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OPTIMIZATION AND EVALUATION OF EFFICIENCY OF A HERBAL

SHAMPOO CONTAINING HIBISCUS ROSA-SINENSIS LEAVES


EXTRACT AND TREATMENT OF ALOPECIA AREATA DISEASE

MUHAMMAD FAWWAZ BIN MOHD ZULKIFLI


55101113096

UNIVERSITY KUALA LUMPUR


JANUARY 2015
OPTIMIZATION AND EVALUATION OF EFFICIENCY OF A HERBAL
SHAMPOO CONTAINING HIBISCUS ROSA-SINENSIS LEAVES
EXTRACT AND TREATMENT OF ALOPECIA AREATA DISEASE

MUHAMMAD FAWWAZ BIN MOHD ZULKIFLI


55101113096

Report Submitted to Fulfill the Partial Requirements


For the Diploma of Chemical Engineering Technology (Process)
Universiti Kuala Lumpur

JANUARY 2015
DECLARATION

I declare this thesis report is my original work and all references have
been cited adequately as required by the University.

Date : Signature :………………….


Full Name : MUHAMMAD
FAWWAZ BIN
MOHD ZULKIFLI
ID Number : 55101113096

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APPROVAL

I have supervised and examined this report and verify that it meets the
programmed and University’s requirements for the Diploma in Chemical
Engineering Technology (Process)

Date : Signature :………………….

Supervisor : Dr. Marmy Roshaidah


Bt. Mohd Salleh

Official Stamp :

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ACKNOWLEDGEMENT

First and foremost I am grateful to Allah S.W.T as He gave me all this


valuable opportunity and strength to experience all the challenge in order to
finish my Final Year Project in 2015. This is a very useful knowledge where it
prepare us to be ready with working experience and all the method can be
used during working later on.
I would like to express my deepest appreciation to my supervisor, Dr.
Marmy Roshaidah Bt. Mohd Salleh, who has the attitude and the ingredient
of a genius. Thank you dr. for all the ups and downs during my Final Year
Project where she always give full attention along my study, give helping
hands, advices, actuation, guidance, suggestion and information which very
useful for my project. Without her guidance and persistent help, this thesis
may not be exists at all.
Next, my gratitude to my beloved mom, Noor Hasmini Bt. Abd Ghani,
who always give uncountable moral support during my Final Year Project.
She willing to go any lengths to make sure that my project is a success.
At long last, I would like thanks you to all my friends especially
Muhammad Eizatul Adhwa’ bin Jalani who are very supportive, give a helping
hands during my needs, go through all the challenge together until we
managed to finish up our own Final Year Project.

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CONTENTS

Page No.

DECLARATION……………………………………..……………………..… iii

APPROVAL PAGE………………………………………………………….. iv

ACKNOWLEDGEMENT……………………………………………………. v

LIST OF TABLES……………………………………..…………………….. ix

LIST OF FIGURES…………………………………………………………... x

LIST OF SYMBOL/ABBREVIATION…………………………………….... xii

ABSTRACT…………………………………………………………………… xiii

ABSTRAK…………………………………………….................................... xiv

CHAPTER 1 : INTRODUCTION

1.1 Alopecia Areata…...……………………………………………………… 1

1.2 Shampoo………………………………………………………………….. 2

1.3 Hibiscus Rosa-Sinensis Linn Leaves………………………………….. 2

1.4 Problem Statement………………………………………………………. 3

1.5 Objective………………………………………………………………….. 4

1.6 Scope……………………………………………………………………… 4

1.7 Thesis Layout……………………………………………………………...5

CHAPTER 2 : LITERATURE REVIEW

2.1 Alopecia Areata…..………………………………………………………. 6

2.1.1 Types of Alopecia………………………………………… 7

2.1.2 Treatment for Alopecia Areata…………………………………...8

2.2 Shampoo…………………………………………………………………. 9
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2.3 Basic Ingredients of Shampoo…..……………………………………… 9

2.3.1 Shampoo Based on Synthetic Ingredients……………………. 12

2.3.2 Shampoo Based on Synthetic Ingredients……………………..16

2.4 Marketing of the Herbal Shampoo……………………………………… 19

2.5 Forms of Shampoo…………….………………………………………… 20

2.6 Hibiscus Rosa-Sinensis………….……………………………………… 21

2.7 Scientific Classification………………………………………………….. 22

2.8 Phytochemical Review in Hibiscus…………………………….. …...….23

2.9 Pharmacological Review in Hibiscus………...………………………… 24

2.10 Origin and Distribution of Hibiscus…………………………………….. 26

2.11 Description of Hibiscus …………………………………………………. 27

2.12 Hibiscus Rosa-Sinensis Leaves as Remedy…………………………. 28

2.13 Application on the Hibiscus Rosa-sinensis……………………………. 29

2.14 Antioxidant………………………………………………….…………….. 29

2.14.1 Total Flavonoid Content (TFC)…………………………………. 30

2.14.2 Function of TFC in the Hibiscus Leaves…...………………….. 32

2.15 Soxhlet Extractor…………….…………………………………………… 33

2.16 Ultrasonic Assistance Extraction………………………………………...34

2.17 Filtration…………………………………………………………………….34

2.18 Ultraviolet-visible spectroscopy (UV-Vis)……………………………….35

2.19 Fourier Transform InfraRed FT-IR….…………………………………...37

2.20 Solvent………………………………………………………………..… 38

2.20.1 Petroleum Ether…………………………………………………...39

2.20.2 Methanol……………………………………………………………40

2.20.3 Ethanol…………………………………………………………… 40

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2.21 Studying TFC Content on Different Parameters………………………41

2.21.1 Effect of Solvents on the Yield of TFC…………………………41

2.21.2 Effect of Extraction Method on the Extraction Yield of TFC….41

2.21.3 Effect of Solid-to-Liquid Ratio on the Yield of TFC……………41

2.21.4 Effect of the Time on the Extraction Yield of TFC……………..41

2.22 Identifying of TFC in the Hibiscus Leaves Using FT-IR……………….42

CHAPTER 3 : METHODOLOGY

3.1 Materials……….………………………………………………………….. 43

3.2 Equipment…………………...……………………………………………. 43

3.3 Procedure………….……………………………………………………… 44

3.4 Pre-treatment Process………...………………………………………… 44

3.5 Preparation for Extraction Process…….………………………………. 45

3.5.1 Soxhlet Extraction Method………………………………………. 45

3.5.2 Ultrasonic Assistance Extraction Method……………………. 46

3.5.2.1 Filtration Process………………………………… 47

3.6 Fourier Transform infrared radiation (FT-ir) analysis………………… 48

3.7 Analysis Using UV-Vis…………………………………………………… 49

3.8 Rotary Evaporator…………..……………………………………………. 49

3.9 Formulation Herbal Shampoo………...………………………………… 50

CHAPTER 4 : RESULT AND DISCUSSION

4.1 Introduction…….…………………………………………………………. 52

4.2 Parameters of the Experiments…...……………………………………. 53

4.3 Effect of Solvent on the Yield of TFC………………………………….. 53

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4.4 Effect of Extraction Methods on the Extraction Yield of TFC…………54

