FORMULATION OF AN ANTIOXIDANT LOTION USING LYOPHILIZED

LEAVES EXTRACT OF Bambusa blumeana (KAUAYAN-TINIK)

A Thesis
Presented to
The Faculty of Pharmacy
University of La Salette, Inc.
Santiago City, Philippines

In Partial Fulfillment
Of the Requirements for the Degree
Bachelor of Science in Pharmacy

Czariana Cassidy Cordova

Jill F. Dagandanan
Trizia Andrea D. Genuino
Efrelyn Mae G. Lagmay
Fanny Lois G. Panaga

July 2023
 ACKNOWLEDGEMENT

The researchers would like to extend their gratitude to all the people who

helped and contributed to complete this research. First, to their respected

adviser, Mrs. Melanie C. Madamba RPh, MSPh, MS Pharm, for being a pillar to

this study, mentoring and guiding the researchers in every step of the way.

Special thanks to Ms. Ma. Clarisse A. Alindada, RPh, for imparting her

knowledge and giving continuous valuable suggestions to the researchers.

Also to Mrs. Laila Sanchez, Director of the Laboratory Services, and the

staffs of the Chemistry Laboratory, for patiently assisting the researchers in the

different experiments done in the laboratory.

Much appreciation is also extended to the researchers’ family, friends

and classmates, who showed their unwavering support and encouragement.

The researchers owe a deep debt to the University of the Philippines,

Los Baños, BIOTECH Department headed by Ms. Maria Katrina Alaon, M.Sc,

for accepting their request for the making of different high yield experiments.

i
 To the Animal Corner Veterinary and Supply, for providing the needed

certification and assisting the animal handling.

Lastly, to the Almighty GOD, for giving a constant knowledge and strength

to complete this research.

The Researchers

ii
 DEDICATION

We, the researchers, would like to dedicate this research study, first of all,

to Almighty God, who gave us strength, courage and enough knowledge to

pursue the goal of the study. Also, to our ever loving and supportive parents, Mr.

Arnold Magbanua and Ms. Cristina Cordova, Mr. Jerry Dagandanan and Mrs.

Josephine Dagandanan, Mrs. Elda Genuino, Mr.Efren Lagmay and Mrs. Marilyn

Lagmay, and Mr. and Mrs. Panaga. Lastly, to the College of Medicine and Allied

Medical Programs, especially to the Pharmacy Department Instructors who

imparted knowledge and guidance through the process.

The Researchers

iii
 ABSTRACT

Name of Institution : University of La Salette, Inc.

Address : Santiago City, Philippines

Title : Formulation of an Antioxidant Lotion using Lyophilized

Leave Extract of Bambusa blumeana (Kauayan-Tinik)

Authors : Czariana Cassidy Cordova

Jill Dagandanan
Trizia Andrea Genuino
Efrelyn Lagmay
Fanny Lois Panaga

Degree : Bachelor of Science in Pharmacy

Date of Completion : July 2023

The antioxidant property of Bambusa blumeana lyophilized leaf extract

was investigated to prove its antioxidant property and formulate a lotion using the

extract.

Confirmatory tests were done to prove the presence of Phenolic

Compounds, Flavonoids and Tannins which are responsible to the antioxidant

activity. In addition, the DPPH Assay was conducted to prove the scavenging

inhibition of the extract compared to Ascorbic Acid. The T-test was used to

compare the scavenging inhibition of samples, B. blumeana extract and Ascorbic

acid, that resulted to having no significant effect. Evaluation of the characteristic

and safety test was done using three male guinea pigs.

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 In conclusion, the Confirmatory tests proved the presence of Phenolic

Compounds, Flavonoids and Tannins. In connection with Phenolic Compounds,

the total phenolic content of the extract has 25.24±0.24 which is suitable for

formulating a lotion. Based on the DPPH assay, the B. blumeana lyophilized leaf

extract has an antioxidant activity in a concentration-dependent manner. The T-

test showed that the action of B. blumeana lyophilized leaf extract and Ascorbic

acid are comparable. B. blumeana lyophilized leaf extract can be used as a main

ingredient and additive to formulate a lotion and a natural source of Antioxidant

property.

v
 TABLE OF CONTENTS

TITLE PAGE
ACKNOWLEDGEMENT
DEDICATION ii
ABSTRACT
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES

INTRODUCTION

Background of the Study 1

Statement of the Problem 3
Hypotheses 6
Significance of the Study 6
Experimental Framework 8
Literature Review
9
Synthesis 46

METHODS

Research Design 48
Study Site 48
Data Gathering Procedures 50
Collection and Identification of Plant Sample 50
Collection and Preparation of Plant Sample 50
Plant Extraction 50
Freeze drying of the Extract 51
Percentage Yield 51
Confirmatory Test
52
DPPH Assay 54
Formulation of Antioxidant Lotion 55
Patch Skin test 56
Evaluation of Characteristic of Formulated Lotion 57
Ethical Considerations 58
Data Analysis
58

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RESULTS

Confirmatory Tests 62
DPPH Assay 67
T-Test 69
Characteristic and Evaluation of Formulated Lotion 71

BIBLIOGRAPHY

DISCUSSION

Summary 72
Conclusion 73
Recommendation 74

APPENDICES

APPENDIX A: Approval Letter for the Dean 75

APPENDIX B: Gantt Chart 76
APPENDIX C: Budget Plan 77
APPENDIX D: Certificate of Authentication 78
APPENDIX E: Communication with UP, Los Baños 79
APPENDIX F: Statement of Account 80
APPENDIX G: Documentation of Collection and Washing 81
APPENDIX H: Concentration, Extraction and Freeze Drying 82
APPENDIX I: DPPH Assay, Confirmatory tests and TPC 83
APPENDIX J: Documentation of Product Formulation 85
APPENDIX K: Documentation of Animal Testing 88
APPENDIX L: Certification of Guinea Pigs 90
APPENDIX M: Documentation for Quality Control Test 91

CURRICULUME VITAE

vii
 LIST OF TABLES

Table Page
Number
1 Evaluation of Skin Reactions 60

2 Sample Log Book Entry for Patch Test 61

3 Results for the Presence of Phenolic Compounds 63

4 Total Phenolic Content 64

5 Results for the Presence of Flavonoids 65

6 Results for the Presence of Tannins 66

7 The Percent DPPH Inhibition 67

8 T-TEST 68

9 Patch Test Results 71

viii
 LIST OF FIGURES

Figure Page
Number
1 Experimental Framework 8

2 Bambusa blumeana (kauayan-tinik) 9

3 Bambusa blumeana Culms 13

4 Bambusa blumeana Branch 13

5 Bambusa blumeana Leaves 14

6 Phytochemical Screening of B. blumeana Leaf Extract 37

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 INTRODUCTION

Background of the Study

Bamboo could very well be considered as just a type of grass, but in

reality it is among the most amazing plants. This came out from recent studies

conducted by the Forest Products Research and Development Institute of the

Department of Science and Technology (DOST-FPRDI) which will perhaps make

bamboo leaves more popular in the Philippines.

According to the research, the leaves of the Bambusa blumeana

(kauayan-tinik) species of bamboo are a promising source of antioxidants and

might therefore be used as a raw material for pharmaceuticals and dietary

supplements. They have strong antioxidant potential, comparable to that of

ascorbic acid, the current gold standard for antioxidants. Bambusa blumeana

(kauayan-tinik), one of the five species of bamboo they have researched, was the

most promising, according to researcher Rebecca B. Lapuz (2020).

According to Tan, B. L. (2018), oxidative stress, which is hypothesized to

result from an imbalance between pro- and antioxidant species, harms cells and

molecules. Oxidative stress can also contribute significantly to age-related

diseases. New research data reveals that antioxidants can limit the production or

spread of free radicals, which would improve immune system function, reduce

oxidative stress, and increase healthy lifespan by regulating autoxidation.

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 Antioxidants are now utilized to stop the oxidation of skin surface cells

caused by free radicals and environmental aggressors such UV radiation and

pollution. Antioxidants are frequently used in the compositions of skin care

products due to their powerful anti-aging benefits.

There is currently a shortage of information regarding the usage of

bamboo leaves as a source of phytochemicals for health and wellness in the

Philippines. Although numerous research have confirmed Bambusa blumeana's

strong antioxidant activity, few of them go into detail about its phytochemical

components and which individual ingredient is responsible for this degree of

antioxidant activity. Given the abundance of many bamboo species throughout

the nation and the sizeable market for items that promote good health, there is a

great need for such knowledge. As a result, the researchers are of the opinion

that bamboo, a rapidly growing plant with a large amount of biomass, can be

used as a substitute for producing natural antioxidants and as a beneficial

ingredient in the creation of beauty products. Thus, the researchers intended to

study extensively the phytochemical compounds found in Bambusa blumeana

(kauayan-tinik) and create an antioxidant lotion out of it.

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Statement of the Problem

Research Gaps

The following are the Research Gaps of the study:

1. Many studies have shown the components that are responsible for

the antioxidant activity of Bambusa blumeana shoots. However,

there is a lack of research that explores the viable antioxidant yield

of Bambusa blumeana leaves.

2. Various studies are currently present about the shoots of Bambusa

blumeana are currently present, specifically its phytochemical

compounds. But there are limited studies about its leaves.

Additionally, there are only a few articles that discuss how the

Bambusa blumeana leaves extract has the highest yield of

antioxidant properties. Thus, this study aims to determine the %

scavenging activity of Bambusa blumeana leaves extract using

DPPH Assay.

3. While Bambusa blumeana's antioxidant activity has been studied in

comparison to other species of Kauyan, there are still limited

studies about the comparison of Bambusa blumeana leaves extract

with ascorbic acid (Vitamin C). Therefore, the researchers aim to

3
 know the difference between the % scavenging activity of these two

samples.

4. The effects of antioxidants on the skin have been the subject of

numerous studies, and nature is a rich source of antioxidant

compounds. While there is potential for plants to support skin care,

more research is needed before any definitive results can be

gleaned. Therefore, to prevent, mitigate, or potentially reverse this

damage, it is necessary to develop natural products with strong

antioxidant property.

Research Questions

The following are the Research Questions of the study:

1. Does B. blumeana lyophilized leaf extract consists of Phenolic

compounds, Flavonoids and Tannins?

2. What is the level of antioxidant activity of B. blumeana lyophilized

leaf extract?

3. How much does the antioxidant activity of B. blumeana lyophilized

leaf extract differ in comparison with Ascorbic acid (Vitamin C)?

4. Can B. blumeana lyophilized leaf extract be used in formulating a

lotion?

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 5. Will the Antioxidant Lotion of B. blumeana lyophilized leaf extract

pass the evaluation of characteristic and safety tests?

Research Objectives

The main objective of this study is to prove the antioxidant property

of B. blumeana (Kauayan-tinik) and formulate a lotion with B. blumeana

lyophilized leaf extract.

Specific Objectives:

The following are the specific objectives of this study:

1. To perform confirmatory tests on the presence of phenolic

compounds, flavonoids, and tannins in B. blumeana lyophilized leaf

extract

2. To identify the % scavenging activity of B. blumeana lyophilized leaf

extract using DPPH Assay

3. To compare % scavenging activity of B. blumeana lyophilized leaf

extract to Ascorbic acid using DPPH Assay

4. To formulate an antioxidant product, specifically a lotion containing

the extract from B. blumeana lyophilized leaf extract

5. To conduct an evaluation of characteristic like Organoleptic test

using the Antioxidant Lotion of B. blumeana lyophilized leaf extract

and evaluation of safety by Patch Skin Test to guinea pigs

5
Hypotheses of the Study

Ho: There is no significant difference in the antioxidant activity of Bambusa

blumeana (Kauayan-tinik) and ascorbic acid (Vitamin C).

