Clinical Nutrition, 72(6),

1424-1435.

du Preez, R., Majzoub, M. E., Thomas, T., Panchal, S. K., & Brown, L. (2020).

Caulerpa lentillifera (Sea Grapes) Improves Cardiovascular and Metabolic

Health of Rats with Diet-Induced Metabolic Syndrome. Metabolites, 10(12),

500. https://doi.org/10.3390/metabo10120500

Ekpo M. A. and P.C. ETIM. (2009). Antimicrobial activity of ethanolic and aqueous

extracts of Sida acuta on microorganisms from skin infections. Journal of

Medicinal Plants Research 3: 621-624

El-Beltagi, H. S., Mohamed, A. A., Mohamed, H. I., Ramadan, K. M. A., Barqawi,

A. A., & Mansour, A. T. (2022). Phytochemical and Potential Properties of

Seaweeds and Their Recent Applications: A Review. Marine drugs, 20(6),

342. https://doi.org/10.3390/md20060342
 47

Estrada, J.L., Dionisio-Sese, M., & Bautista, N.S. (2020). Morphological variation

of two common sea grapes (Caulerpa lentillifera and Caulerpa racemosa)

from selected regions in the Philippines. Biodiversitas Journal of Biological

Diversity, 21, 1823-1832. DOI:10.13057/biodiv/d210508.

Estrada, J.L., Arboleda, M.D.M. & Dionisio-Sese, M.L. Current status of sea grapes

(Caulerpa spp.) farming and wild harvesting in the Philippines. J Appl

Phycol 33, 3215–3223 (2021). https://doi.org/10.1007/s10811-021-02533-

Fajriah S, Rizki IF, Sinurat E (2021) Characterization and analysis of the

antidiabetic activities of sulphated polysaccharide extract from Caulerpa

lentillifera. Pharmacia 68(4): 869–875.

https://doi.org/10.3897/pharmacia.68.e73158

Fakhrulddin, I. M., Muta Harah, Z., Shiamala, R. D., & Azrie, A. M. (2021). Effect

of salinity, light and fertilizer on Caulerpa lentillifera under culture conditions.

AIP Conference Proceedings, 2347(1), 020074.

https://doi.org/10.1063/5.0051569

Ganzon-Fortes, E. T. (2015). Caulerpa lentillifera (Bryopsidales, Chlorophyta) and

its potential as a tropical aquaculture crop. Journal of Applied Phycology,

27(5), 2007-2018.

Gao, Y., Zhai, Y., Huang, Y., Li, Z., Li, L., Li, X., ... & Yu, G. (2021). Seaweed

polysaccharides: structure, bioactivity, and application. Critical Reviews in

Food Science and Nutrition, 1-18.

 48

Garcia-Vaquero, M., Mora, L., & Hayes, M. (2019). In Vitro and In Silico

Approaches to Generating and Identifying Angiotensin-Converting Enzyme

I Inhibitory Peptides from Green Macroalga Ulva lactuca. Marine drugs,

17(4), 204. https://doi.org/10.3390/md17040204

Garcia-Vaquero, M., Rajauria, G., Miranda, M., Sweeney, T., Lopez-Alonso, M., &

O'Doherty, J. (2021). Seasonal Variation of the Proximate Composition,

Mineral Content, Fatty Acid Profiles and Other Phytochemical Constituents

of Selected Brown Macroalgae. Marine drugs, 19(4), 204.

https://doi.org/10.3390/md19040204

Gengmao, Z., Yu, H., Xing, S., Shihui, L., Quanmei, S., & Changhai, W. (2015).

Salinity stress increases secondary metabolites and enzyme activity in

safflower. Industrial crops and products, 64, 175-181.

Goraya, R. K., Bhatia, R. K., & Bansal, V. (2017). Seaweeds: a sustainable

functional food for complementary and alternative therapy. Trends in Food

Science & Technology, 66, 1-10.

Gosch, B. J., Magnusson, M., Paul, N.A. and Nys, R (2012). Total lipid and fatty

acid composition of seaweeds for the selection of species for oil-based

biofuel and bioproducts. Glob. Chang. Biol. Bioenergy, 4(6): 919-930.