4.5 Effect of Solid-to-Liquid Ratio on the Yield of TFC…………………….56

4.6 Effect of Time on the Extraction Yield of TFC………………………… 57

4.7 Confirmation of TFC in the Hibiscus Leaves Using FTIR…………… 58

4.7.1 Confirmation TFC Using Solvent Extraction of

Petroleum Ether………………............................................... 59

4.7.2 Confirmation TFC Using Solvent Extraction of Methanol……. 59

4.7.3 Confirmation TFC Using Solvent Extraction of Ethanol……… 60

CHAPTER 5 : CONCLUSION AND RECOMMENDATIONS

5.1 Conclusion…………………………………………………………….. 61

5.2 Recommendation……………………………………………………….. 62

REFERENCES…………………………………………………………………. 63

APPENDICES…………………………………………………………………… 68

APPENDIX A: Table for Raw Material………………………………….. 68

APPENDIX B: Figure of Functional Groups of

Compound Inside the Sample……………………….......72

APPENDIX C: Figure of Standard Calibration of

Ferrous Sulphate (FeSO4)………………………………75

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LIST OF TABLE

Page No.

Table 2-1 Distribution in Groups with Alopecia Areata…………………… 7

Table 2-2 Type of Synthetic Ingredients and Its Own

Effects on Human Body……………………………………………13

Table 2-3 The List of Plants Which Are Commonly Used in Shampoos

Along With Their Common Names and Reported Functions

Or Uses………………………………………………………….. 17

Table 2-4 Scientific Classification of Hibiscus Rosa-sinensis…………… 22

Table 2-5 Phytochemical Review in Hibiscus Leaves……………………. 23

Table 2-6 Pharmacological Review in Hibiscus………………………… 25

Table 2-7 Types of Vitamin………………………………………………… 30

Table 3-1 Chemical Formulation of Herbal Shampoo……………………. 50

Table 4-1 the functional groups of the purified flavonoid compound

from IR-spectrum……………………………………………….. 58

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LIST OF FIGURES

Page No.

Figure 2-1 Stages Alopecia Areata Occur from Beginning to the End 8

Figure 2-2 Ingredients Shampoo…………………………………………. 12

Figure 2-3 Synthetic Shampoos…………………………………………... 12

Figure 2-4 Herbal Shampoo………………………………………………. 16

Figure 2-5 Herbal Shampoo Usages in Past and Present in India…….. 19

Figure 2-6 the Most Preferred Brand Shampoo in India………………….. 19

Figure 2-7 Flower of Hibiscus Rosa-sinensis……………………………… 21

Figure 2-8 Basic Structures of Flavonoids………………………………… 31

Figure 2-9 Biosynthesis of Flavonoids……………………………………. 31

Figure 2-10 Chemical Structures of Different Types of Flavonoids…….. 32

Figure 2-11 Schematic Diagram of UV-Vis…………………………………. 36

Figure 2-12 Schematic Diagram of FT-IR…………………………………… 38

Figure 2-13 Solvent Polarity Chart……………………………………………. 39

Figure 3-1 Pre-treatment Process of Hibiscus Leaves……………………. 45

Figure 3-2 Figure 3.2 Process of Soxhlet Extraction Method……………. 46

Figure 3-3 Process Ultrasonic Assistant Extraction Method…………….. 48

Figure 3-4 Flow of Formulation of Herbal Shampoo………………………. 51

Figure 4-1 Flavonoids Contents (mg FeSO4 per 100 gram of sample)

against Type of Solvents………………………………………. 54

Figure 4-2 Flavonoids Contents (mg FeSO4 per 100 gram of sample)

against Type of Methods……………………………………….. 55

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Figure 4-3 Flavonoids Contents (mg FeSO4 per 100 gram of sample)

against Material per Solvent (gram per ml)…………………. 56

Figure 4-4 Flavonoids Contents (mg FeSO4 per 100 gram of sample

against Time (hour)………………………………………….. 58

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LIST OF ABBREVIATIONS

TFC Total Flavonoid Content

M Meter

nm Nanometre

ml Millilitre

mm milimetre

µl Microlitre

% Percent

°C Degree Celcius

g gram

M Mole

ppm Part per milion

nm Nanometre

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ABSTRACT

Alopecia areata is the diseases that affect part of the skin from which hairs grow
which are hair follicles and there is no cure for it. The hibiscus leaves have
active substance that can treat the alopecia disease formulated in the herbal
shampoo that act as the medium. The objectives of experiments are to
determine the active ingredient in Hibiscus rosa-sinensis that can improve blood
circulation to nourish the hair follicle and thereby promoting hair growth, to
determine the yield of flavonoids content on different parameters which are
solvents, mass ratio between samples and solvents and time of extraction and
also to obtain the optimization of flavonoid content on different parameter for
extraction process. There are two types of method had been used for this
experiment which is soxhlet extraction method and ultrasonic assistant
extraction. After the extraction had done, the sample must through the rotary
evaporator to get the oil and use it as main ingredients of formulation herbal
shampoo. For discussion, the solvent of ethanol is the highest extract flavonoid
content in hibiscus leaves due to highest polarity compare to others. Moreover,
the best method for extract the flavonoid content is soxhlet extraction because of
longer extraction time compare to the ultrasonic assistant extraction. The
optimizations of yield flavonoid content are ethanol for solvents, 240 minutes of
extraction time and soxhlet extraction for method. The recommendation of the
experiment is keep the sample extracted in cold place as the flavonoid content is
sensitive to temperature. Invent new product which is pills from hibiscus leaves
as main source as the leaves can be consumed.

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ABSTRAK

Alopecia areata adalah penyakit yang memberi kesan kepada bahagian kulit
dimana rambut tumbuh iaitu folikel rambut dan ia tidak ada ubat untuk penawar.
Daun bunga raya mempunyai bahan aktif yang boleh merawat penyakit alopecia
dengan dirumuskan dalam syampu herba yang bertindak sebagai medium.
Objektif eksperimen ini adalah untuk menentukan bahan aktif dalam Hibiscus
rosa-sinensis yang boleh meningkatkan peredaran darah untuk menyuburkan
folikel rambut dan dengan itu menggalakkan pertumbuhan rambut, untuk
menentukan hasil kandungan flavonoid pada parameter yang berbeza yang
pelarut, nisbah jisim antara sampel dan pelarut dan masa pengekstrakan dan
juga untuk mendapatkan pengoptimuman kandungan flavonoid pada parameter
yang berbeza untuk proses pengekstrakan. Terdapat dua jenis kaedah telah
digunakan untuk eksperimen ini iaitu kaedah pengekstrakan soxhlet dan
pembantu pengekstrakan ultrasonik. Selepas pengekstrakan yang dibuatnya,
sampel harus melalui penyejat berputar untuk mendapatkan minyak dan
menggunakannya sebagai bahan utama rumusan syampu herba. Pada
bahagian perbincangan, pelarut etanol adalah ekstrak tertinggi kandungan
flavonoid dalam daun bunga raya kerana ia mempunyai kekutuban tertinggi
berbanding dengan pelarut yang lain. Selain itu, kaedah terbaik untuk ekstrak
kandungan flavonoid adalah adalah kaedah pengekstrakan soxhlet kerana
masa pengeluaran yang lebih lama berbanding dengan pembantu
pengekstrakan ultrasonik. Pengoptimuman hasil kandungan flavonoid adalah
etanol untuk pelarut, 240 minit masa pengekstrakan dan pengekstrakan soxhlet
bagi kaedah. Syor percubaan daripada ekperimen ini adalah menyimpan
sampel yang diekstrak di tempat sejuk kerana kandungan flavonoid sensitif

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kepada suhu. Mencipta produk baru iaitu pil dari daun bunga raya sebagai
sumber utama kerana daunnya adalah sejenis daun yang boleh dimakan.