Ha:There is a significant difference in the antioxidant activity of Bambusa

blumeana (Kauayan-tinik) and ascorbic acid (Vitamin C).

Significance of the Study

By conducting the confirmatory tests to B. blumeana lyophilized leaf

extract and a DPPH Assay to prove its antioxidant property, it can become

beneficial for people who want to take care of their skin.

Specifically, this study will be significant to the following:

The Department of Health (DOH) can recommend the use of B. blumeana

lyophilized leaf extract as an antioxidant and can help in promoting the use of

natural products for disease prevention.

The Department of Science and Technology can make use of this study of

B. blumeana lyophilized leaf extract as a source of antioxidants.

The Community will be able to obtain and utilize the benefits of B.

blumeana lyophilized leaf extract that are useful to one’s health. The study will

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also promote the buying and purchasing of our own local products in the

Philippines.

The University of La Salette, Inc. will exhibit well-written and established

new and timely research studies.

Future researchers can use the study as their review of related literature

to support their claim and improve this study.

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Experimental Framework

Gathering and Identification of Plant

Sample

Extraction of Plant Sample

Freeze Drying of Extract

Confirmatory Tests

DPPH Assay

Formulation of Product

Skin Sensitivity Testing Quality Control

Test

Statistical Method of Data

Figure 1. Experimental Framework

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Literature Review

Bambusa blumeana (Kauayan-Tinik)

Botanical Classification

An article from Guadua Bamboo (2022) states that Bambusa

blumeana, also known as Spiny Bamboo or Thorny Bamboo, is a tropical

clumping bamboo native to Indonesia and Malaysia.

Figure 2. Bambusa blumeana (Kauayan-tinik)

Taxonomical Classification

According to Integrated Taxonomic Information System - Report

(2009), B.blumeana has the following data:

Taxonomical Hierarchy

Kingdom: Plantae – plantes, planta, vegetal, plants

Subkingdom: Viridiplantae – green plants

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 Infrakingdom: Streptophyta – land plants

Superdivision: Embryophyta

Division: Tracheophyta – vascular plants, tracheophytes

Subdivision: Spermatophytina – spermatophytes, seed plants,

phanérogames

Class: Magnoliopsida

Superorder : Lilianae – monocots, monocotyledons,

monocotylédones

Order: Poales

Family: Poaceae – grasses, graminées

Genus: Bambusa Schreb. – bamboo

Species: Bambusa blumeana Schult. & Schult. f.

Characteristics of Bambusa blumeana

It is a bamboo with a sympodial shape that is densely tufted and

has spiny basal branches that grow to a height of 2 to 3 meters; Culm

upright, 15–25 m tall, up to 20 cm in diameter, with a wall thickness of

0.5–3 cm; internodes typically hollow, 25–60 cm long, glabrous, and

green; nodes protruding, with the lowest ones supporting aerial roots.

Branches protrude from almost every node, with the upper branches

ascending and branching at the base, while the lower branches spread

horizontally and have strong straight or curved spines. Culm sheath up to

30 cm 22 cm, with the lowest ones being short and narrow and getting

longer and narrower as they go up, coriaceous, dull, and coated in stiff,

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appressed, short, dark-brown hairs that eventually fall out. The apex is

narrowly acute and glabrescent abaxially. young shoots with sheaths and

blades that are yellowish green. Linear-lanceolate leaf blade, 15-20 cm x

1.5–2 cm. The base is rounded, the margins are scabrous, and the apex is

narrowly acute. The auricles are small and contain a few bristles that are

about 3 mm long. The sheath is striate. Inflorescence with spikelets up to

5 cm long that are compressed laterally and contain 2-3 empty glumes

and 5–12 florets is borne on both leafy branches and branches of a culm

without leaves (PlantUse English contributors 2022).

Compared to plants grown in Indonesia and Malaysia, plants grown

in the Philippines typically have longer internodes. B. blumeana is very

similar to B. bambos (L.) Voss, yet their culm sheaths make it simple to tell

them apart from one another. In B. blumeana, the auricles of the culm

sheath are not as noticeable and have less curled bristles along the

borders. Unlike auricle-like structures, B. bambos are developed as

extensions of the blade's base and are covered with dark brown hairs. B.

blumeana doesn't expand as much as B. bambos. Additionally, less

resilient are bambos and their culms.

B.blumeana is still regarded as the best species for shoots in the

Philippines and is one of the seven primary species highly recommended

for shoot production in Yunan, China (Rojo 1999). It may be harvested

four years after planting as a grass, has a far shorter growth cycle than

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wood, and regenerates more quickly. In a 2003 study by Decipulo et

al. that compared three indigenous bamboo species in Malaybalay,

Bukidnon, a mean yield and harvest of 2.87 culms/clump per year and

4.87 culms/clump per year, respectively, Bambusa blumeana species

emerged as the species with the highest clump productivity and

sustainability.

Morphological Characteristics of B. blumeana

The findings of the measurements of Bambusa blumeana's

morphological characteristics are stated as follows by Aguinsatan et al.

(2019): the mean pole length of B. Shorter than blumeana. The number of

internodes in the middle and at the bottom indicated that B. blumeana, in

comparison, had one more internode but two less at the summit. When

comparing the middle section to the bottom and top sections, the

internode length was the longest. From bottom to top, the culm diameter

showed a trend toward reduction. Culm wall thickness demonstrated that

there is a downward trend from bottom to top. Only the bottom of each

bamboo pole was utilized to evaluate the physical and mechanical

characteristics of the control and thermally treated bamboos because

there is intrinsic variance within each pole.

Culms: Bambusa blumeana, a thorny bamboo with 15–25 m-tall,

slightly arched, green culms. The internodes are 25–35 cm in length, 8–15

cm in diameter, and have walls that are typically 2–3 cm thick. Particularly

12
in dry places or on poor soils, the wall thickness at the base of the culms

is usually solid. A ring of aerial roots can be seen around the lower culm

nodes, and the sheath scar is surrounded by a gray or brown ring.

Figure 3. blumeana Culms

Branches: Branches often grow from the center of the culm to the

top and are clustered several to many times, with one to three larger

dominating branches that are noticeably thicker and longer. Lower node

branches are solitary and covered in a dense web of hard, angular thorns.

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 Figure 4. blumeana Branch

Leaves: The lance-shaped leaves are typically 10–20 cm long and

12–25 mm broad.

Figure 5. blumeana Leaves

Origin of Bambusa blumeana

Bambusa blumeana is described as a species native to Indonesia

and Malaysia in an article from Guadua Bamboo (2022), although it has

been widely spread in Southeast Asia, including Thailand, the Philippines,

Vietnam, China, and Japan. Salzer, C. (2018) claims that in the early

1900s, it was brought to the Philippines; since then, it has naturalized and

may be found all over the populated areas at low and medium altitudes an

all of the Philippine archipelago's regions, from Northern Luzon over the

Visayas to Southern Mindanao, grow the B. blumeana species the most.

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Beyond the Philippines, it is grown in Southern China, Peninsular

Malaysia, the Moluccas, Sumatra, Borneo, India, and Indochina. It is

native to Java, Indonesia, and Eastern Malaysia.

Habitat: This type of bamboo can be found growing along

riverbanks, on hillside slopes, and in freshwater creeks in wet or dry

tropical regions. It is common at low or moderate altitudes, typically up to

300 m (up to 1,000 m in Taiwan). Bambusa blumeana grows close to solid

stems (at the base) in heavy or poor soil and can withstand flooding.

Heavy saline soils are unsuitable because the species likes a low pH (5-

6.5).

Uses of Bambusa blumeana

Bamboo is closely linked to the cultural, social, and economic

circumstances of people in many Asian countries. It is regarded as the

fastest-growing, most versatile woody plant with a wide range of industrial

and domestic purposes. In fact, according to the Department of Science

and Technology (2020), Bambusa blumeana (Kauayan-tinik) is among the

top 12 economically significant bamboo species in the Philippines. The

Bambusa blumeana species is one of the most significant bamboo

resources for the rural people of the Philippines, claims an article from

Agrimag (2019).

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 Every component of the bamboo plant, including the rhizome, culm

shavings, leaves, roots, shoots, and seeds, has medical uses. It has taken

some time for bamboo, a versatile plant mostly used in industry, to gain

recognition as a potential source of bioactive substances and natural

antioxidants. Bamboo is currently attracting attention on a global scale for

its nutritional and medicinal possibilities and is crucial to the food,

pharmaceutical, and cosmetic industries. According to a paper by Bisht

(2017), bamboo shoots and leaves have excellent therapeutic potential

and can offer a sustainable, natural approach to health care.

In fact, enterprises like Nestlé have begun to recognize the

remarkable advantages of bamboo. This new forestry effort from Nestlé

and EcoPlanet Bamboo Group, which promotes the industrialization of

bamboo as a sustainable fiber source, focuses on worldwide replanting.

Throughout keeping with this, they started a new program to plant 2.5

million natural bamboo bunches and 1 million trees throughout the

Philippines over the next three years.

Similar to this, the Department of Science and Technology's Forest

Products Research and Development Institute (DOST-FPRDI) has just

begun a project that aims to maximize the utilization of the nation's

abundant bamboo resources by learning more about the fundamental

characteristics of lesser-used native bamboos. The study will examine

16
each species' shape and organization, its cells and tissues, as well as its

mechanical and physical characteristics.

The DOST-Philippine Council for Agriculture, Aquatic and Natural

Resources Study and Development, in partnership with the Department of

Environment and Natural Resources Ecosystems Research and

Development Bureau, is funding this Php 5 million study project.

Woodcraft

Building, parquet, basketry, furniture, concrete

reinforcement, cooking items, handicrafts, chopsticks, headgear,

and toys all use culms. If wood is in short supply, bambusa

blumeana is also employed as a fuel source and a source of paper

pulp. Additionally, this variety of bamboo can be used to border

agricultural fields as living fences, as a windbreak, or to stop

erosion along streams. It also has tremendous promise for the

rehabilitation of marginal regions.

Food

Many bamboo species are consumed as vegetables, salads,

pickles, and in preparation of different types of curries and dishes in

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many Asian countries such as China, Korea, Japan, Thailand,

Malaysia and India. In countries like Korea, there are many

traditional foods in which bamboo shoots are used as a prominent

ingredient for their many health benefits.

Floods

Bamboo appears to be more resistant to wind and rain than

hardwood trees. Additionally, bamboo can act as a natural barrier

to landslides and combat soil erosion in flood-prone locations.

Additionally, bamboo is simple to grow—even in the wild—needs

little irrigation, and may be harvested in three to five years. The

coconut tree is a good substitute (Banzuelo, 2020).