Guiry, M.D. and Guiry, M.D. (2019). Algaebase. Worldwide electronic publication,

National University of Ireland, Galway. Available from:

https://www.algaebase.org/.
 49

Guo, H., Yao, J., Sun, Z. et al. (2015). Effects of salinity and nutrients on the growth

and chlorophyll fluorescence of Caulerpa lentillifera . Chin. J. Ocean.

Limnol. 33, 410–418. https://doi.org/10.1007/s00343-015-4105-y

Hanani, M.T. (2021). Phytochemical Constituents of Three Species of Marine

Macro-Algae from Sitio Usadda, Pangutaran, Sulu, Philippines. American

Journal of Applied Chemistry.Vol. 9, No. 4, 2021, pp. 97-101

Handa, S.S., et al. (2008). Extraction technologies for medicinal and aromatic

plants. International Center for Science High Technology.

Hanson, J.R. (2003). Natural products the secondary metabolites. The Royal

Society of Chemistry 1-27. Cambridge, U.K

Kapali J, Sharma K R. (2021). Estimation of phytochemicals, antioxidant,

antidiabetic and brine shrimp lethality activities of some medicinal plants

growing in Nepal. J. Med. Plants; 20 (80) :102-116 URL: http://jmp.ir/article-

1-3207-en.html

Khairy, H.M., & El-Sheikh, M.A. (2015). Antioxidant activity and mineral

composition of three Mediterranean common seaweeds from Abu-Qir Bay,

Egypt. Saudi Journal of Biological Sciences, 22(5), 623–630.

doi:10.1016/j.sjbs.2015.01.010

Labiaga, A. S., Montano, M. E., Ybanez, A. P., & Ples, M.B. (2021). Phytochemical

profiling of Eucheuma denticulatum (NL Burman) Collins and Hervey, 1917

and Padina minor (Yamada, 1925) seaweeds for compounds of potential

biomedical and pharmaceutical applications. DLSU Research Congress

 50

Lee, S. H., Jeong, S. J., Kim, B. H., Kim, O. H., Lee, J. H., & Choi, K. H. (2013).

"Inhibitory effect of Caulerpa lentillifera extract on nitric oxide production in

lipopolysaccharide-stimulated RAW 264.7 cells." Journal of food

biochemistry, 37(4), 453-458. DOI: 10.1111/jfbc.12008

Liu, H. (2011). Extraction and Isolation of Compounds from Herbal Medicines. In:

J. Willow and H. Liu (eds.) Traditional Herbal Medicine Research

Methods. John Wiley and Sons, Inc.

Lunagariya Jignesh, Bhadja Poonam, Zhong Shenghui , Vekariya Rohit and Xu

Shihai* (2019). Marine Natural Product Bis-indole Alkaloid Caulerpin:

Chemistry and Biology, Mini-Reviews in Medicinal Chemistry; 19 (9)

. https://dx.doi.org/10.2174/1389557517666170927154231

Mæhre, H.K.; Malde, M.K.; Eilertsen, K.E.; Elvevoll, E.O.J. (2014).

Characterization of protein, lipid and mineral contents in common

Norwegian seaweeds and evaluation of their potential as food and feed. J.

Sci. Food Agrc., 94, 3281–3290.

Mary, A., Mary, V., Lorella, A., & Matias, J.R. (2009). Rediscovery of naturally

occurring sea grape Caulerpa lentillifera from the Gulf of Mannar and its

mariculture. Curr Sci 97:1418–20

Matanjun P, Mohamed S, Muhammad K, Mustapha NM. (2010) Comparison of

cardiovascular protective effects of tropical seaweeds, Kappaphycus

alvarezii, Caulerpa lentillifera, and Sargassum polycystum, on high-

 51

cholesterol/high-fat diet in rats. J Med Food. Aug;13(4):792-800. doi:

10.1089/jmf.2008.1212. PMID: 20482284.