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CHAPTER 1: INTRODUCTION

1.1 Alopecia Areata

Alopecia areata is a general, inflammatory disease that difficult to be cure, which


occurs in 0.01 to 0.1% of the Caucasian population. (Meidan et al., 2001). It is a
disease that affects part of the skin from which hairs grow which is hair follicles.
In most cases, hair falls out in small, round patches about the size of a quarter.
Most of the patients with the disease get only a few bare patches while some
may lose more hair. Rarely, the disease causes total loss of hair on the head or
complete loss of hair on the head, face, and body. Everybody can have alopecia
areata. It commonly begins in childhood. There is a slightly increased risk of
having the disease if you have a close family member with the disease Alopecia
areata is an autoimmune disease or medical condition is one that is caused by
substances that usually prevent illness.

Generally the immune system protects the body against infection and
disease. In an autoimmune disease, the body’s immune system mistakenly
attacks some part of your own body. In alopecia areata, the immune system
attacks the hair follicles. The cause is unknown. Scientists think that a person’s
genes may play a role. For people whose genes put them at risk for the disease,
some type of trigger starts the attack on the hair follicles. The triggers may be a
virus or something in the person’s environment. There is neither cure nor drugs
for alopecia areata to approve to treat it. Doctors may use medicines approved
for other diseases to help hair grow back. Fortunately, since the immunological
events of alopecia areata do not lethally damage extremely important of the
elements of the hair follicle, symptomatic treatment of the condition is possible.
(Meidan et al., 2001)

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1.2 Shampoo

The definition of shampoo by Harry is preparation of a surfactant (surface active


material) in a suitable form such as liquid, solid, and powder. Unfortunately, it is
very harmful for the youth and our environment when use it for long period.
Numerous side effects that cause various skin diseases can be proved by
various synthetic compounds, chemicals, dye, and their derivative. Herbal is the
word that means of safety in contrast to the synthetic one which has adverse
effects on human health. Thus, herbal cosmetic become higher attract to the
society and now generally tremendous range of herbal products is available to
the public. (Namita et al.,2013). One of the herbal product is made from hibiscus
leaves that have active substance to act as the hair growth promoter.

1.3 Hibiscus Rosa-Sinensis Linn Leaves


Hibiscus rosa-sinensis known as Chinese hibiscus, Japa and at the tropical and
subtropical regions of East Asia the Hibiscus has widely grown as ornamental
plant. The genus is varied and wide because it consist about 250 different
species and not all tropical as native hibiscus species are represent on all
continents except Antartica. The annual or perennial herbs contained from some
of these species while the others are small trees or flowering shrubs. Hibiscus
rosa-sinensis has great potential for investigation to know more about various
biological activities and it also useful developing more therapeutic value and
new formulations. (Kumar et al.,2012).
One of the results from alcoholic and chloroform extract of Hibiscus rosa-
sinensis leaves is flavonoids. The quantity of flavonoid is much higher in the
leaves compare to the other parts. (Mak et al.,2013). Flavonoids is a hair growth
promoting activity by strengthening the capillary wall of the smaller blood
vessels supplying hair follicle, promote blood circulation to nourish the hair
follicle and thereby make the hair become growth. (Awe and Makinde, 2009).

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1.4 Problem Statement

Hibiscus rosa-sinensis L. can easily grow anywhere in Malaysia as the climate in


Malaysia is suitable for its growth. It does not need extra care and supervision
as it only need sun light and water to grow better. The content of flavonoids is
abundance found in the leaves.

Common scenario in Malaysia, Hibiscus rosa-sinensis is planted just for


decoration to make the place become attractive and as the symbol of national
flower of Malaysia and the State of Hawaii. Some of the Malaysian is not aware
about the importance of the other part of the Hibiscus including its leaves. Its
leaves are commonly served as waste and it will be thrown away when the tree
is cut off. Moreover, hair loss can cause by many factor such as pregnancy, lack
of protein, heredity and more. Although the hair loss has medicine to cure but it
cost is really expensive and contained harmful chemicals. Knowing that the
leaves of Hibiscus rosa-sinensis have valuable phytochemical content that can
be extracted and give profit, it is the best opportunity to create wealth from the
waste and the most highlight opportunity is to create health from the waste.

Extraction process from Hibiscus leaves is done by using analytical grade


solvent to obtain the valuable phytochemical content. The analytical grade
solvent is used as it is safe to consume and no harm will bring to the consumer.
Hence, a research was done to produce hair loss medicine as herbal shampoo
for future generation.

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1.5 Objective

The purpose research was studied to achieve the following objectives:

1. To determine the active ingredient in Hibiscus rosa-sinensis that can


improve blood circulation to nourish the hair follicle and thereby
promoting hair growth.
2. To determine the yield of flavonoids content on different parameters.
3. To obtain the optimization of flavonoid content on different parameter for
extraction process

1.6 Scope

In the extraction of flavonoids from Hibiscus rosa-sinensis leaves, it focused on


four scopes to obtain the optimum condition for the extraction.

i. Two methods are used to determine the optimum method for extraction
process which are by using Soxhlet extraction method and Ultrasonic
bath method.
ii. Three solvents are used in the experiments which are petroleum ether,
methanol and ethanol. The usage of three solvents will be compare to
see which of the solvent affect the amount of the flavonoids content when
doing analysis.
iii. To determine the optimum mass for the extraction process. Three
different mass are chosen which are 10 gram, 20 gram and 30 gram.
iv. To determine the optimum time taken for the extraction process. For
method using Soxhlet extraction, each and every sample undergoes
extraction process for 120 minutes, 240 minutes, and 360 minutes.
Meanwhile for the method using ultrasonic bath, each and every sample
undergoes extraction process for 10 minutes, 20 minutes, and 30
minutes.