Folkloric use of Bambusa blumeana

Chinese traditional medicine has detailed the use of several

bamboo plant components, including leaves and rhizomes, to cure

a variety of ailments, according to Wróblewska, Oliveira, Guaratini,

and Moreno (2018). Modern scientific research has shown that

bamboo extracts are quite effective biologically in terms of their

antioxidant properties. Theoretically, this action may also be

associated to the treatment of a variety of pathologies, including

anticancer, cardiovascular protection against neurodegenerative

18
 illnesses, resistance to free radicals, and protection against

neurodegenerative diseases.

Since ancient times, bamboo leaves have been used as a

herbal remedy to treat a variety of ailments, including fever,

hypertension, arteriosclerosis, detoxification, respiratory conditions,

chest inflammation, oedema, diarrhea, vomiting, excessive thirst,

as well as to improve the flavor and color of food (Liu et al., 2016).

Numerous in-vitro and in-vivo studies have demonstrated the

significant biological and therapeutic benefits of bamboo leaf

extracts, including their anti-oxidant, anti-microbial, anti-

inflammatory, anti-helmintic, antidiabetic, and anti-ulcer

characteristics (Daswad et al., 2017).

Pytochemical Components of B. blumeana

Using a standard method, the chemical test was carried out on

various solvent extracted fractions of Bambusa blumeana leaves.

Benzoic and hydrocyanic acids are abundant in leaves. Alkaloids,

carbohydrates, steroids, tannins, glycosides, and flavonoids were

discovered by pharmacognostic analysis of different leaf extracts. The

total ash found in the leaf after physiochemical analysis was 11.46%. Oil

ether extractive 1.85%, chloroform extractive 2.11%, ethyl acetate

19
 extractive 2.98%, ethanol extractive 26.77%, and water extractive 18.56%.

Acid-soluble ash 5.81%, water-soluble ash 2.66%, sulphated ash 9.25%,

loss on drying 15.77%.

SKIN AND THE IMPORTANCE OF ANTIOXIDANTS

Definition of Skin

The skin is the body's largest organ, according to Sullivan & Myers

(2022), and it covers the entire body. It is a vital organ that aids in

defending the body against harmful substances, physical and biological

threats, and the environment. The skin also plays a role in maintaining

body hydration.

According to Lopez-Ojeda et al. (2020), the skin also regulates

body temperature and provides protection from UV rays, wounds,

pathogens, germs, and poisons. The skin also plays a role in

immunological defense, sensory perception, controlling undetectable fluid

loss, and maintaining overall homeostasis. The skin is also remarkably

versatile, fulfilling particular functions at different body regions at varied

thicknesses.

Skin serves as an effective barrier, however some toxins can

penetrate deeper skin layers and enter the bloodstream, according to

Dbrowska (2018). This weakness in the skin barrier qualities creates an

opportunity for novel medicine delivery methods when exposed to

20
hazardous chemicals. Since skin qualities vary so widely, individualized

factors must be taken into account when modeling skin or developing

transdermal delivery-based therapy approaches.

Humphrey et al. (2021) also noted how social interactions, one's

sense of oneself, quality of life, and emotional health are all significantly

impacted by an improvement in skin condition. Even while skin quality

enhancements are routinely assessed as an end point in cosmeceutical

studies, they are rarely assessed in clinical investigations of other

aesthetic services and products.

Definition of Antioxidant

The ability of antioxidants to lessen any harmful or corrosive

compounds that could harm our body's cells or structure is known as

having antioxidant property or activity. Antioxidants are defined as "any

substance that, when present in low concentrations relative to those of an

oxidizable substrate, significantly prevents oxidation of that substrate," by

Halliwell and Gutteridge.

Free radicals are substances that are a constant threat. They have

the capacity to harm cells and genetic material at very high

concentrations. Free radicals are created by the body as an inevitable

result of converting food into energy. Additionally, free radicals are

produced as a result of physical activity, exposure to cigarette smoke, air

pollution, and sunshine. We also extract "antioxidants," which are

21
chemicals that liberally donate electrons to free radicals rather than

becoming themselves electron-scavenging compounds. They participate

in processes that preserve cell health and repair DNA. In defense

systems, antioxidants are essential. A careful equilibrium between

oxidants and antioxidants allows healthy organisms to resist the damaging

effects of reactive oxygen species. Antioxidants, whether they are

enzyme- or non-enzyme-based, work to stop free radicals from forming

and to find, neutralize, or repair the harm they do (Clark et al., 1985)

Oxidative Stress and its effect on the skin

Humans require oxygen to breathe, but according to Nakai &

Tsuruta (2021), 50% of the population creates extremely reactive oxygen

species (ROS), which can be used as energy and eventually transform

into water. ROS production causes the skin to age and become

inflammatory, and it also has an impact on human homeostasis.

Additionally, Gu et al.'s (2020) research explores how oxidative stress

contributes to skin aging. This is because an excess of reactive oxygen

species in the skin can harm lipids, proteins, nucleic acids, and cells.

Additionally, it may cause wrinkles and skin age spots. The skin is a

delicate organ that is susceptible to both inherent and extrinsic aging. UV

rays may hasten the natural aging process.

According to Nahhas et al. (2019), complex processes involving

UVR can lead to a variety of skin cancers and photoaging, some of which

22
are mediated by UV-induced reactive oxygen species. UVR exposure

outweighs the skin's built-in antioxidant defenses, which leads to DNA

damage, intracellular lipid and protein peroxidation, and the deregulation

of pathways that govern inflammatory and apoptotic responses. Natural

products with potent antioxidant qualities have been produced to stop,

lessen, or reverse this damage.

Importance of Antioxidant on Skin

Below is some proof that antioxidant-based skin treatments may be

successful in treating skin issues like age-related skin pathology,

according to Petruk et al. (2018). In this setting, numerous secondary

metabolites are produced by various plant species that can shield the skin

from UV radiation, lessen inflammation and oxidative stress, and influence

a number of survival signaling pathways. Additionally, oxidants are crucial

for cell metabolism and can boost the effects of xenobiotic and

environmental substances that affect skin photoaging.

According to McDaniel et al.'s (2019) study, mixtures of

antioxidants can give thorough skin protection by promoting their

synergistic interaction and offering all-encompassing defense against

various forms of ROS at all skin cellular levels. The skin's natural

antioxidant defenses can be strengthened by using topical antioxidants to

restore depleted antioxidant levels. Topical antioxidants help to protect

and restore the skin while also offering many other advantages for good

23
skin health. Topical antioxidants occur in a number of forms and are

regarded to have special qualities that are good for the skin and offer

varied degrees of assistance in the fight against free radicals. Hydrophilic

antioxidants like vitamin C shield the interstitial fluid, water-containing

parts of cells, and internal cell structures.

Enzymatic antioxidants like superoxide dismutase and ubiquinone,

for instance, support the body's natural defense mechanism and

safeguard mitochondria. Lipid-rich cell components, like the cell

membrane, are shielded by vitamin E and other hydrophobic antioxidants.

According to Rathore et al. (2021), having healthy skin has a

significant impact on how people perceive their general health. Skin is

crucial for immunity because it protects the body from pathogens,

maintains the electrolyte and water balance, and regulates body

temperature. Eating foods high in antioxidants may help you stay younger

and healthier for longer, according to a number of studies. Many skincare

products incorporate antioxidants into their formulas to give the benefits of

these ingredients.

Benefits and uses on skin

Skin acts as a barrier against harmful UV radiation and other

external environmental stressors. It also has the innate ability to defend

itself against aging, harmful UV radiation, and exposure to other

24
environmental factors like ozone (O3) through a sophisticated antioxidant

defense system (Pecorelli, 2020).

The skin's defense mechanism against oxidative damage, the

antioxidant system, is crucial. The skin has defense mechanisms that

work to stop radical reactions and neutralize free radicals. In comparison

to the dermis, the epidermis has a higher concentration of antioxidants.

Collagen and elastin are supported by antioxidants; nevertheless,

when your body is subjected to oxidative stress, production of collagen

and elastin slows down, and these priceless structural skin cells even

begin to degrade. Although pores cannot be shrunk, as you age, they do

increase. Antioxidants help reduce pore size. The pore lining begins to

droop as a result of the collagen in your skin breaking down. You then

develop skin that resembles orange peel. Additionally, antioxidants

brighten skin tone in two different ways. The first is that it reduces UV

damage, which is the main culprit for discolouration. The second is that

certain antioxidants have the ability to act as pigment inhibitors2, which

means they prevent the development of dark spots altogether.

Additionally, it lessens inflammation, which is brought on by oxidative

stress and free radicals. Although inflammation is a systemic problem, it

can manifest on the skin as a number of symptoms, such as acne,

redness, sensitivity, rashes, and so forth. Antioxidants reduce

inflammation by scavenging free radicals. It also delays photo-aging

because antioxidants can stop the formation of free radicals, a process

25
that causes UV rays and other harmful substances like blue light to cause

damage.

Mechanism of Action

A substance that lowers radicals in a test tube does not always act

as an antioxidant in a living organism. This is because to how easily FR

diffuses and spreads. It is challenging for the antioxidant to be present at

the moment and location where oxidative damage is being caused

because some have incredibly short lives, on the range of nanoseconds. A

second order reaction also occurs when antioxidants and FR interact. As a

result, in addition to factors linked to the concentration of antioxidants and

free radicals, they also depend on the medium, the reaction conditions,

and factors connected to the chemical structure of both reagents (Santos-

Sánchez, 2019).

There are several different ways that antioxidants work, such as

preventive antioxidants, free radical scavengers, free radical producing

enzyme inhibitors, lipid peroxidation inhibitors, DNA damage inhibitors,

and protein modification inhibitors (Aziz, 2019).

According to Chongtham et al. (2018), antioxidants found in

bamboo leaves have the power to suppress nitrile compounds, chelate

metal ions in a temporary state, chelate chain reactions of lipid auto-

oxidation, and prevent the synthesis of nitrosamine.

26
Categories of Antioxidants

There are various ways to categorize antioxidants (Aziz, 2019). On

the basis of their activity, size, solubility, and occurrence, they might be

grouped kinetically or otherwise.

They can be divided into enzymatic and nonenzymatic antioxidants

based on their action. Antioxidants can be categorized as either lipid-

soluble or water-soluble antioxidants based on how they are soluble.

Nutritionally speaking, an informative distinction between endogenous and

exogenous classes of antioxidants between enzymatic and non-enzymatic

classes can be made (Banafsheh and Sirous, 2016). Because the

vegetables and fruits that contain these antioxidants also include water,

the water soluble antioxidants are best absorbed in the body. Polyphenols

and vitamin C are both examples of water soluble antioxidants (Lazzarino

et al., 2019).

Antioxidants that are absorbed in the presence of lipids are known

as liposoluble or fat-soluble antioxidants. As a result, the body is unable to

absorb and utilize these antioxidants in the absence of lipids. It is crucial

to keep in mind, though, that they are difficult to eliminate from the body

and can build up over time, exceeding the appropriate amount. An

illustration of a fat-soluble antioxidant is vitamin E (Lazzarino et al., 2019).

27
 Liposoluble antioxidants, fat-soluble antioxidants are those that are

absorbed in the presence of fats. Therefore, in the absence of fats, the

body cannot absorb and use these antioxidants. It is important to note,

however, that they are not easily removed from the body and can

accumulate over time, exceeding the healthy level. Vitamin E is an

example of a fat-soluble antioxidant (Lazzarino et al., 2019).