Mehra, R., Bhushan, S., Bast, F., & Singh, S. (2019). Marine macroalga Caulerpa:

role of its metabolites in modulating cancer signaling. Molecular Biology

Reports. doi:10.1007/s11033-019-04743-5

Mehmood, A., Javid, S., Khan, M.F. et al. In vitro total phenolics, total flavonoids,

antioxidant and antibacterial activities of selected medicinal plants using

different solvent systems. BMC Chemistry 16, 64 (2022).

https://doi.org/10.1186/s13065-022-00858-2

Nguyen, T. M. H., Ueng, J. P. Y., & Tsai, G. J. (2011). "In vitro antioxidant activities

of Caulerpa lentillifera." Journal of Food Science, 76(1), C115-C121. DOI:

10.1111/j.1750-3841.2010.01953.x

https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1750-3841.2010.01953.x

Nurkolis F, Taslim NA, Qhabibi FR, Kang S, Moon M, Choi J, Choi M, Park MN,

Mayulu N, Kim B. (2023). Ulvophyte Green Algae Caulerpa lentillifera:

Metabolites Profile and Antioxidant, Anticancer, Anti-Obesity, and In Vitro

Cytotoxicity Properties. Molecules. 2023 Jan 31;28(3):1365. doi:

10.3390/molecules28031365. PMID: 36771032; PMCID: PMC9919714.

Palaniyappan, S., Sridhar, A., Kari, Z. A., Téllez-Isaías, G., & Ramasamy, T.

(2023). Evaluation of Phytochemical Screening, Pigment Content, In Vitro

Antioxidant, Antibacterial Potential and GC-MS Metabolite Profiling of

 52

Green Seaweed Caulerpa racemosa. Mar. Drugs, 21(5), 278.

https://doi.org/10.3390/md21050278

Percutaneous permeation enhancement by terpenes: Mechanistic view. The

AAPS Journal, Vol. 10, issue 1 pp 120.1

Pechroj, S.., Potiparsat, K., Nangam, O., Athipornchai, A., Suriyaphan, J,

Muangham, S. ., & Srisook, K. (2020). Comparative evaluation of

antioxidant and anti-Inflammatory activities of four seaweed species from

the east coast of the Gulf of Thailand. Naresuan Phayao Journal, 13(3), 11–

21. Retrieved from https://li01.tci-

thaijo.org/index.php/journalup/article/view/245541

Peña-Rodriguez, A., Mawhinney, T. P., Ricque-Marie, D., Cruz-Suarez, L. E., &

Rodríguez-Padilla, C. (2011). Chemical composition of two tropical

seaweeds, Gracilaria domingensis and Ulva lactuca, as a feed source for

the abalone Haliotis asinina. Journal of Applied Phycology, 23(4), 567-570.

Ponnanikajamideen, M., Malini, M., Malarkodi, C., & Rajeshkumar, S. (2014).

Bioactivity and phytochemical constituents of marine brown seaweed

(Padina tetrastromatica) extract from various organic solvents. Int. J.

Pharm. Ther, 5, 108-112

Rabia, N. A. (2016). Seaweeds and its potential applications in the Philippines.

Journal of Global Innovations in Agricultural and Social Sciences, 4(2), 96-

101.
 53

Raffo, M. P., Dellatorre, F., & Ciancia, M. (2022). Seaweed resources of Argentina

(S W Atlantic): production, bio-ecological, applied research and challenges

for sustainable development. Pages 383-421. Retrieved from

https://www.tandfonline.com/doi/full/10.1080/26388081.2022.2076260

Rahul, M., Suresh, N., Anil, T., Arun, M., Nivedita, G., & Mv, B. (2014). GC-MS

analysis of phytocomponents of seaweed, Caulerpa racemosa. Int. J.

Pharma Res. Rev, 3(11), 39-45

Saito H, Xue CH, Yamashiro R, Moromizato S, Itabashi Y. (2010). High

polyunsaturated fatty acid levels in two subtropical macroalgae,

Cladosiphon okamuranus and Caulerpa lentillifera. J Phycol 46: 665-673.