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1.7 Thesis Layout

Thesis layout for this final year project is state on the above:

I. The chapter 1 is introduction about the Alopecia areata disease, herbal


shampoo and Hisbiscus Rosa-sinensis. Moreover, it also tells about the
problem statement and also objectives of this final year project.
II. The chapter 2 is about the all literature review that involve the experiment
including equipment for testing, solvents, materials, and disease
III. The chapter 3 is the methodology to preparation of sample includes
extraction of process with two different method which are soxhlet
extraction method and ultrasonic assistant extraction method and also
formulation of herbal shampoo. Moreover, it tells the procedure using UV-
Vis and FT-iR to identify the flavonoid content and determine the
concentration of flavonoid of the sample.
IV. The chapter 4 is for discussion about the differences parameters used
that affect the flavonoid content. The parameters are solvents, mass ratio
between sample and solvents and time of extraction.
V. The chapter 5 is for conclusion of optimization flavonoid concentration of
the sample and recommendation to improve about the research involve
flavonoid content and also Hibiscus Rosa-sinensis

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CHAPTER 2 :LITERATURE REVIEW

2.1 Alopecia Areata

2% of the population experiences the Alopecia areate disease throughout their


life which is a common type of hair loss. Alopecia areata is seen at different
ages which from breast-fed children until the elderly. However, at the ages of 40
to 50 the Alopecia areata mostly occur and it is more common at ages of 30 to
50. Alopecia is caused by an inflammatory or autoimmune process which it is a
non-scaring hair loss, and is mostly seen in the scalp. The genetic background
is an important issue and it is specifically targets hair follicles. However,
evidences indicate the importance of melanocytic antigens as a motivator. The
aetiology of Alopecia areata is unknown. Nonetheless, according to the studies,
stress could provoke the onset of the disease. Alopecia areata is an
unpredictable disease. In some people, hair grows back and remains. In others
hair grows back but falls out again later. Each case is unique. Even if someone
loses all of his or her hair, there is a chance that it will grow back.(Arbabi et al.
2013). Table 2.1 shows the distribution in groups with Alopecia areata.

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Table 2.1 Distribution in Groups with Alopecia Areata

Ages of 15-20 21-30 31-45 >45


Groups

Number of 7 20 8 8
males with
Alopecia
areata

Number of 4 5 5 3
females with
Alopecia
areata

2.1.1 Types of Alopecia

There are five different types of this disease such as Alopecia


Multilocularis which it results into multiple area of hair loss. Next is
Alopecia Totalis that can cause the loss of all the hair on the scalp.
Thirdly is Alopecia Universalis that can loss of all body hair including
public hair. Fourthly is Alopecia areata barbae if it is the loss of hair only
to the beard area. Lastly, Alopecia Areata which is the kind of alopecia
that refers to hair loss that occurs in rounded patches. These patches can
appear anywhere on the body. (P. Jyoti, 2012)

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2.1.2 Treatment for Alopecia Areata

Alopecia areata cannot be cured. However, it can be treated and hair can
grow back. In many cases, alopecia areata is treated with drugs that are
used for other conditions. Moreover, some researcher have found other
way to treat alopecia disease which is by using shampoo formulated with
extracted herbs such as hibiscus leaves that act as hair growth promoter
and do not have side effects (van den Biggelaar, Smolders, and Jansen
2010). Figure 2.1 shows the stages of alopecia areata begins until the
end.

Figure 2.1 Stages Alopecia Areata Occur from Beginning to the End

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2.2 Shampoo

The definition of shampoo is the problem solver that is used to removed dirt, oil,
skin particles and dandruff from hair which had been build up in hair frequently
and need to be discharge to maintain the practice of keeping yourself and your
living and working areas clean in order to prevent illness and disease. It is
important to remove these macroganism from hair by exclude stripping the scalp
of sebum which is natural oil. (Mottram and Lees, 2000). It is hair care product
that can be form into liquid, creamy and gel. Shampooing is the most frequent
treatment of hair form. The main function of shampoo to achieved at cleansing
of the hair necessitated due to accumulated sebum, dust, scalp debris and
more. The quality of preparation that always in the same way is depends on the
inclusion of traditional soaps saturated with glycerides and herbal or synthetic
fatty alcohols or thickening agents. (Suriyaprakash et al., 2011)

2.3 Basic Ingredients of Shampoo

A product of shampoo is combined of 10 to 30 ingredients and divides into 4


classifications such as cleansing agents (surfactants), special care ingredients,
conditioning agents and additives. First as for surfactant, the surface activity of
shampoo detergents is affected the cleansing ability of it. The function of
surfactants is to facilitate the removal of environmental dirt by reducing surface
tension between water and dirt. Dirt is the particles that suspended in liquid
phase adsorption prevented. This is achieved by a special molecular structure
consisting of a hydrophilic and lipopholic group. The center of a micelle structure
with the hydrophilic molecule ends pointing outward is the place where sebum
and dirt are bound and surrounded. The very small pieces of dirt turn into water

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soluble and can be take away from the hair shaft. Surfactant can be divide into
hydrophilic polar group as anionic, cationic, amphoteric (zwitterionic) and
nonionic. (Trueb, 2007)

Anionic surfactants act as negatively-charged hydrophilic polar group in a


solution. These surfactants are the most widely used type of surfactant for
preparing shampoos because of its excellent cleaning properties and high hair
conditioning effects. The best known anionic surfactants are sulfated fatty
alcohols, alkyls sulfates, and their polyethoxylated analogues, alkyl ether
sulfates that act as builders (Ca per Mg sequestrants) in hard water. Still, they
can react in the wash water with the positively charged water hardness ions
(calcium and magnesium), which can lead to partial deactivation. So, the more
calcium and magnesium molecules in water, the more anionic surfactants
suffers from deactivation. To prevent this occur, anionic surfactants need help
from other ingredients such as builders and more detergent should be dosed in
hard water. (Mishra et al. 2009)

Nonionic surfactants do not have any electrical charge, which makes


them repel to water hardness deactivation. They are less irritant compare other
anionic or cationic surfactants. The polyoxyethylene, polyoxypropylene or polyol
derivatives is contained in the hydrophilic part while the saturated or
unsaturated fatty acids or fatty alcohols is contained in the hydrophobic part. In
combination with amphoteric surfactants, they give to further improve the
tolerability in very mild cleansers such as baby shampoo. (Mishra et al. 2009)

Additives are important for shampoo product to possess stability and to


have an appealing quality. Some additive is to modify of surfactants effects. It
helps to less the skin irritation when the surfactant is select and combine with
other surfactants. Moisturizers such as natural oils, fatty acid esters and
alkanolamides will combined with humectant include propylene glycol,
polyethylene glycol, glycerin, sorbitol, and lactate to make the hair silkier.
Additives also make shampoo stabilization. Preservative is required to protect

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against bacterial contamination and hence ensuring shampoo stability. UV
absorber to stabilize dyes against light, antioxidant to protect oxidation-sensitive
substances and buffers to ensure pH stability. In addition, co-solvents to
maintain conditioning oils, dispersing agents to keep otherwise insoluble agents
such as silicone oils and anti-dandruff agents in suspension and fragrances
clear in solutions. (Trueb, 2007)

Next, the conditioner, the primary function of conditioning agents in a


shampoo is to keep the natural condition of newly grown hairs for as long as
possible. Conditioning agents are contained in all standard shampoo used today
virtually. Nevertheless, it is possible to become greater shine and make hair
more manageable and make it simple with a high proportion of conditioning
agents. Fatty substances such as vegetables oils, wax, lecithin and lanolin
derivatives, protein hydrolysates, quaternary ammonium and silicon are
conditioning agents. There is increasing use of cationic polymers in place of
monomer quaternary ammonium compounds in which the cationic groups are
integrated in a polymer structure. These adhere more firmly to the hair more
than monomer bonds and leave the film that coats the surfaces of the hair fiber,
making it appear to be soft and smooth while improving shine and color by
altering the refractive index. (Trueb, 2007). The figure 2.2 show simplified of
ingredients of shampoo on the next page.