Natural Antioxidants

Phenolic chemicals, vitamins, and carotenoids are the three main

types of plant-based natural antioxidants. In addition to being the main

plant components with antioxidant action, several phenolic compounds

also exhibit antibacterial and antifungal properties (Lourenço et al., 2019).

The production of polyphenols, a significant class of secondary

plant compounds, typically occurs only in these species (Sanjust et al.,

2008; Salehi et al., 2019). Flavonoids, phenolic acids, stilbenes (including

resveratrol), tannins, coumarins, curcuminoids, and lignans are the types

of polyphenols that are most well-known.

Due to its ability to scavenge free radicals and reactive oxygen

species, redox potential, and avoidance of oxidative damage to lipids and

other macromolecules, vitamin C (ascorbic acid) is a powerful antioxidant

that occurs naturally (Macan)

28
 A large family of tetraterpenes known as carotenoids is found in a

wide variety of plant species. Carotenes are precursors to vitamin A.

Photosynthesis and vision are two processes that are particularly

interested in the anti-/pro-oxidant functions of carotenoids and

xanthophylls.

The antioxidant epigallocatechin-3-gallate (EGCG) is well known.

By acting as an antioxidant, EGCG shields your cells from oxidative

stress-related cell damage and inhibits the activity of pro-inflammatory

substances your body produces, such as tumor necrosis factor-alpha

(TNF-alpha) (Hill, 2019).

Strong antioxidants have also been proposed for organosulfur

compounds. Many of the sulfur-containing metabolites found in garlic have

likely undergone the most research (Kimura et al., 2017). According to

Miltonprabu et al. (2017) and Borek (2001), their antioxidant effects

include scavenging ROS and reducing lipid peroxidation. A few minerals

are also necessary in tiny concentrations for various enzymatic antioxidant

actions. As a result, they are occasionally thought of as antioxidants.

29
 Effects of Phenolic compounds, Flavonoids and Tannins

According to Boo, Y. C. (2019), Plant-derived phenolic antioxidants

can alleviate oxidative stress and inflammatory skin diseases.

As botanic secondary metabolites, flavonoids and phenols are two

of the most important antioxidant elements in plants. Particularly bamboo

flavonoids have been employed in human health protection because to

their proven antibacterial, blood pressure management, immunological

modulation, and anti-aging properties (Chongtham, 2022).

Tannins effect on the skin on the other hand, will manifest as

shrinking of pores and wrinkles disappearing. At the same time, tannins

help to draw out all irritants from the skin (ElmaSkinCare, 2018).

Demands for Lotion

The Philippines' growing demand for natural and organic cosmetics and

skincare products is being driven by young adults with more discretionary

income.

As young adults in the Philippines place more importance on preserving a

youthful appearance, the market for personal care products is still growing. 110

million people, or 2% to 5% of the overall population, can buy higher-end goods.

30
These affluent customers will use third-party agents who import goods to conduct

their shopping (Domingo, 2022).

Antioxidant property of Bambusa blumeana Leaves Extract

The DOST completed a study that demonstrates bamboo's

potential health advantages (2020). According to the research done by

Ms. Mariluz Dionglay's team, the bamboo species "kauayan-tinik"

(Bambusa blumeana J.A. & J.H. Schultes) leaves are a promising source

of antioxidants and antimicrobials, making them potential raw materials for

medications and dietary supplements. In contrast to bolo, enormous

bamboo, kauayan-kiling, and buho, researcher Rebecca B. Lapuz claimed

that "Kauayan-tinik was the most promising of the five species."

Kauayan-tinik (Bambusa blumeana J.A. & J.H. Schultes) leaf

extract had the highest total phenolic content and antioxidant or free-

radical scavenging property when compared to bolo, gigantic bamboo,

kauayan-kiling, and buho. Similar research was conducted by Dionglay

(2018) on the phytochemical analysis and assessment of the antibacterial

and antioxidant capabilities of Philippine bamboo during the dry season.

The objective of this study was to identify the phytochemical, antioxidant,

and antibacterial properties of bamboo leaves. The province of Laguna

was where the fresh bamboo leaves were collected.

These were sieved, allowed to air dry, then put through the Wiley

mill before being put into polyethylene containers for storage. To create

31
ethanolic extracts, 500 g of powdered leaf samples were fully steeped in

80% ethanol for three days. The mixture was filtered, and the remaining

particles were taken out. The filtrate was concentrated using a rotary

evaporator, and the ethanol solvent was extracted. The extracts were

duplicated three times and stored in sealed bottles until they were

required. Aqueous extracts were made by fully steeping 500 g of

powdered leaf samples in distilled water for 5 days, keeping them in a

cooling cabinet. The filtrate was dried by evaporation in a steam bath.

Extracts were created in three duplicates, just like preparations, and kept

in sealed containers until needed. The effectiveness of antioxidant

capabilities in scavenging DPPH radicals was assessed.

Dimerthylsulfoxide was used as the blank, and ascorbic acid was used as

the standard. Kauayan-tinik had the greatest phenolic component

concentration (38.62%) and DPPH inhibition value (84.20%). The study

advises performing fractionation tests to identify the components important

for the antioxidant property of bamboo leaf extracts and developing

antioxidant products employing kauayan-tinik bamboo leaf extracts

(Dionglay et al., 2018).

Bambusa blumeana shoots

The research was divided into three substudies. Sub-Study 2

examined the anti-oxidant activity through total phenolic content and

DPPH free radicals scavenging activity, and Sub-Study 3 carried out a

screening of the phytochemical components of the bamboo shoot extracts.

32
Using the procedure described by Kolak et al., the ethanol and hot water

extracts from the shoots of Bambusa blumeana were diluted in methanol

to a final concentration of 500 ppm and then examined for their capacity to

scavenge DPPH radicals. 0.1 mM of DDPH in methanol was freshly

created by adding 100 ml of methanol to 1 ml of DPPH stock solution

(3.49 mg DPPH in 10 ml methanol). 1 cc of the extract and 4 cc of the

DPPH solution are combined the mixture was incubated at 37 degrees

Celsius in the dark for 30 minutes. Each extract was examined three

times. The ethanol extract of Bambusa blumeana had the highest level of

radical scavenging activity, 56.66%. Antioxidant-rich extracts from

Bambusa blumeana shoots are available.

The antioxidant qualities of four fractions of the bamboo shavings

extract (BSE) and each of their unique antioxidant components were

investigated in this work. On ABTS, DPPH, FRAP, and total antioxidant

capacity studies, the antioxidant capacities of BSE and four fractions may

be seen in the following descending order. Among the discovered phenolic

compounds, caffeic acid showed the best antioxidant properties in DPPH,

FRAP, and total antioxidant capacity experiments. An incredibly strong

positive correlation between the antioxidant activity and the quantities of

total flavonoids, total phenolic acids, or total phenolics was discovered in

this study.

The results indicated that the bamboo shaving extract and its

solvent fractions might function as natural antioxidants due to their strong

33
 antioxidant properties. Bamboo shavings' antioxidant ability was assessed

using the DPPH free radical scavenging activity method, which uses

hydrogen donation to quench reactive species. Because it is a rather

stable free radical that can receive an electron or hydrogen radical to

produce a stable diamagnetic molecule, the DPPH is commonly used to

research the radical scavenging activity. The BSE and its constituent

elements, or its main bioactive components, have a sizable capacity to

scavenge free radicals, claims this study. The broad range of antioxidant

activity of the extracts suggests that bamboo shavings may be used as a

natural source of antioxidants or nutraceuticals that may be used to

reduce oxidative stress and provide health benefits. However, more

investigation is needed to identify additional naturally occurring

substances with unique features in the diethyl ether fraction and assess

the in vivo antioxidant efficiency of these substances (Gong et al., 2016).

Correlational Analysis

According to earlier studies by Hasegawa et al. (2008) and Kweon

et al. (2001) on the content of bamboo leaves and its potential as an

antioxidant, bioactive chemicals such flavonoids and phenolics are the

primary antioxidant component present in bamboo leaves. Bamboo leaves

contain a variety of flavonoid compounds, including the flavone C-

glycosides orientin, isoorientin, vitexin, and homovitexin, as well as

phenolic acids such as p-coumaric acid, chlorogenic acid, caffeic acid, and

ferulic acid .

34
 In 2021, Wahyuni et al.'s study aims to ascertain the value of the

correlation between the content of bioactive compounds (total phenolic

and total flavonoids) and their antioxidant activity, as well as the effect of

temperature and maceration time on the content of bioactive compounds

and antioxidant activity of thorn bamboo leaf extract. The link between

phenolic chemicals and antioxidant activity had the greatest Pearson

correlation analysis results (r=0.981). The results of a multiple correlation

analysis between bioactive compounds (total phenolic and total flavonoid)

and the antioxidant activity of thorn bamboo leaf extract reveal a positive

and linear relationship with the solutions of the regression equation Y =

1445 - 2,941 X1 - 4,940 X2; the correlation coefficient (R) and coefficient

of determination (R2) are both positive and linear (0.983 and 0.967),

respectively.

Confirmatory test for Antioxidant activity

For Phenolic compounds, here are the Confirmatory Tests

according to Admin (2023).

Litmus Test: Place the drop of given organic solution or a

small crystal on moist blue litmus paper. Observe the change in

colour, if it changes to red then the phenolic group may be present.

Ferric Chloride Test: Dissolve the given organic compounds

in water. Add a neutral solution of ferric chloride slowly dropwise.

35
Observe the change in colour. A red, blue, green or purple

colouration indicates the presence of phenol

Alkaloids. To extract the alkaloid base from the prepared

solution, chloroform was added, and the mixture was agitated.

Glacial acetic acid was used to remove the layer of chloroform.

Dragendorff's reagent was used on one portion, and Mayer's

reagent on the other. Alkaloids can be detected by the precipitation

of a cream (Mayer's) or a reddish brown precipitate (Dragendorff's).

Saponins (test for foaming). In a test tube, 5 mL of distilled

water were added to 0.5 g of the extract. The solution was briskly

agitated and examined for lingering foam. Three drops of extra

virgin olive oil were added to the ensuing foam, which was then

forcefully agitated. The existence of saponins is confirmed by the

appearance of a creamy substance with tiny bubbles.

Tannins. The extracts were boiled in water and filtered.

Ferric chloride solution was added to the filtrates, which were

observed for brownish green or blue-black coloration that indicates

the presence of tannins.

Flavonoids. Magnesium strips were added to the

concentrated ethanolic and aqueous extracts. The appearance of

pink scarlet, crimson red, green or blue-black precipitates shows

the presence of flavonoid upon addition of hydrochloric acid.

36
 Steroids. Acetic anhydride was added to the extract prior to

adding sulfuric acid. The transformation of the violet solution to blue

or green points to the presence of steroids.

TerpeK++noids (Salkowski method). Sulfuric acid was

carefully added to the extract mixtures with chloroform to form a

layer. A reddish brown color of the interfere indicates the presence

of terpenoids.

Cardiac glycosides (Keller-Killiani Test). About 2 mL glacial

acetic acid with one drop of ferric chloride solution was mixed to 0.5

g of leaf extract diluted in 5 mL water. To this was added 1 mL of

concentrated sulfuric acid . A brown ring at the interface shows the

presence of deoxysugar characteristic of cardenolides. A violet ring

may form below the brown ring, while in the acetic acid layer a

greenish ring may appear just above the brown ring and slowly

diffuse throughout this layer.