Sakunthala, M., & Paul, J. J. P. (2019). GC-MS Analysis of Ethanolic Extract of

Caulerpa Mexicana Sonder Ex Kuetzing from Manapad Coast, Tamil Nadu,

India. J. Emerg. Technol. Innov. Res, 6, 244-247.

Seigler, D.S. (1998). Triterpenes and Steroids. In: Plant Secondary Metabolism.

Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-4913-0_23

Sharanabasappa, G. K., Santosh, M. K., Shaila, D., Seetharam, Y. N., &

Sanjeevarao, I. (2007). Phytochemical Studies on Bauhinia racemosa Lam.

Bauhinia purpurea Linn. and Hardwickia binata Roxb. E-Journal of

Chemistry, 4(1), 21-31.

Sharma, B. R., & Rhyu, D. Y. (2014). Anti-diabetic effects of Caulerpa lentillifera:

stimulation of insulin secretion in pancreatic beta-cells and enhancement of

 54

glucose uptake in adipocytes. Asian Pacific Journal of Tropical

Biomedicine, 4, 575-580.

Shevchenko, N.M., Burtseva, Y.V., Zvyagintseva, T.N. et al. (2009).

Polysaccharides and sterols from green algae Caulerpa lentillifera and C.

sertularioides . Chem Nat Compd 45, 1–5. https://doi.org/10.1007/s10600-

009-9223-3

Silva, A. M., Vidal-Novoa, A., Batista-González, A. E., Pinto, J. R., Portari

Mancini, D. A., Reina-Urquijo, W., & Mancini-Filho, J. (2012). In vivo and

in vitro antioxidant activity and hepatoprotective properties of polyphenols

from Halimeda opuntia (Linnaeus) Lamouroux. RedoxReport, 17(2), 47–

53.doi:10.1179/1351000212y.0000000003

Sun Y, Gong G, Guo Y, Wang Z, Song S, Zhu B, Zhao L, Jiang J. (2017).

Purification, structural features and immunostimulatory activity of novel

polysaccharides from Caulerpa lentillifera. Int J Biol Macromol. 2018

Mar;108:314-323. doi: 10.1016/j.ijbiomac.2017.12.016. Epub 2017 Dec 6.

PMID: 29222013.

Sun, Y. and Lin, FC. (2003). Analysis of genetic diversity in natural germplasm of

Lentinula edodes in China using RAPD technique. Mycosystema, 22: 387-

393.

Syakilla, N., George, R., Chye, F. Y., Pindi, W., Mantihal, S., Wahab, N. A., Fadzwi,

F. M., Gu, P. H., & Matanjun, P. (2022). A Review on Nutrients,

Phytochemicals, and Health Benefits of Green Seaweed, Caulerpa

 55

lentillifera. Foods (Basel, Switzerland), 11(18), 2832.

https://doi.org/10.3390/foods11182832

Tong, Y., Ma, H., Hu, T., Liu, Q., Li, Q., Chen, X., Yang, C., Peng, M., Zhu, W., &

Zhao, Y. (2022). Research Progress in the Components and Functional

Characteristics of Caulerpa lentillifera. Shipin gongye ke-ji, 43(7), 400-406

Trono Jr, G. C. (1997). The marine green algae of the Philippines: systematics,

ecology, and biogeography. University of the Philippines Press.

Trono, G.C., Jr. (1987) Studies on the pond culture of Caulerpa. Phil. Jour. Sci.,

Monogr. 17: 83–98.