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Figure 2.2 Ingredients Shampoo (Trueb, 2007)

2.3.1 Shampoos Based On Synthetic Ingredients

Nowadays, people are more concerned about various brands of shampoo


that have caused various conditions such as scalp irritation, hair loss and
severe hair damage. Figure 2.3 shows example of synthetic shampoos.

Figure 2.3 Synthetic Shampoos

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Most synthetic ingredients that available in shampoos are loaded
with chemicals that are hazardous to skin and health. Many people do not
know the side effects associated with these harmful synthetic ingredients.
Generally to most synthetic ingredients based shampoos is an ingredient
called a surfactant, which has ability to reduce surface tension of water.
The result is that, the sebum will wash away as the hair is rinsed.
Lathering of chemical shampoo also is the caused by the activity of
surfactant. Some shampoos contain surfactant with strong lathering
properties although they may not be ideal in terms of conditioning or
irritant potential.(Arora, Nanda, and Karan, 2011). Table 2.2 show the
type of synthetic ingredients that has its own effect on the human body.

Table 2.2 Type of Synthetic Ingredients and Its Own Effects on Human Body

Name of Synthetic Ingredients Effect on Human Body

DEA (Diethanolamine), MEA This foam booster is a skin and eye


(Monoethanolamine), and TEA irritant and causes contact dermatitis.
(Triethanolamine) Easily absorbed through skin to
accumulate in body organs and the
brain.

Dioxin Act as emulsifier that can causes


cancer, reduced immunity, nervous
system disorders, miscarriages and
birth deformity.

DMDM Hydantoin and Urea 2 preservatives that release


(lmidazolidinyl) formaldehyde which may cause joint
pain, cancer, skin reactions, allergies,
depression, headaches, chest pain,
ear infections, chronic fatigue,

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dizziness and insomnia.

FD and C Color and Pigment Synthetic colors from coal tar contain
heavy metal salts that deposit toxins in
skin, causing skin sensitivity or
irritation. Absorption can cause
depletion of oxygen and death. Animal
studies show almost all are
carcinogenic.

Parabens (Methyl, Butyl, Ethyl, Propyl) Used as preservatives. Have been


found in breast cancer tumors. May
contribute to sterility in males,
hormone imbalance in females and
early puberty.

PEG (Polyethylene glycol) Made by ethoxylating Propylene


Glycol. Dangerous levels of dioxin
have been found as a by-product of
the ethoxylation process. PEGs are in
everything including personal care,
baby care and sunscreens.

Phthalates Usually not listed on labels. Health


effect includes damage to liver and
kidney, birth defects, decreased sperm
counts and early breast development
in girls and boys.

Propylene Glycol (PG) and Butylene Petroleum plastics. EPA considers PG


Glycol so toxic requires gloves, clothing,
goggles and disposal by burying. EPA
wams against skin contact to prevent

14 | P a g e
brain, liver, and kidney abnormalities.

Sodium Lauryl Sulfate (SLS) and Used in car washes, garage floor
Sodium Laureth Sulfate (SLES) cleaners, engine degreasers and 90%
of personal-care products that foam.
Eye damage, depression, labored
breathing, diarrhea, skin irritation, and
death.

Sunscreen chemicals Avobenzone, benzphenone,


ethoxycinnnamate, PABA are
commonly used ingredients that are
known free radical generators and are
believed to damage DNA or lead to
cancers.

Tridosan Synthetic antibacterial ingredient. EPA


registers it as pesticide, posing risk to
human health and environment.
Classified as a chloropherol,
chemicals suspected of causing
cancer in humans

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2.3.2 Shampoos Based On Herbal Ingredients

From the past few decades, the use of natural product in cosmetic has
been dramatic increase. The precautions and need for cosmetics with
herbs is on the rise as it believes that these products are safe and free
from side effects. Figure 2.4 shows example of herbal shampoo

Figure 2.4 Herbal Shampoo

Now-a-days, most of the herbal shampoos are available in the


market which contains herbal ingredients such as plant extracts and
essential oils. The plants which are commonly used in shampoo as the
main ingredient are reported to have beneficial effects on hair. Table 2.3
shows the list of plants which are commonly used in shampoos along with
their common names and reported functions or uses. (Arora, Nanda, and
Karan 2011).

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Table 2.3 the List of Plants Which Are Commonly Used in Shampoos Along With
Their Common Names and Reported Functions or Uses. (Arora, Nanda, and
Karan 2011)

Botanical name Common name Reported functions/uses.

Used in shampoos and hair


Berberis vulgaris Barberry
rinses for hair growth.

Used in shampoos, hair


Symphytum officinale Comfrey
rinses, hair creams.

It is traditional component of
hair care, used for its
Trigonella foenum-graecum Fenugreek
cleaning and softening
activity.

Used as antidandruff agent


Eucalyptus sp. Eucalyptus
in shampoos.

Relieve itchy scalp and


Larrea divaricata Creosote bush dandruff, also used as hair
tonic.

Conditioning agent, provide


Lawsonia alba or Lawsonia body and bounce to the
Henna
inermis hair, makes hair
manageable.

Used in shampoos and


Pilocarpus jaborandi Jaborandi herbal hair rinses, act as
stimulant for hair growth.

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Used in shampoo for
antidandruff property, act as
natural cleanser, used in
Citrus limon Lemon
herbal mixtures as
decoction for normal to oily
hair.

Used in shampoos and hair


rinses, stimulates growth of
Urtica dioica Nettles
hair and improves the
condition of scalp.

Used in shampoos, it is a
Rosmarinus officinalis Rosemary
good hair conditioner.

Its oil is used in shampoos,


Santalum album Sandalwood act as antidandruff agent
and anti-microbial agent.

Used in shampoo, it is good


Quillaja saponaria Soapbark
for itchy scalp or dandruff.

Used in shampoo as it
produces good detergent
Saponaria officinalis Soapwort
lather this is good for itchy
scalp or dandruff.

Used in herbal shampoos


Hibiscus Rosa-Sinensis Hibiscus and hair rinses, it act as hair
growth stimulant.

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2.4 Marketing of the Herbal Shampoo

Based on the country India, the sales of herbal shampoo is increase with greatly
due to the herbal shampoo brings a lot of benefits not only for the hair but also in
the skin. One of the brand shampoo that successfully market around the world is
“DOVE”. Figure 2.5 shows of herbal shampoo usage in past and present in India
market while figure 2.6 shows the most preferred brand shampoo in India.
(Bhumi, 2010).