The result of the Phytochecmical Screening showed that Bambusa

blumeana (Kauayan-Tinik) in ethanolic extract has positive remarks with

Saponins, Tannins, Flavonoids, Steroids, Terpenoids and Reducing Sugar

(Ramos, 2018).

37
 Figure 6. Phytochemical Screening of B. blumeana leaf extract

Extraction

Solvent

A mixture of phenolics that are soluble in the solvent will be

extracted from plant materials depending on the solvent system that is

employed during exaction. Another effective and safe for human

consumption solvent for polyphenol extraction is ethanol. An acidified

organic solvent, most frequently methanol or ethanol, is used to prepare

phenolic-rich extracts from plant sources.

Temperature

The likelihood of phenolics oxidizing is increased by prolonged

extraction times and high temperatures, which reduces the yield of

phenolics in the extracts.

38
 For instance, standard flavonoid extraction and concentration are

normally carried out between 20 and 50 °C because to the fast

deterioration that has been proven to occur at temperatures above 70 °C.

Lyophilized Extract

Natural antioxidants are vulnerable to deterioration after extraction

because of their great susceptibility to environmental factors (such as

temperature, pH, light, oxygen, and moisture). The degradation rate

should be kept to a minimum in order to increase their stability and

prevent nutritional and functional losses (Lourenço et al., 2019). In

general, freeze-drying preserves more phenolics than simple air-drying

does in plant samples. The low temperature and oxygen partial pressure

created by freeze drying, which might limit polyphenol oxidase activity and

lessen oxidation, may be the cause. Because of this, it is indicated that

lyophilization is a better drying procedure for the preservation of highly

bioactive and valuable extracts (Liu et al., 2022).

Natural extracts that are sensitive to high temperatures, like

anthocyanins, can be preserved by encapsulating them using freeze

drying. This process increases their shelf life at room temperature and

makes handling and shipping easier. To protect the target product from

harmful environmental factors (including temperature, light, moisture, and

oxygen), this technique involves encasing it in a carrier. However, due to

39
the differences in each carrier's structure and properties, different carriers

offer powders with various qualities.

Encapsulating Agent

Polysaccharides, lipids, and certain proteins are typical

encapsulation carriers. Maltodextrin and gum arabic are frequently used

as carriers because of their high water solubility and low viscosity. By

hydrolyzing starch, maltodextrins are oligosaccharides made up primarily

of B-D-glucose units connected by glycosidic linkages.

The results of a study by Adetoro (2020), which examined the

physicochemical and technofunctional properties and antioxidant capacity

of freeze-dried "Wonderful" pomegranate juice powder (PJP) with different

carrier agents, revealed that their powder was produced with maltodextrin

and had the highest yield (46.6%), followed by gum arabic and having the

lowest yield (40.6%), and waxy starch. Additionally, compared to gum

arabic (28.45 mM TE/g DM) and waxy starch (26.96 mM TE/g DM), the

powder made with maltodextrin had better radical scavenging activity

(33.19 mM TE/g dry matter; DM). Maltodextrin emerged as the most

effective carrier agent overall.

Similarly, Lachowicz (2020) found that using maltodextrin increased

the antioxidant potency of the product because it protected total

40
 anthocyanins and their derivatives, total flavan-3-ols and their derivatives,

total phenolic acids and their derivatives, and total flavonols and their

derivatives. This study examined the effect of maltodextrin on the

protection of natural antioxidants in powders.

In terms of concentration, Lachowicz (2020) discovered that the

dilution effect with the carrier had an impact on the content of phenolics;

the lower the level of bioactive compounds, the greater the carrier

concentration. Similar findings regarding the impact of a carrier's

concentration on the amount of polyphenols were made in the instance of

Eugenia dysenterica DC powders. Polyphenol content was highest in

samples made with 10% of carriers, while phenol content was lowest in

powders made with 30% of carriers. Individual polyphenolic components

(anthocyanins, phenolic acids, flavonols, and fla-van-3-ols) and

antioxidant activity show a high positive association, which supports this.

Total Phenolic Content

It is well recognized that phenolic plant components play a significant role

as antioxidants. Because of their functions as free radical scavengers and their

potential applications in the treatment of numerous diseases, interest in naturally

occurring and food-derived phenolics has surged. One of the best sources of

phenolic chemicals in plants are bamboo shoots (Chongtham et al., 2018).

41
 The correlation coefficient between TF, TP, and TT and the antioxidant

ability as determined by the FRAP assay is TP > TF > TT, according to a study

that examined the total phenolic (TP), total flavonoid (TF), and total triterpenoid

(TT) content of bamboo leaves. This indicates that the TP in bamboo leaves is

the main source of TP for FRAP, which assesses antioxidant power.

In a related investigation by Mapoung (2021), there has been an increase

in recent years in the usage of natural extracts and antioxidants in the cosmetic

cream business to generate whitening effects. In Thailand, there has likewise

been a consistent pattern. All samples that were compared had total phenolic

levels that fell between 0.46-47.92 mg GAE/30 g cream.

DPPH ASSAY

The total antioxidant capacity of the extracts can be determined using the

free radical scavenging technique known as the DPPH test. In fact, according to

Romulo's (2020) research, DPPH is the free radical that is employed the most

frequently to study in vitro antioxidants. Additionally, as of July 2019, the PubMed

database revealed that over 13000 papers have used DPPH.

The antioxidant assay known as the 2,2-diphenyl-1-picryl-hydrazyl-

hydrate (DPPH) in vitro method is based on DPPH scavenging by antioxidants.

In its radical state, DPPH displays an active absorption band at a maximum of

515–517 nm. A more stable molecule is created when the reactive free radical

DPPH receives an electron or hydrogen. This process is based on the idea that

42
antioxidants, which may be present in a solution (such as an extract), might

interact with the free radical, a-diphenyl--picrylhydrazyl (deep violet in color), and

change it into a more stable form, a. a-diphenyl-picrylhydrazine, which has a light

yellow tint. 2017; Austria et al. This discolouration shows the antioxidant

scavenging abilities of the material, specifically phenolic chemicals (Ramos,

2018).

In a study by Ni (2012), the total phenolic (TP), total flavonoid (TF), and

total triptein (TT) content of bamboo leaves were examined. They discovered that

the correlation coefficient between TP, TF, and TT and the ability to scavenge

DPPH radicals was TF > TP > TT, indicating that TF in bamboo leaves plays a

key role in this capacity.

Scavenging activity of B. blumeana

In a study by Ni (2012), the total phenolic (TP), total flavonoid (TF), and

total triptein (TT) content of bamboo leaves were examined. They discovered that

the correlation coefficient between TP, TF, and TT and the ability to scavenge

DPPH radicals was TF > TP > TT, indicating that TF in bamboo leaves plays a

key role in this capacity.

Formulation of Lotion

Based on the study of Sungkar et al. (2019) on the "Skin Lotion Formula

R. mucronata extract in 100 mL", as an emulsifier, stearic acid and cetyl alcohol

are employed. As an emollient or humectant, glycerin is employed.

43
Triethanolamine is employed as an emulsifier, an alkalizing agent, and a

surfactant. Preservatives like methyl paraben are utilized. The final solvent is

water.

pH

The normal pH range for skin care products, according to Abelman (2018),

is 4–7 because somewhat acidic products are favoured for improved

complexions and low pH is closer to the skin's pH level, allowing it to work

effectively with the skin.

Viscosity

For orgnoleptic properties D Fransiska et al. (2021), stated that the

standard viscosity for a lotion ranges from 2000-50000 mPa.

Spreadability

Tchienou (2018) states that the topical cream is advantageous if

spreadability is reduced since it is simple to apply to the skin. Depending on the

substance of each phase, the spreadability values were discovered to be in the

range of 9.0 to 31.02 gcm/s. based on a study by Hadisoebroto, G. and Saptarini,

N. M. This is the spreadability test formula for the year 2020.

spreadability=weight tied ¿ upper side x length of glass slide ¿ separate the slide
time taken ¿

44
Patch Test

Sensitization experiments are carried out on animals prior to the

approval of product formulations to prevent sensitization reactions in

humans. Sensitivity assessments can be conducted using a variety of

methods, the majority of which employ guinea pigs. These studies are

carried out using patch tests, in which a small patch of naked animal skin

is repeatedly exposed to a substance to gauge its reaction.

Guinea pigs

The history of diagnostic patch testing dates back to the 19th

century. It took a long time before the first reliable guinea pig prediction

test was released. And for many years now, the preferred animal utilized

in predicative sensitization experiments has been the guinea pig. Healthy

young adult animals, whether male or female, can be used in this test.

Females should be nulliparous and incapable of becoming pregnant. Each

animal should be healthy and weigh between 300g and 500g (32nd edition

of The United States Pharmacopeia-National Formulary).

According to numerous studies, male guinea pigs are preferable to

females as test animals since they are more manageable. Although the

outcomes of guinea pig skin tests may not be different for males and

females.

45
Synthesis

Topical antioxidants can help prevent damage as well as partially heal it

because of their capacity to protect skin from UVA and UVB rays and their

utilization by the body to counteract free radicals. Because of this, topical

antioxidants are frequently used to boost the skin's built-in antioxidant defenses

by replenishing the skin's depleted antioxidant levels.

Plants contain phenolic chemicals, which are well-known to be potent

antioxidants. Because of their abilities to scavenge free radicals, interest in

naturally-occurring and food-derived phenolics have grown over time

(Chongtham et al., 2018). The primary objective of such therapies in clinical

practice is to improve skin quality, and clinical investigations of aesthetic

treatments increasingly focus on this objective.

Bamboo leaves include a variety of bioactive substances that function

superbly as bioantioxidants. According to further investigation, flavonoids,

phenolic acids, and phenols and their derivatives are the primary antioxidant

components of bamboo leaf extract (Indira et al., 2022).

According to the related research that were given, species B. blumeana

had the highest phenolic component concentration (38.62%) and DPPH inhibition

value (84.20%). According to earlier studies' citations, Bambusa blumeana

(Kauayan-Tinik) phytochemical screening results revealed that Flavonoids, are

abundant in blumeana and contribute to their antioxidant properties. The months

46
of January through June and November through April saw the highest

concentration of total phenolics.

In line with this, the researchers aim to further examine the antioxidant

activity of B. blumeana leaves extract to prove its antioxidant activity and that it

has the potential to be tapped as a natural source of antioxidant. The

researchers also aim to formulate a lotion using B. blumeana lyophilized extract

to produce a topical antioxidant and test the extract’s safety with the use of a skin

sensitivity test.

47
 THE RESEARCH METHODS

This chapter focuses on the methods used by the researchers to perform

the collection, extraction and freeze-drying of plant extracts, the comparison of

the plant extract to the standard antioxidant sample, the Formulation of lotion,

Patch Skin Test and Quality Control test of the lotion.

Research Design

In the study, the researchers conducted an In vitro Experimental Research

specifically the Post Test Only Control Group Design. This design helped in the

determination of the percentage (%) inhibition of free radicals of B. blumeana

lyophilized leaf extract and formulation of lotion.