Wichachucherd, B. (2019). Correlation between Growth, Phenolic Content and

Antioxidant Activity in the Edible Seaweed, Caulerpa lentillifera in Open

Pond Culture System. Journal of Fisheries and Environment, 43(2), 66-75

Yap, W.-F.; Tay, V.; Tan, S.-H.; Yow, Y.-Y.; Chew, J. (2019). Decoding Antioxidant

and Antibacterial Potentials of Malaysian Green Seaweeds: Caulerpa

racemosa and Caulerpa lentillifera. Antibiotics. 8, 152.

https://doi.org/10.3390/antibiotics8030152

Zhang, Q., Chen, Y., Sun, X., & Chen, J. (2021). The Biological Activities and

Applications of Marine Polysaccharides: A Review. Marine Drugs, 19(3),

 56

APPENDICES

APPENDIX A

APPROVAL SHEET (PROPOSAL)

Republic of the Philippines

CEBU TECHNOLOGICAL UNIVERSITY
MAIN CAMPUS
M.J. Cuenco Avenue Cor. R. Palma St., Cebu City,
Philippines
(SUC Level IV & AACUP Accredited)
url: http://www.ctu.edu.ph email: information@ctu.edu.ph
Tel. No. 402-4060 local 1120

COLLEGE OF EDUCATION

CERTIFICATE OF PANEL APPROVAL

The research proposal attached hereto, entitled, “PHYTOCHEMICAL PROFILE OF THE

SEA GRAPE CAULERPA LENTILLIFERA (J. AGARDH) FROM CENTRAL VISAYAS,
PHILIPPINES” was prepared and submitted by JEZELLE MAE MARIE P. ALQUIZALAS, GLEIN
G. CENIZA, JILL JAVA, and ISABEL MARIE D. TANUCO is hereby recommended for approval.

ALITA S. LABIAGA June 13, 2023

Member

DARLENE KEZIAH GRACE MOLATO-RECAMARA June 13, 2023

Member

MARIA SALUD M. DELOS SANTOS June 13, 2023

Chairman

This research paper is approved in partial fulfillment of the requirements of SCIM

3215.

APPROVED BY:

REYLAN G. CAPUNO Dev. Ed. D., Ph. D

Dean, College of Education

 57

APPENDIX B

ETHICS CERTIFICATE
58
 59

APPENDIX C

WORKPLAN
 60

APPENDIX D

GAD SCORE
61
62
 63

APPENDIX E

TRANSMITTAL LETTERS
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
 79

APPENDIX F

CERTIFICATE OF AUTHENTICATION
80
 81

APPENDIX G

ACCESSION RECORDS
82
 83

APPENDIX H

PHYTOCHEMICAL SCREENING RESULTS

Figure 5 Test for Alkaloids (Wagner's-reagent test)-

Medellin to determine the presence of reddish-brown
precipitate, CTU-Main Chemistry Laboratory, January 11,
2023 (5:30 P.M)

Figure 6 Test for Carbohydrates (Molisch’s-reagent Test)-

Medellin to determine the presence of red/dull violet color,
CTU-Main Chemistry Laboratory, January 11, 2023 (5:35
P.M)
 84

Figure 7 Test for Cardiac glycosides (Keller Kelliani’s-

reagent Test)- Medellin to determine the presence of brown
ring at interface of layers, CTU-Main Chemistry Laboratory,
January 11, 2023 (5:43 P.M)

Figure 8 Test for Flavonoids (Alkaline reagent test)-

Medellin to determine the presence of intense yellow color
(turns colorless upon addition of dilute HCl), CTU-Main
Chemistry Laboratory, January 11, 2023 (5:43 P.M)
 85

Figure 9 Test for Phenols (Ferric Chloride-reagent test)-

Medellin to determine the formation of bluish-black color,
CTU-Main Chemistry Laboratory, January 11, 2023 (5:45
P.M)

Figure 10 Test for Proteins (1% ninhydrin solution in

acetone) - Medellin to determine the formation of purple
color, CTU-Main Chemistry Laboratory, January 11, 2023
(5:48 P.M)
 86

Figure 11 Test for Saponin (Foam Test) - Medellin to

determine the persistent foam, CTU-Main Chemistry
Laboratory, January 11, 2023 (5:48 P.M)

Figure 12 Test for Sterols (Liebermann-Burchard test) -

Medellin to determine the formation of dark pink/red color,
CTU-Main Chemistry Laboratory, January 11, 2023 (5:51
P.M)
 87

Figure 13 Test for Steroids - Medellin to determine the

formation of violet to blue/bluish-green color, CTU-Main
Chemistry Laboratory, January 11, 2023 (5:55 P.M)