Figure 2.5 Herbal Shampoo Usages in Past and Present in India

Figure 2.6 the Most Preferred Brand Shampoo in India

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2.5 Forms of Shampoo

There are several form of shampoo that had been produced in the world such as
solid shampoo and also known as shampoo bars. It is use as their surfactant
soaps and other surfactants that the quality is being suitable for formulated as
solids. The advantage that they have is spill-roof. Meanwhile the disadvantages
is slowly applied because of need to be dissolved in use. Moreover gel
shampoo, it is stiff, non-pourable clear gels to be squeezed from a tube were
once popular forms of shampoo, and can be produced by increasing a
shampoo’s viscosity. It is cannot be spilled. It is different from solid because it
still can be lost down the drain by sliding off wet skin or hair. Soap jelly was
formerly made at home by dissolving sodium soap in hot water before being
used for shampooing or other purpose as an alternative to synthetic detergent
gels and to avoid the problem of slow application of solid shampoo. (Mottram
and Lees, 2000).

Next is cream shampoo, there are several of cream shampoo especially


came from natural source. It were formerly marketed in jars or tubes. The cream
shampoo are wet content but not completely dissolved. They would apply faster
than solid and dissolved quickly. Jar content were prone to contamination by
users and hence had to be very well preserved. Lastly is dry shampoo that been
designed to work without water. They are typically based on powders such as
starch, silica or talc and are intended to physically absorb excess sebum from
the hair before being brushed out. Brown powders such as cocoa or carob
powder is preferred to be used with dark hair. (Mottram and Lees 2000)

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2.6 Hibiscus Rosa-Sinensis

Carolus Linnaeus discovered and named the Hibiscus rosa-sinensi. It is an


evergreen flowering shrub native to East Asia. Hibiscus rosa-sinensis is known
colloquially as the Chinese hibiscus. It is also known as national flower of
Malaysia. In the tropics and subtropics, it is widely grown as an ornamental
plant. Hibiscus rosa-sinensi also known as China rose and shoe flower. The
flowers generally lack any scent but firm, red in the original varieties and large.
Numerous varieties, cultivars and hybrids are available with flower colors
ranging from white through yellow and orange to scarlet and shade of pink, with
both single and double sets of petals. Although their size and red color are
attractive to nectar-feeding birds but they are not visited regularly by
hummingbirds when grown in the Neotropics. (A. Kumar et al., 2012). Figure 2.7
shows flower of Hibiscus Rosa-sinensis

Figure 2.7 Flower of Hibiscus Rosa-sinensis

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2.7 Scientific Classification

The hibiscus scientific classification shows from kingdom until species in table
2.4

Table 2.4 Scientific Classification of Hibiscus Rosa-sinensis (Pekamwar,


Kalyankar, and Jadhav 2013)

Kingdom Plantae

Subkingdom Tracheobionta

Super division Spermatophyta

Division Magnoliophyta

Class Magnolipsida

Subclass Dilleniidae

Order Malvales

Family Malvaceae

Genus Hibiscus

Species Hibiscus rosa-sinensis

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2.8 Phytochemical Review in Hibiscus

Different extract of Hibiscus rosa-sinensis plant will sees the presence of


alkaloids, glycosides, fatty materials, reducing sugars, resin, sterols and not
having enough of tannins and Saponins. Separating of β-sitosterol, taraxeryl
acetate and four uncharacterized compounds which included an alkaloid and
three sterols has been information that have done in the leaves. The leaves of
Hibiscus rosa-sinensis were also investigated for their fatty alcohol, fatty acids
and hydrocarbon content. Two cyclic acids viz., malvalic and sterculic are also
recognized. Flowers contain vitamins, flavonoids, ascorbic acid, niacin,
riboflavin, thiamine and cyaniding diglucoside. Quercetin-3 diglucoside, cyanidin-
3-sophoroside-5-glycosides, 3, 7- diglucoside, cyanidin-3, 5-diglucoside have
been isolated from deep yellow flowers. (Pekamwar, Kalyankar, and Jadhav
2013). Table 2.5 shows phytochemical review in hibiscus leaves.

Table 2.5 Phytochemical Review in Hibiscus Leaves (Kumar et al., 2012)

No. Plant part Constituent reported

Thiamine, Riboflavin, Niacin and Ascorbic


acid, Apigenidin, citric acid, fructose,
1 Flowers
glucose, oxalic acid, pelargonidin and
quercetin

Alkaloids, glycosides, reducing sugars,


fatty materials, resin and sterols, fatty
2 Leaves
acids, fatty alcohol, hydrocarbon, sterculic
and malvalic acid

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Teraxeryl acetate, ß-sitosterol and the
3 Stems
cyclicacids sterculic and malvalic acids

Glycosides, tannins, phytosterols, fixed


4 Roots oils, fats, proteins, amino acids, flavonoid,
Saponins, gums and mucilage

2.9 Pharmacological Review in Hibiscus

Hibiscus rosa sinensis (Malvaceae) is a perennial ornamental shrub available


throughout India. Most of the parts of this plant such as flowers, leaves and
roots have been available to possess medicinal properties like oral
contraceptive, laxative, aphrodisiac, menorrhagic and more. Several articles and
ancient literature have reported that the flowers of this plant possess antifertility
activity. The aqueous ethanolic extract of aerial parts of this plant has
information for its use in constipation and diarrhea. In traditional medicine, the
leaves of the plant are used in fatigue and skin disease such as bitten from
insect. Powdered root of the plant is given for menorrhagia and the fresh root
juice for gonorrhea. Flowers of the plant are used in diabetes, epilepsy,
bronchial catarrh and leprosy. An infusion of the petal is widely used in
Ayurvedic medicine in India as a demulcent refrigerant drink in fever and
decoction is given in bronchial catarrh. Previous studies showed that the plant
possess anti-phologistic, anti-diarrhetic and anti-complementary activity. (A.
Kumar et al. 2012). Table 2.6 shows pharmacological review in hibiscus.

24 | P a g e
Table 2.6 Pharmacological Review in Hibiscus (L. Kumar et al. 2012)

Plant Activity
No Model used Remark
part reported

Steptozotocin

Found to be active
Antidiabetic induced
1 Flower
Activity

Alloxan induced Found to be active

Hair growth In vivo and in vitro


Exits potency
potential methods

Active on
Antifungal Inhibition found to be
2 Leaves Rhizoctonia solani
Activity 34.50%
Mycelial inhibition

Anti-
Carragenin induced
inflammatory Found to be active
pedal edema of rats
activity

25 | P a g e
Hair growth In vivo and in vitro Found to be more potent
potential methods than flower extract.

Juvenile
Dysdercus
3 Steam Hormone Found to be active
cingulatus
Activity

Antipyretic
4 Root Swiss albino rats Found to be active
activity

2.10 Origin and Distribution of Hibiscus

The precise origin of the plant Hibiscus rosa -sinensis is not identical. It is
generally thought to have originated in South China, though it has been in
cultivation in China, Japan and the Pacific islands for an equally long time. Asian
origin is believed have the plant with deep-red flowers, hence the meaning of
name rosa-sinensis is 'rose of China'. However, two white-flowered hibiscus
species, namely Hibiscus arnottianus and Hibiscus waimeae are believed to be
native to Hawaii. In the tropics throughout the world there are around 300
related species of hibiscus founded. One species, Hibiscus tiliaceus or the Sea
Hibiscus, is commonly found in Singapore. Over a thousand hybrid varieties
have been obtained and propagated for either a selected purpose or for simply
ornamental value.