Study Sites

The process of gathering related literature and studies was conducted at

the library of University of La Salette, Inc. The collection of the sample, Bambusa

blumeana (Kauayan-Tinik), took place at Jones, Isabela. Soon after, the plant

sample was sent to the Department of Agriculture in Santiago City, Isabela for

authentication. Based on the study of Ramos (2018), the plant samples used

were extracted during the dry season around January to June. Hence, gathering

of plant sample was carried out around February to align to the said study. The

extraction, freeze drying of extract and DPPH Assay was conducted by the

University of the Philippines, Los Baños. Confirmatory Test, Product Formulation,

48
Skin Sensitivity Testing, Quality Control were performed at the Chemistry

Laboratory of University of La Salette, Inc.

Guinea pigs, specifically male, were used for the Skin Sensitization test.

Due to different factors including pregnancy and non reproductivity, the

researchers have decided to opt for male guinea pigs over female. Additionally,

multiple articles have stated that male guinea pigs are easier to handle for their

behavior rather than females. Although results of skin testing of guinea pigs may

not differ for females or males.

49
Data Gathering Procedures

The data gathering procedures that supported and confirmed the

Antioxidant property of B. blumeana lyophilized leaf extract were the following

procedures:

Collection and Identification of the Plant Sample

One (1) kilogram of Bambusa blumeana (Kauayan-Tinik) leaves

was collected at Jones, Isabela. The leaves were washed thoroughly and

then weighed once air dried.

The plant samples were sent and duly authenticated by The

Department of Agriculture in Santiago City, Isabela.

Collection and Preparation of the materials

One (1) kilogram and three hundred seventy eight (378) grams of

Bambusa blumeana (Kauayan-Tinik) was collected at the land of Mrs.

Nicolas in Jones, Isabela. According to the owner, the bamboo was 4-5

years old. It was prepared through washing and pat drying then shipped

out to University of the Philippines, Los Baños.

Plant Extraction

This procedure was done at the University of the Philippines, Los

Baños BIOTECH. The washed bamboo leaves were oven-dried at 60°C

overnight (convective air dryer), then ground and sieved to recover fine

50
bamboo leaves powder. The powder was mixed with 2 liters of 80%

ethanol for 2 hours and centrifuged at 9,000 rpm for 20 minutes at 25°C.

Freeze drying of the Extract

This procedure was done at the University of the Philippines, Los

Baños BIOTECH. The extract was subjected to rotary evaporation at 50°C

for 20 minutes to remove the ethanol. Maltodextrin at 8% was then

dissolved in the concentrated extract, mixed for three hours and freeze

dried for 48 hours.

The Percentage Yield of the Lyophilized Leaf Extract

The percentage yield of all solvent extracts of the bamboo leaves

on dry weight basis was calculated as follows:

Yield (%) = [ EW / DW ] ×100

Where EW is the weight of the extract after solvent evaporation and

DW is the dry weight of the plant material used for extraction (Tripathi et

al., 2015)

total recovered freez dried (g)

% yield = x 100
total sample (g)

14.77 grams
% yield = x 100
300 grams

= 4.92%

51
Confirmatory Test

Phenolic compounds, Flavonoids and Tannins are known for their

antioxidant property. Hence, the researchers used the following

Confirmatory Tests to prove the presence of the flavonoids in B.

blumeana lyophilized leaf extract (Ramos, 2018).

Preparation of the Freeze-dried Extract

An equivalent of one (1) gram of the freeze-dried extract was

diluted with 10 mL of distilled water.

Test for Flavonoids

Alkaline reagent test: Two to three drops of sodium

hydroxide were added to 2 mL of extract. Initially, a deep yellow

colour appeared but it gradually became colourless by adding few

drops of dilute HCL, indicating that flavonoids were present.

Ferric chloride test: Two milliliters of 5% neutral ferric

chloride solution were added to 1 mL of extract, the dark blue

colouring indicating the presence of Flavonoids.

Shinoda Test: Magnesium strips were added to 1 mL extract.

The appearance of pink, scarlet, crimson red, green or blue-black

precipitates shows the presence of flavonoid upon addition of 10

drops of hydrochloric acid.

52
 Lead-acetate test: A few drops of aqueous basic lead

acetate solution was added to 1 mL of extract using a test tube.

The appearance of a reddish brown bulky precipitate indicated the

presence of flavonoids.

Zinc-HCl reduction test: A pinch of zinc dust and few drops

of concentrated HCl were added to 1 mL of an extract in a test

tube. The appearance of magenta color indicated the presence of

flavonoids.

Test for Phenols

Litmus Test: The extract was placed in a test tube and 1-2

drops of it was dropped on a blue litmus paper. The colour of litmus

paper changing from blue to red is a positive indication.

Ferric Chloride Test: Two milliliters of 5% neutral ferric

chloride solution were added to 1 mL of extract. The colour change

was noted. If the colour of the solution becomes blue, green, violet,

or red, this indicates the presence of a phenol group. Ortho, meta

or para – cresol, resorcinol gives violet or blue colour. β - naphthol

gives green colour and α - naphthol gives pink color. Formic acid

and acetic acid gives a deep red colour.

53
 Test for Tannins

Gelatin Test: To a 1% gelatin solution, a little of 10% sodium

chloride was added to 1 mL extract. Tannins cause a precipitation

of gelatine from solution.

Vanillin-hydrochloric acid test: 1 mL Extract and added

vanillin-hydrochloric acid reagent (Vanillin 1 gm, alcohol 10 ml,

concentrated hydrochloric acid 10 ml) were mixed together. A pink

or red colour is formed due to formation of phloroglucinol.

Match stick test (Catechin test): A match stick was dipped in

extract, dried near burner, and moistened with concentrated

hydrochloric acid. Upon warming near flame, the matchstick wood

turns pink or red due to formation of phloroglucinol.

Lead acetate test : A few drops of aqueous basic lead

acetate solution was added to 1 mL of extract using a test tube.

Reddish-brown bulky precipitate indicated the presence of tannins.

DPPH Assay

This method was done at the BIOTECH Laboratory of University of

the Philippines, Los Baños.

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging

activity was analyzed according to the method of Ribeiro et al. (2008) with

54
modifications. The DPPH is a stable free radical with purple color

(absorbed at 517nm). If free radicals have been scavenged or inhibited by

the phenolic compounds in the sample, DPPH will change its color to

yellow. Briefly, 100 μL of bamboo leaves powder at different phenolic

concentrations (10, 15, 20, 25 and 30 µg GAE) were mixed to 5 mL of

0.1mM DPPH in methanol and left to stand for 20 min. The absorbance

was determined at 517 nm using distilled water to zero the instrument.

The DPPH reagent and water served as control and DPPH inhibition was

calculated using the formula below.

Formulation of Antioxidant Lotion for 300 mL

Procedure:

Water was placed in a container and heated up to 75 degree

celsius. Then 3 grams of 2% carbomer, 15 grams of stearic acid, 0.5

grams of titanium dioxide, were added into the container. In a separate

container, 3 mL of dimethicone, 3 mL of sodium hydroxide, 15 mL of

glycerin, 6 mL of triethanolamine and 3 mL of cetyl alcohol were mixed

together and heated till 75 degree celsius. Then, the oil phase (container

2) and the water phase (container 1) was slowly mixed together. After, 1

mL of extract was added and mixed for 30 minutes The mixture was left

alone to cool at 25 degree celsius. Once the mixture reached <40 degree

55
celsius 5 drops of the fragrance was added to the mixture (Sungkar et al.,

2019). .

Patch Skin Test

Based on the Guidebook to Plant Screening: Phytochemical and

Biological (Guevera B., Rev. Ed., 2005). The male guinea pigs were

prepared by shaving bilateral their spinal trench (2 inches). The left side of

the groove in the animal was utilized as the negative control site and the

right side as the test drug site. Both sites were cleaned with 70% alcohol.

The test drug and the negative drug control were delivered on the

inoculation sites respectively. Both sites were covered with sterilized

gauze (2x2 cm in size). Surgical tape was used to keep the gauze in

place. The guinea pigs were left undisturbed for 24 to 72 hours. The

patches were removed after 24 hours of exposure and the reactions were

evaluated according to the scores. The patches were returned and

another reading or scoring was made after 72 hours. The average scores

of the 24 and 72 hours reading are computed.

2.3.10. Evaluation of Characteristic of the Formulated Antioxidant Lotion

The procedures were based on the research study of formulating

and evaluation of a lotion (Saptarini, 2022).

Organoleptic Characteristics:

56
 From color and aroma, organoleptic qualities were

evaluated.

Homogeneity:

Visual inspection was used to assess homogeneity for the

presence and appearance of any clogs.

pH Evaluation:

Using a pH paper, the pH of the formulated lotion of B.

blumeana lyophilized leaf extract was determined. The standard pH

for lotion is 4-7.

Viscosity:

The viscosity determination was carried out using a NDJ-5S

Viscometer with rotor 4 at 60 rpm at a temperature of 25°C. The

standard viscosity for lotion is 2000-50000 mPa.

Spreadability:

The recipe was pressed to create a consistent layer

thickness between two glass slides. The top plate was then pulled

with the aid of a line tied to the hook after a weight (10 g) was

added to the pan. The spreadability (S) is calculated using the

57
 formula by noting the amount of time it takes the upper glass slide

to move across the lower plate to cover a distance of 10 cm.

s preadability=weight tied ¿ theupper side x length of glass slide ¿ seperate the slide ¿
time taken¿

Ethical Considerations

In this study, the ethical considerations that were taken seriously were

honesty, accuracy and cruelty free to animals. The researchers aim to show the

results and interpretations that were gathered during experiments to the readers.

Hence, these results are only based on the truths and facts of the said

experiments. Lastly, the researchers made sure of the safety of the test animals

before, during and after the study.

Data Analysis

The values taken from the results of the experimentation were analyzed

using the T-Test: Two-Sample assuming equal variances because there are two

samples from the plant extracts that were compared to the positive control which

is Ascorbic acid (Vitamin C). This method is considered as the method of choice

in analyzing data after the Quantitative DPPH (2,2-Diphenyl-1-pierylhydrazyl)

free radical scavenging assay is conducted by the Registered Chemist of the

Institute of Pharmaceutical Sciences, UP Manila.

The data that were collected in the study are: (1) % scavenging activity of

B. blumeana leaves; (2) % scavenging activity of ascorbic acid (Vitamin C).

58
Basis of Interpretation:

If T critical is greater than T-stat

1. Accept the null hypothesis

2. Reject the alternative hypothesis

59
Table 1. Evaluation of Skin Reactions

The basis of describing the extent of visually observed erythema and

edema formation at the back of male guinea pigs observed 24 and 72 hours after

application was shown based on the Guidebook to Plant Screening:

Phytochemical and Biological (Guevara et. al, 2005),

Erythema Edema Formation

Formation

No Erythema 0 No Edema 0

Very Slight 1 Very Slight Edema (barely 1

Erythema perceptible)

Well-defined 2 Well-defined Edema (edges of 2

Erythema area well-defined by definite
raising)

Moderate 3 Moderate Edema (raised 3

Erythema approximately)

Severe Erythema 4 Severe Edema 4

60
Table 2. Sample Log Book Entry for Patch Test

The score table shows for patch test after 24 hours and 72 hours of observation.

Patch Test

After 24 hours After 72 hours

Erythema

Edema

Total

Average

61
 RESULTS

This chapter presents the overall results of Experimental Research by

Confirmatory Tests that confirmed the identification of Phenolic compounds,

Flavonoids and Tannins, comparing the Antioxidant property of B. blumeana

lyophilized leaf extract with Ascorbic acid using DPPH Free Radical Scavenging

Assay and T-test, and Evaluation of Characteristic and Safety of the formulated

lotion obtained by the researchers.