Figure 14 Test for Tannins (Braymer’s-reagent test) -

Medellin to determine the formation of violet to blue or
greenish color, CTU-Main Chemistry Laboratory, January
11, 2023 (5:58 P.M)
 88

Figure 15 Test for Terpenoids (Salkowski's-reagent test) -

Medellin to determine the formation of reddish-brown
precipitate, CTU-Main Chemistry Laboratory, January 11,
2023 (6:00 P.M)

Figure 16 Test for Coumarin- Medellin to determine the

formation of yellow color, CTU-Main Chemistry Laboratory,
January 11, 2023 (6:05 P.M)
 89

Figure 17 Test for Alkaloids (Wagner's-reagent test)-

Kalawisan to determine the presence of reddish-brown
precipitate, CTU-Main Chemistry Laboratory, January 11,
2023 (5:30 P.M)

Figure 18 Test for Carbohydrates (Molisch’s-reagent Test)-

Kalawisan to determine the presence of red/dull violet
color, CTU-Main Chemistry Laboratory, January 11, 2023
(5:35 P.M)
 90

Figure 19 Test for Cardiac glycosides (Keller Kelliani’s-

reagent Test)- Kalawisan to determine the presence of
brown ring at interface of layers, CTU-Main Chemistry
Laboratory, January 11, 2023 (5:43 P.M)

Figure 20 Test for Flavonoids (Alkaline reagent test)-

Kalawisan to determine the presence of intense yellow
color (turns colorless upon addition of dilute HCl), CTU-
Main Chemistry Laboratory, January 11, 2023 (5:43 P.M)
 91

Figure 21 Test for Phenols (Ferric Chloride-reagent test)-

Kalawisan to determine the formation of bluish-black color,
CTU-Main Chemistry Laboratory, January 11, 2023 (5:45
P.M)

Figure 22 Test for Proteins (1% ninhydrin solution in

acetone) - Kalawisan to determine the formation of purple
color, CTU-Main Chemistry Laboratory, January 11, 2023
(5:48 P.M)
 92

Figure 23 Test for Saponin (Foam Test) - Kalawisan to

determine the persistent foam, CTU-Main Chemistry
Laboratory, January 11, 2023 (5:48 P.M)

Figure 24 Test for Sterols (Liebermann-Burchard test) -

Kalawisan to determine the formation of dark pink/red color,
CTU-Main Chemistry Laboratory, January 11, 2023 (5:51
P.M)
 93

Figure 25 Test for Steroids - Kalawisan to determine the

formation of violet to blue/bluish-green color, CTU-Main
Chemistry Laboratory, January 11, 2023 (5:55 P.M)

Figure 26 Test for Tannins (Braymer’s-reagent test) -

Kalawisan to determine the formation of violet to blue or
greenish color, CTU-Main Chemistry Laboratory, January
11, 2023 (5:58 P.M)
 94

Figure 27 Test for Terpenoids (Salkowski's-reagent test) -

Kalawisan to determine the formation of reddish-brown
precipitate, CTU-Main Chemistry Laboratory, January 11,
2023 (6:00 P.M)

Figure 28 Test for Coumarin- Kalawisan to determine the

formation of yellow color, CTU-Main Chemistry Laboratory,
January 11, 2023 (6:05 P.M)
 95

APPENDIX I

COMPUTATIONS

A. Percentage Yield of C. lentillifera Extract

𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑎𝑓𝑡𝑒𝑟 𝑑𝑟𝑦𝑖𝑛𝑔

𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑦𝑖𝑒𝑙𝑑 = × 100
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑏𝑒𝑓𝑜𝑟𝑒 𝑑𝑟𝑦𝑖𝑛𝑔
For C. lentillifera (Kalawisan)
35𝑔
𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑦𝑖𝑒𝑙𝑑 = × 100
3000 𝑔
Percentage yield = 1.17%

For C. lentillifera (Medellin)