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Singapore uses the hibiscus shrubs that made to grow into trees between
two until three meter tall as road dividers. Hybrid hibiscus flowers are today
available in pink, yellow, orange, purple, lavender or in multicolour forms.
Hibiscus rosa sinensis is the national flower of Malaysia. (Naidu, 2010)

2.11 Description of Hibiscus

The height of tropical hibiscus is often in range of 5’ to 6’ but it can be reached


until 15’. Plants can be used in a tree form or as a medium which is large shrub.
Containers also are the place where the tropical hibiscus can be grown. The
glossy, green leaves are arranged alternately and many cultivars have toothed
margins. The dominating characteristics are flowers and it have many colors and
it can reach up to 6” in diameter. Hibiscus has bell-shaped flowers with stamen
spirally arranged along a distinct pistil. Bloom may have single or double rows of
petals, with either smooth or scalloped edges. (Robledo 2004). The roots about
cylindrical of 5 to 15 cm length and 2 cm in diameter off white in color light
brown transverse lenticies. The roots taste sweet and mucilaginous and its
fracture is fibrous. Hibiscus leaves are simply ovate or ovatelancolate and wholly
at the base and coarsely toothed at the apex. Their taste is mucilaginous. The
flowers are pedicillate, actinomorphic, pentamerous and complete. 5 petals
contained in corolla, color in red and diameter is about 3 inches. The fruit is very
rarely formed and it is a capsule about 3 cm long. (L. Kumar et al. 2012).

Plant ability to tolerate cold temperature can be described by term of


Hardiness. The USDA Hardiness Map divides the US into several Hardiness
Zones based on a range of average low temperatures. Galveston County and
the Texas Upper Gulf Coast are located in Zone 9, with a minimum temperature
range of 20 °F to 30 °F. These are average lows and it should be noted that on
occasion temperatures can dip below the 20 °F mark. From zones 9 to 10, the
Hibiscus is already hardy but it can occur cold damage on some cultivars at
temperature above 20 °F.

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At the extremely cold years along the Gulf Coast, Hibiscus frequently
succumbs to low temperatures. Damage plants may require pruning to reshape
the canopy and to remove dead limbs or branches. Hibiscus prefers well drained
soils and does best under slightly acidic conditions. To adjust the aeration,
drainage and water holding characteristic of the soil, liberal amount of sandy soil
are used thoroughly decompose organic matter. In heavy clay soils consider the
use of raised beds to avoid periods of excessive moisture. Moreover, Hibiscus
requires full sun but will tolerate partial shade. Plants will become tall and leggy
if grown too shady. Inadequate light will also limit flowering. Many established
hibiscus compete for light in the landscape because they were not provided
enough room at planting to reach their mature size. Accommodation plants as
they grow and mature must carefully review the size specifications of select
cultivars before planting and provide adequate space. (Robledo 2004).

2.12 Hibiscus Rosa-Sinensis Leaves as Remedy

It is accepted that leaves of Hibiscus rosa-sinensis have hair growth promoting


and anti-greying properties based on the traditional text. Moreover, herbal
products in the market have a plan for hair growth include the extract of various
parts of Hibiscus rosa-sinensis in India. Hence, the present gaining knowledge is
aiming to the scientific investigation of the hair growth developed of the herb
Hibiscus rosa-sinensis. (Adhirajan et al. 2003). The activity learning of
pharmacological and clinical give people information about the present review
confirm the therapeutic value of Hibiscus rosa-sinensis. It is important source of
various type of compounds with very different from each other chemical
structures as well as pharmacological activities.

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Hibiscus rosa-sinensis consist phytochemical content that have a lot of
function which can prevent variety of diseases such as preventing woman from
becoming pregnant, controlling uterine bleeding, menorrhagia, venereal disease,
and cough, a medical condition in which a person has a temperature that is
higher than normal, refrigerant and vitaliser in palpitation. Phytochemical that
consist in the Hibiscus rosa-sinensis are anthocyanins, flavonoids, cyclopeptide,
alkaloid and vitamins. Presence of such wide range of chemical compounds
show that the plant could to be useful as lead for the development of novel
agents having good efficacy in several different disorder in the year that going to
happen soon. (L. Kumar et al. 2012)

The phytochemicals that produce from Hibiscus rosa-sinensis leaves for


the flavonoids can be used as oxygen free radical quenching and inhibition of
lipid peroxidation and studies has shown that it has antioxidant properties.
(Tiliaceus et al., 2014).

2.13 Application on the Hibiscus Rosa-sinensis

Hibiscus Rosa Sinensis helps in inducing abortion, provide treatment for


headache. Young leaves are sometimes used as a spinach substitute. Flowers
can also be made into a kind of pickle or used as a purple dye for coloring foods
such as preserved fruits and cooked vegetables. Root is edible but very fibrous.
It’s also good for hair treatment. The leaves and flowers are beaten into a paste
and poultice onto cancerous swellings and mumps. The leaves are anodyne,
aperient, emollient and laxative. A decoction is used as a lotion in the treatment
of fevers.

2.14 Antioxidant

Antioxidants help to neutralize and eliminate free radicals. Free radicals can
cause damage to cells and it is believed to play a central role in the aging
process and in disease progression. Antioxidants are the first line of defense

29 | P a g e
against free radical damage, and it is important for maintaining optimum health
and wellbeing. The need for antioxidants have becomes even more critical with
increased exposure to free radicals. There has been extensive scientific
research done on the antioxidants found in vitamins especially vitamin C and
vitamin E. Table 2.7 shows the types of vitamin C and E and their functions.

Table 2.7 Types of Vitamin (Wuand Hansen, 2008)

Vitamin Type Function

 neutralizing reactive oxygen


species in the aqueous phase
 water-soluble antioxidant
C
 capable of regenerating vitamin E
in extracellular fluids
 increase collagen production

 protects membrane fatty acids


from lipid peroxidation
 protect cell membranes from
E  lipid-soluble antioxidant
oxidative damage and from the
early stages of ultraviolet light
damage

Fruits and vegetables are major sources of vitamin C and carotenoids, while
whole grains and high quality, properly extracted and protected vegetable oils
are major sources of vitamin E. Several components of fruits and vegetables
have been shown to have antioxidant activity, like ascorbic acid, tocopherol, b-
carotene, flavonoids, and phenolic compounds (Wuand Hansen, 2008).