The data gathered provide an essential outcome in discerning the

antioxidant property of B. blumeana lyophilized leaf extract.

Identification of the B. blumeana lyophilized leaf extract.

Confirmatory Tests

The presence of Phenolic compounds was determined using

Litmus Test and Ferric Chloride Test. For Flavonoids, it was determined

using 5 tests namely: Alkaline Reagent Test, Ferric Chloride Test,

Shinoda Test, Lead-Acetate Test and Zinc-HCL reduction test. Lastly, for

Tannins Test: Gelatin Test, Vanillin-hydrochloric acid test, Match stick test

(Catechin test) and Lead acetate test.

62
Table 3. Results of Confirmatory Tests for the presence of Phenolic

Compounds in B. blumeana lyophilized leaf extract

TEST EXPECTED ACTUAL INTERPRETATION

RESULT RESULT

Litmus Test Litmus paper (+) Positive for

changes from acidity
blue to red Blue to red

 Phenol group
- Blue, green,
violet, or red

 Ortho, meta
or para –
cresol,
resorcinol-
Ferric Chloride violet or blue Green (+) Positive for the
Test colour. presence of
phenolic
 β - naphthol compounds
gives green particularly phenol
colour and α - group
naphthol
gives pink
color.

 Formic acid
and acetic
acid give a
deep red
colour.

Table 3 shows that both the Litmus Test and Ferric Chloride Test

confirmed the presence of Phenolic Compounds in B. blumeana lyophilized leaf

extract.

63
Table 4. Total Phenolic Content of B. blumeana Lyophilized Leaf Extract

Table 4 shows that the total phenolic content of B. blumeana lyophilized

leaf extract is 25.24 ± 0.24. In a study by S. Mapoung (2021), cosmetic products

in the market possess a TPC ranging from 0.46-47.92 ug GAE. This signifies that

the total phenolic contents of the B. blumeana lyophilized leaf extract met the

standard range suitable for the formulation of lotion.

64
Table 5. Results of Confirmatory Tests for the Presence of Flavonoids

TEST EXPECTED ACTUAL INTERPRETATION

RESULT RESULT

Alkaline Initial deep color Initial deep color (-) Negative for
Reagent Test that turns but doesn’t turn flavonoids
colorless colorless

Ferric Chloride Dark blue Dark yellow (-) Negative for

Test flavonoids

Shinoda Test Pink scarlet, (+) Positive for

crimson red, flavonoids
green or blue- Crimson red
black precipitates

Lead-Acetate Reddish brown Reddish brown (+) Positive for

test bulky precipitate bulky precipitate flavonoids

Zinc-HCL Magenta colour Magenta colour (+) Positive for

reduction Test flavonoids

Table 5 shows that Shinoda Test, Lead-Acetate Test and Zinc-HCL

reduction Test confirmed the presence of flavonoids. But, Alkaline Reagent Test

and Ferric Chloride Test showed a negative remark for the presence of

flavonoids. Out of 5 tests, 3 have positive remark that signifies the presence of

flavonoids. Based on Ramos (2018), flavonoids have positive remark for the

confirmatory test in ethanolic solvent.

65
Table 6. Results of Confirmatory Tests for the Presence of Tannins

TEST EXPECTED ACTUAL INTERPRETATION

RESULT RESULT

Gelatin Test Precipitation of Gelatinous (+) Positive for

gelatine from precipitate Tannins
solution

Vanillin- Pink or red colour Red color (+) Positive for

hydrochloric Tannins
acid test

Match stick test Matchstick wood Match stick turns (+) Positive for
(Catechin test) turns pink or red red due Tannins

Lead acetate Reddish-brown Brownish bulky (+) Positive for

test bulky precipitate precipitate Tannins

Table 6 shows that all the tests conducted for the presence of Tannins

gave results in the B.blumeana lyophilized leaf extract. This signifies the

confirmation of the presence of Tannins. Based on Ramos (2018), the study

confirmed the presence of Tannins in ethanolic solvent.

66
DPPH Radical Scavenging Assay Result

Table 7. The Percent DPPH Inhibition

The table below shows the % scavenging activity of B. blumeana

lyophilized leaf extract compared to Ascorbic acid in different concentrations.

The Percent DPPH inhibition confirmed that B. blumeana lyophilized leaf

extract possesses antioxidant property based on the scavenging activity using

different concentrations.

From the tabulated values of the percentage inhibition presented, it

showed that the % scavenging activity is in a concentration-dependent manner.

Both of the samples exhibit the same trend of action, wherein as the

concentration increases the inhibitory activity also increases.

67
Table 8. T-test: Two-sample assuming equal variances

Variable 1 Variable 2
B. blumeana
lyophilized leaf Ascorbic acid
extract
Mean 28.67 41.62
Variance 107.00445 324.5186
Observations 5 5
Pooled Variance 215.761525
Hypothesized
Mean Difference 0
df 8
t Stat -1.393967894
P(T<=t) one-tail 0.1004131344
t Critical one-tail 1.859548033
P(T<=t) two-tail 0.2008262688
t Critical two-tail 2.306004133

Table 8 shows the result of T-Test of two sample assuming equal

variances. This proves that the scavenging activity of B. blumeana lyophilized

leaf extract and Ascorbic acid do not exhibit a significant difference. Based on the

statistics, there is no comparable difference between the scavenging inhibition of

both samples.

68
Characteristics and Evaluation of the Formulated Antioxidant Lotion of B.

blumeana Lyophilized lLeaf Extract

Characteristic of the Formulated Antioxidant Lotion

Organoleptic Test:

The formulated lotion has a white and shiny appearance. It

has a scent of fresh bamboo extract with a soft texture.

Homogeneity:

There are no appearance of any clogs.

Evaluation Test of the Formulated Antioxidant Lotion

pH Evaluation:

The pH of the Antioxidant Lotion of B. blumeana lyophilized

leaf extract shows to be 6 using a pH paper. The standard pH for

skin care products is 4-7. According to Abelman (2018), slightly

acidic products is preferred for better complexion. Because low pH

is closer to skin’s pH level, it will effectively work with the skin.

Viscosity:

The result shows that the viscosity of antioxidant lotion of B.

blumeana lyophilized leaf extract has 9350 mPa. According to D

Fransiska et al. (2021), the standard viscosity for a lotion is 2000-

69
 50000 mPa. The formulated antioxidant lotion met the standard

range.

Spreadability:

The formula for the spreadability test is:

spreadability=weight tied ¿ the upper side x length of glass slide ¿ seperate the slide ¿
time taken¿

8 grams x 3.5 cm
spreadability=
3 seconds

spreadability=9.33 grams /cm/seconds

According to Saptarini, N. M. & Hadisoebroto, G. (2020), the

standard spreadability is of 9.0 to 31.02 g·cm/s. The spreadability

of the formulated antioxidant lotion met the standard range and

passed the specification. If spreadability decreases, the topical

product is good because applying it to the skin is easy (Tchienou,

et. al., 2018).

70
 Evaluation of the Safety of Formulated Antioxidant Lotion

Patch Test

The results presented in the succeeding pages show the qualitative

assessment of Skin Irritation and Sensitivity Test among animals applied

with the formulated lotion with the extract of B. blumeana lyophilized leaf

extract (Guevara et. al., 2005).

Table 9. Patch Test Results of the Formulated Lotion

Patch Test
After 24 hours After 72 hours
Female Guinea Pig 1 2 3 1 2 3
Number
Erythema 0 0 0 0 0 0
Edema 0 0 0 0 0 0
Total 0 0 0 0 0 0
Average 0 0 0 0 0 0

Legend: 0 = No Erythema and Edema; 1 = Very Slight Erythema and Edema; 2 = Moderate
Erythema and Edema and 3 = Severe Erythema and Edema

Table 9 shows the results obtained in the Patch Test. The extract from B.

blumeana lyophilized leaf extract in lotion form shows no signs for erythema and

edema to the 3 male guinea pigs.

71
 DISCUSSION

This chapter sums up the gathered results and data from the development

of the study from its objectives, hypotheses, and method of confirmation. The

conclusion is based on the result of the previous chapters. This chapter may help

or guide the future researchers for deeper investigation of the study and may get

an idea for researches that are parallel to this study.

Summary

The extraction, DPPH Assay and Freeze Drying of B. blumeana leaf

extract were done at University of the Philippines, Los Baños. The Confirmatory

Test, Product Formulation, Patch Skin Test and Quality Control were done at

University of La Salette, Inc. The findings and results drawn from the different

tests were summarized and were presented in this chapter.

The Confirmatory Tests of B. blumeana lyophilized leaf extract determined

that the Phenolic Compounds, Flavonoids and Tannins are present in the

sample. The Total Phenolic Content also showed to be suitable for the

formulation of lotion because 25.25± 0.24 passed the standard range.

The DPPH Assay confirmed and showed the scavenging activity of B.

blumeana lyophilized leaf extract in different concentrations: 10 ug GAE (15.63

± 0.32), 15 ug GAE (21.96 ± 0.51), 20 ug GAE (28.58 ± 0.21), 25 ug GAE

(35.75 ± 0.13) and 30 ug GAE (41.43 ± 0.16).

72
 The T-test showed that the difference between both samples which are B.

blumeana lyophilized leaf extract and Ascorbic Acid is not significant. It signifies

that the scavenging activity is comparable.

B. blumeana lyophilized leaf extract can be used as an active component

for further formulation of lotion.

The antioxidant dotion of B. blumeana lyophilized leaf extract showed

acceptable range in evaluation of characteristic and safety tests.

Conclusion

Based on the findings obtained, the researchers conclude that the

presence of Phenolic Compounds, Flavonoids and Tannins in B. blumeana

lyophilized leaf extract proves that the plant contains antioxidant property. The

total phenolic content of the B. blumeana lyophilized leaf extract has 25.24±0.24

ug GAE which is suitable in formulating a lotion. The result of the DPPH Assay

also confirmed the Scavenging activity of B. blumeana lyophilized leaf extract

and Ascorbic acid have the same trend of action as concentration-dependent.

The T-test showed that the scavenging activity B. blumeana lyophilized leaf

extract and Ascorbic acid are not significant. The action of both samples are

comparable. B. blumeana lyophilized leaf extract can be a natural source of

antioxidant for the formulation of lotion and other cosmetic products.

73
Recommendations

Considering the limitations of the study, the following are highly

recommended.

 Use other plant parts of Bambusa blumeana (Kauayan-Tinik), such

as stem and roots in determining its antioxidant property.

 Isolate and identify the specific antioxidant component of B.

blumeana leaf extract using HPLC.

 Formulate other product aside from lotion using Bambusa blumeana

(Kauayan-Tinik).

 Further assess the antioxidant property of Bamboos.