170 𝑔
𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑦𝑖𝑒𝑙𝑑 = × 100
3000 𝑔
Percentage yield = 5.67 %
 96

CURRICULUM VITAE

JEZELLE MAE MARIE P. ALQUIZALAS

Dapdap, Pilar, Camotes, Cebu
Contact Number: 0910-413-2693
jezellemaemariepalquizalas@gmail.com

PERSONAL INFORMATION
Date of Birth : May 23, 2002
Place of Birth : Dapdap, Pilar, Camotes Island, Cebu
Age : 21
Gender : Female
Nationality : Filipino
Civil Status : Single
Religion : Roman Catholic

EDUCATIONAL BACKGROUND
Tertiary Level
2020-Present Cebu Technological University-Main Campus
M.J. Cuenco Avenue Cor. R. Palma St., Cebu City
Bachelor of Secondary Education major in Science

Secondary level
 97

Junior High School

2014-2018 Dapdap National High School
Dapdap, Pilar, Camotes Island, Cebu
Senior High School
2018-2020 Dapdap, National High School
Dapdap, Pilar, Camotes Island, Cebu

Primary level
2008-2014 Dapdap Elementary School
Dapdap, Pilar, Camotes Island, Cebu
 98

GLEIN G. CENIZA
Anapog, San Remigio, Cebu
Contact Number: 0919-944-8396
gleinceniza@gmail.com

PERSONAL INFORMATION
Date of Birth : November 14, 2001
Place of Birth : Anapog, San Remigio, Cebu
Age : 22
Gender : Male
Nationality : Filipino
Civil Status : Single
Religion : Roman Catholic

EDUCATIONAL BACKGROUND
Tertiary Level
2020-Present Cebu Technological University-Main Campus
M.J. Cuenco Avenue Cor. R. Palma St., Cebu City
Bachelor of Secondary Education major in Science

Secondary level
Junior High School
 99

2014-2018 San Remigio National High School

Goopio St., Poblacion, San Remigio, Cebu
Senior High School
2018-2020 San Remigio National High School
Goopio St., Poblacion, San Remigio, Cebu

Primary level
2008-2014 Anapog Integrated School
Anapog, San Remigio, Cebu
 100

JILL JAVA
Casanova, Pondol, Balamban, Cebu
Contact Number: 0919-337-9908
jilljava18@gmail.com

PERSONAL INFORMATION
Date of Birth : October 21, 2001
Place of Birth : Vicente Sotto Memorial Medical Center
Age : 22
Gender : Female
Nationality : Filipino
Civil Status : Single
Religion : Roman Catholic

EDUCATIONAL BACKGROUND
Tertiary Level
2020-Present Cebu Technological University-Main Campus
M.J. Cuenco Avenue Cor. R. Palma St., Cebu City
Bachelor of Secondary Education major in Science

Secondary level
Junior High School
 101

2014-2018 Buanoy National High School

Buanoy, Balamban, Cebu
Senior High School
2018-2020 Buanoy National High School
Buanoy, Balamban, Cebu

Primary level
2008-2014 Bactas Elementary School
Bactas. Catmon, Cebu
 102

ISABEL MARIE D. TANUCO

Sagay, Borbon, Cebu
Contact Number: 0906-962-4837
tanucoisabelmarie@gmail.com

PERSONAL INFORMATION
Date of Birth : September 1, 2001
Place of Birth : Cebu Velez General Hospital
Age : 22
Gender : Female
Nationality : Filipino
Civil Status : Single
Religion : Roman Catholic

EDUCATIONAL BACKGROUND
Tertiary Level
2020-Present Cebu Technological University-Main Campus
M.J. Cuenco Avenue Cor. R. Palma St., Cebu City
Bachelor of Secondary Education major in Science

Secondary level
Junior High School
 103

2014-2018 Don Emilio Osmena Memorial High School

Sagay, Borbon, Cebu
Senior High School
2018-2020 City of Bogo Science and Arts Academy
Saint Joseph Village, Cogon, Bogo City, Cebu

Primary level
2008-2014 Dona Mary Elementary School
Sagay, Borbon, Cebu