74
 APPENDIX A

APPROVED LETTER FOR THE DEAN

75
 APPENDIX B

GANTT CHART

76
 APPENDIX C

BUDGET PLAN

MATERIAL/PROCEDURE BUDGET

Printing Materials / Bookbind P1,500

Research Procedure
Extraction (drying, extraction and P2750
concentration)
Freeze Drying of Extract P1500
DPPH Assay P2500
Reagents/Ingredients
Carbomer P195
Dimethicone P85
Scent P95
Animal Testing
Certificate of the Guinea Pigs P200
3 Guinea Pigs P1,250
Packaging Materials
Bottles P238
Printing of sticker P150

77
 APPENDIX D

CERTIFICATE OF AUTHENTICATION

78
 APPENDIX E

COMMUNICATION THROUGH EMAIL WITH UNIVERSITY OF THE

PHILIPPINES, LOS Baños

79
 APPENDIX F

STATEMENT OF ACCOUNT

80
 APPENDIX G

DOCUMENTATION OF COLLECTION AND WASHING

Bambusa blumeana (Kauayan-tinik). leaves collection

Bambusa blumeana (Kauayan-tinik) leaves washing

81
 APPENDIX H

DOCUMENTATION FOR CONCENTRATION, EXTRACTION AND FREEZE

DRYING

Bambusa blumeana (Kauayan-tinik) leaves drying and pounding

Bambusa blumeana (Kauayan-tinik) leaves seiving, concentration with 80%

ethanol and centrifuge to recover the extract

Bambusa blumeana (Kauayan-tinik) leaves extraction of using rotary

evaporation, freeze drying the extract and grouding the lyophilized extract

82
 APPENDIX I

DOCUMENTATION OF DPPH ASSAY, CONFIRMATORY TESTS AND TOTAL

PHENOLIC CONTENT

DPPh Assay

Confirmatroy Tests for the Presence of Flavonoids

83
 Confirmatory Tests for the Presence of Phenolic Compounds

Total Phenolic Content of Bambusa blumeana (Kauayan-tinik) Lyophilized

Leaves Extract

Confirmatory Tests for the Presence of Tannins

84
 APPENDIX J

DOCUMENTATION OF PRODUCT FORMULATION

Product Formula for 300 mL

Ingredients Amount in batch Use

300 mL

Bambusa bluemana 1 mL Active Ingredient

(kauayan-tinik)
leaves extract

Distilled Water 250 mL qs ad Solvent

Carbomer (2%) 3 grams Thickener

Stearic Acid 15 grams Emulsifier

Titanium Dioxide 0.5 grams Colorant

Dimethicone 3 mL Occlusive

Sodium Hydroxide 3 mL pH adjuster

Glycerin 15 mL Humectant/Emollient

Triethanolamine 6 mL Surfactant, Alkalizing

and Emulsifying agent

Cetyl alcohol 3 mL Emulsifier

Fresh Bamboo Scent 5 drops Fragrance

85
Methyl Paraben 0.1 gram Preservative

86
 Formulation of the Lotion

87
 Sample Label

88
 APPENDIX K

DOCUMENTATION OF ANIMAL TESTING

Weighing of the 3 guinea pigs

Shaving the fur of the 3 guinea pigs

Applying the lotion to the right side of the shaved area

89
Covering the applied lotion using a wire gauze

The shaved area after 24 hours

The shaved area after 72 hours

90
 APPENDIX L

CERTIFICATION OF THE GUINEA PIGS

91
 APPENDIX M

DOCUMENTATION OF QUALITY CONTROL TEST

Determination of pH level of the formulated lotion

Determination of the viscosity of the formulated lotion

Determination of spreadability of the formulated lotion

92
CZARIANA CASSIDY CORDOVA
#154 Bayaua street, Purok 3, Centro West, Santiago City
Contact Number: 0967-305-2570
Email Address: czarianacassidy11@gmail.com

PERSONAL INFORMATION

FULL NAME: Cordova, Czariana Cassidy

BIRTHDATE: June 11, 2001
AGE: 22
PLACE OF BIRTH: Echague, Isabela
SEX: Female
HEIGHT: 5'4
CIVIL STATUS: Single
NATIONALITY: Filipino
RELIGION: Roman Catholic
LANGUAGE SPOKEN: Tagalog, English

EDUCATION

College University of La Salette, Inc.

Dubinan West, Santiago City
S.Y. 2019 - present

Senior High School University of La Salette, Inc.

Dubinan West, Santiago City
S.Y. 2017-2019

Junior High School Santiago City National High School

Calaocan, Santiago City
S.Y. 2013-2017

Elementary Santiago North Central School

Calao West, Santiago City
S.Y. 2007-2012

93
 INTERNSHIP

Community Internship June 1, 2022 - July 5, 2022

Loven’s Drug.
Santiago City, Isabela

Hospital Internship July 26, 2022 - August 31, 2022

Lung Center of the Philippines
Quezon City, Manila

Manufacturing Internship January 24, 2023 - February 28, 2023

Eurasia Research Pharma Corp.
San Leonardo, Nueva Ecija

Major Internship March 1, 2023 - May 11, 2023

Herbs and NutriPharm Laboratories.
San Leonardo, Nueva Ecija

I do hereby certify that the above information is true and correct to the best of my
knowledge and belief.

CZARIANA CASSIDY CORDOVA

94
JILL F. DAGANDANAN
#023 Quezon St., Bugallon Proper, Ramon, Isabela
jilldagandanan003@gmail.com
0966-630-5206

PERSONAL INFORMATION

FULL NAME: Dagandanan, Jill Flores

BIRTHDATE: July 3, 2000
AGE: 23
PLACE OF BIRTH: Echague, Isabela
SEX: Female
HEIGHT: 5'2
CIVIL STATUS: Single
NATIONALITY: Filipino
RELIGION: Roman Catholic
LANGUAGE SPOKEN: Tagalog, English

EDUCATION

College University of La Salette, Inc.

Dubinan West, Santiago City
S.Y. 2019 - present

Senior High School La Salette of Ramon, Inc.

Ramon, Isabela
S.Y. 2018-2019

Junior High School La Salette of Ramon, Inc.

Ramon, Isabela
S.Y. 2012-2018

Elementary La Salette of Ramon, Inc.

Ramon, Isabela
S.Y. 2006-2012

95
 INTERNSHIP

Community Pharmacy Internship June 13, 2022 – July 15, 2022

PJR Pharmacy, San Mateo Isabela

Hospital Pharmacy Internship February 1, 2023 – March 7, 2023

Southern Integrated Medical Hospital,
Santiago City, Isabela

Manufacturing Pharmacy Internship August 8, 2022 – September 8, 2022

Sydenham Laboratories, Inc.,
Dasmarinas, Cavite

Major Pharmacy Internship March 20, 2023 – May 19, 2023

Farmacia Navarro, Santiago City, Isabela

I do hereby certify that the above information is true and correct to the best of my
knowledge and belief.

JILL F. DAGANDANAN

96
TRIZIA ANDREA D. GENUINO
Brgy. 2 San Mateo, Isabela
trizia.andrea18@gmail.com
0955-621-1001

PERSONAL DATA

FULL NAME: Trizia Andrea D. Genuino

BIRTHDATE: June 18, 2001
AGE: 22
PLACE OF BIRTH: San Mateo, Isabela
SEX: Female
HEIGHT: 5'0
CIVIL STATUS: Single
NATIONALITY: Filipino
RELIGION: Roman Catholic
LANGUAGE SPOKEN: Tagalog, English

EDUCATIONAL BACKGROUND

College University of La Salette, Inc.

Dubinan West, Santiago City
S.Y. 2019 - present

Senior High School La Salette of San Mateo

Brgy. 1, San Mateo Isabela
S.Y. 2017-2019

Junior High School La Salette of San Mateo

Brgy. 1, San Mateo Isabela
S.Y. 2013-2017

Elementary La Salette of San Mateo

Brgy. 1, San Mateo Isabela
S.Y. 2007-2012

97
 INTERNSHIP

Community Pharmacy Internship June 8, 2022 – July 10, 2022

PJR Pharmacy, San Mateo Isabela

Hospital Internship July 26, 2022 - August 31, 2022

Lung Center of the Philippines
Quezon City, Manila

Manufacturing Internship January 24, 2023 - February 28, 2023

Eurasia Research Pharma Corp.
San Leonardo, Nueva Ecija

Major Internship March 1, 2023 - May 11, 2023

Herbs and NutriPharm Laboratories.
San Leonardo, Nueva Ecija

I do hereby certify that the above information is true and correct to the best of my
knowledge and belief.

TRIZIA ANDREA D. GENUINO

98
EFRELYN MAE G. LAGMAY
Lagmay St., Zone 4, Sagat, Cordon, Isabela
lagmayefrelyn@gmail.com
0917-637-2355

PERSONAL BACKGROUND

FULL NAME: Efrelyn Mae G Lagmay

BIRTHDATE: August 20, 2002
AGE: 19
SEX: Female
HEIGHT: 5'2
CIVIL STATUS: Single
NATIONALITY: Filipino
RELIGION: Roman Catholic
LANGUAGE SPOKEN: Tagalog, English

EDUCATION HISTORY
College University of La Salette Inc.
Dubinan West, Santiago City
S.Y. 2019 - Present

Senior Highschool Cagasat National High School-Main

Gayong, Cordon, Isabela
S.Y 2013-2019
Junior Highschool Cagasat National High School-Main
Gayong, Cordon, Isabela
S.Y 2013-2019
Elementary Sagat Elementary School
Sagat, Cordon, Isabela
S. Y 2007-2013

INTERNSHIP

Community Internship June 22, 2022 - July 26, 2022

Farmacia Navarro, Santiago City, Isabela

99
Hospital Pharmacy Internship February 1, 2023 – March 7, 2023
Southern Integrated Medical Hospital,
Santiago City, Isabela

Manufacturing Pharmacy Internship August 8, 2022 – September 8, 2022

Sydenham Laboratories, Inc.,
Dasmarinas, Cavite

Major Pharmacy Internship March 20, 2023 – May 19, 2023

Farmacia Navarro, Santiago City, Isabela

I do hereby certify that the above information is true and correct to the best of my
knowledge and belief.
.

EFRELYN MAE G. LAGMAY

100
FANNY LOIS G. PANAGA
Brgy. 02, Jones, Isabela
fannypanaga@gmail.com
0955-308-0244

PERSONAL DATA

FULL NAME: Fanny Lois G. Panaga

BIRTHDATE: August 21, 2001
AGE: 22
SEX: Female
HEIGHT: 5'2
CIVIL STATUS: Single
NATIONALITY: Filipino
RELIGION: Baptist
LANGUAGE SPOKEN: Tagalog, English

EDUCATIONAL BACKGROUND

College University of La Salette, Inc.

Dubinan West, Santiago City
S.Y. 2019 - present

Senior High School Jones Rural School

Brgy. 02, Jones, Isabela
S.Y. 2017-2019

Junior High School Jones Rural School

Brgy. 02, Jones, Isabela
S.Y. 2013-2017

Elementary Jones West Central School

Brgy. 02, Jones, Isabela
S. Y. 2007-2012

101
 AFFILIATIONS:

Community Pharmacy Internship June 13, 2022 – July 15, 2022

Bingking’s Pharmacy, Jones, Isabela

Hospital Internship July 26, 2022 - August 31, 2022

Callang General Hospital
Santiago City, Isabela

Manufacturing Internship January 24, 2023 - February 28, 2023

Eurasia Research Pharma Corp.
San Leonardo, Nueva Ecija

Major Internship March 1, 2023 - May 11, 2023

Herbs and NutriPharm Laboratories.
San Leonardo, Nueva Ecija

I do hereby certify that the above information is true and correct to the best of my
knowledge and belief.

FANNY LOIS G. PANAGA

102
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