2.14.1 Total Flavonoid Content (TFC)

Flavonoids are low molecular weight bioactive polyphenols which play an


essential role in photosynthesizing cells. The active substance of
flavonoid research apparently began when Hungarian scientist Albert
Szent-Gyorgi was uncovering a synergy between pure vitamin C and as

30 | P a g e
yet unidentified co-factors from the peels of lemons in 1936, which he first
called "citrin," and, later, "vitamin P". Flavonoids are secondary
metabolites characterized by flavan nucleus and carbon-skeleton which
are C6-C8-C6. These are group of structurally related compounds with a
chromane-type skelton having phenyl substituent in C2-C3 position. The
basic structural feature of flavonoid is 2-phenyl-benzo-γ-pyrane nucleus
containing of two benzene rings (A and B) linked through a heterocyclic
pyran ring (C) as shown in figure 2.8 while the figure 2.9 shows the
biosynthesis of flavonoid. (Sandhar et al. 2011)

Figure 2.8 Basic Structures of Flavonoids

Figure 2.9 Biosynthesis of Flavonoids

Arrangement of flavonoid is different of hydroxyl, methoxy and glycosidic


side groups and in the conjunction between A and B rings. A variation in
C ring will give division of subclasses. According to their molecular
structure, they are divided into eight classes that is shown in figure 2.3. In
plants, flavonoids are often present as O-glycosides or C-glycosides. The
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O-glycosides possess sugar substituent bound to –OH of aglycone,
usually at position 3 or 7, whereas, C-glycosides possess sugar. groups
bound to Carbon of a glycone usually 6-C or 8-C. (Sandhar et al. 2011).
The figure 2.10 shows chemical structure of different types of flavonoid.

Figure 2.10 Chemical Structures of Different Types of Flavonoids

2.14.2 Function of TFC in the Hibiscus Leaves

TFC in the hibiscus are Anthocyanins and Cyaniding-3,5-diglucoside,


Cyaniding-3 sophoroside-5-glucoside, Quercetin-3,7-diglucoside,
Quercetin-3-diglucoside. It is a glabrous shrub widely cultivated in the
tropics. It is well accepted and promoted by researcher that the leaves
and flowers of Hibiscus rosa-sinensis have hair growth promting and
antigreying properties. In India, the herbal products in the market
intended for hair growth include the extract of various parts of Hibiscus
rosa-sinensis but most of the highest of TFC is part of the leaves. The
leaf extract of Hibiscus rosa-sinensis has a potential effect on maintaining
the hair growth in-vivo and in-vitro methods. (Jadhav et al. 2009).

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2.15 Soxhlet Extractor

In 1879 by Franz von Soxhlet invented a soxhlet extractor for a piece of


laboratory apparatus. (M. Harwood, 1879) It was originally designed for the
extraction of a lipid from a solid material. However, a Soxhlet extractor is not
limited to the extraction of lipids. Typically, a Soxhlet extraction is only required
where the desired compound has not very great in amount of solubility in a
solvent, and the impurity is insoluble in that solvent. If the desired compound
has a significant solubility in a solvent then a simple filtration can be used to
separate the compound from the insoluble substance. Basically, a solid
substances containing some of the desired compound is put inside a thimble
made from thick filter paper, which is carried into the main chamber of the
Soxhlet extractor. The Soxhlet extractor is placed onto a flask containing the
extraction solvent. The Soxhlet is then mounted with a condenser. The solvent is
heated to reflux. The solvent vapour goes up a distillation arm, and floods into
the chamber housing the thimble of solid. The condenser ensures that any
solvent vapour cools, and drips back down into the chamber housing the solid
material. The chamber containing the solid material moves slowly and fills with
warm solvent.

Some of the desired compound will then dissolve in the warm solvent.
When the Soxhlet chamber is almost full, the chamber is automatically emptied
by a siphon side arm, with the solvent running back down to the distillation flask.
This cycle may be allowed to repeat many times, over hours or days. During
each cycle, a portion of the non-volatile compound dissolves in the solvent. After
many cycles the desired compound is concentrated in the distillation flask. The
advantage of this system is that instead of many portions of warm solvent being
passed through the sample, just one batch of solvent is recycled. After
extraction the solvent is removed, typically by means of a rotary evaporator,
yielding the extracted compound. The non-soluble portion of the extracted solid
remains in the thimble, and is usually discarded.

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2.16 Ultrasonic Assistance Extraction

The application of for plant materials or herbs (Vinatoru, 2001) and its
opportunities in the food processing industry (Vilkhu et al., 2008) has proved
many advantages. Its effectiveness in increasing extraction yield is proven as
the ultrasonic wave generates supersonic liquid micro jet that has the ability to
disrupt plant cells when the cavitating bubbles collapse at close proximity.

The improvement of bioactive compound extraction yield in the


ultrasound-assisted extraction depends on the collapse of cavitation bubbles
which produces micro jets that disrupt plant cell membrane and subsequently
provoke the release of phenolic compounds. The degree of yield enhancement
depends largely on the cavitation bubbles formed within the solvent. The
formation or threshold of cavitation bubble is influenced by the ultrasound
frequency and intensity, sonication exposure time on the solvent, physical
properties of solvent which include its surface tension, viscosity, and the
presence of gas or particulate matters in the solvent. (Mason and Lorimer,
2002). Sun et al. (2011) conducted studies to evaluate the effects of various
extraction factors, including particle size, extraction solvent, solid/solvent ratio,
temperature, extraction time, electrical acoustic intensity, liquid height and duty
cycle of ultrasound exposure on the extraction yield of all-trans- β-carotene from
citrus peels (Cheok et al., 2013).

2.17 Filtration

The definition of filtration as given by Coulson and Richardson is the separation


of solids from a suspension in a liquid by means of a porous medium or screen
which retains the solids and allows the liquid to pass. Based on the definition it
can conclude that the main element in filtration is the porous medium or also
known as screen. Without this device, the process of filtration could not be
carried out. Generally the process of filtration starts when the mixture of solids
and liquids or can be called slurry is allowed to flow across a porous or a filter
medium. The Filter Medium has some value of porosity or openings which is

34 | P a g e
bigger than the particle size of liquid to be removed but smaller than the particle
size of the solid to be obtained At the same time, after liquid will pass through
the filter medium which is recycled or removed, depends on the requirement of
the process it will call filtrate. (Fadzli, 2013).

Initially during the filtration process, the solid which deposited in the
surface of the Filter Medium will form the true filtering medium as mentioned by
Coulson and Richardson. This is the first layer of cake on the filter medium and
is formed at a high filtration rate. As Filtration Cycle continues the rate of
filtration decrease because of the resistance of the layer of cake or can be called
as filter cake. At the end of Filtration Cycle, the pressure from the filter cake is
very high due to the thickness of the cake has increase and cause in the rate of
filtration to stop and no more filtration happens. The solid or filter cake is then
removed from the filter medium, collected and send to other equipment such as
Drying which is also depends on the process involved but for this case, the
filtrate will be collected to do the analysis. (Fadzli, 2013).

2.18 Ultraviolet–visible spectroscopy (UV-Vis)

UV spectroscopy is type of absorption spectroscopy in which light of ultra-violet


region from 200-400 nm. is absorbed by the molecule. Absorption of the ultra-
violet radiations results in the excitation of the electrons from the ground state to
higher energy state. The energy of the ultra-violet radiation that are absorbed is
equal to the energy difference between the ground state and higher energy
states (deltaE = hf). For the principle of UV spectroscopy, UV spectroscopy
follows the Beer-Lambert law. The definition of law is when a beam of
monochromatic light is passed through a solution of an absorbing substance, the
rate of decrease of intensity of radiation with thickness of the absorbing solution
is proportional to the incident radiation as well as the concentration of the
solution. From the Beer-Lambert law it is obviously that higher the number of
molecules capable of absorbing light of a given wavelength, the greater the
extent of light absorption. This is the basic principle of UV spectroscopy. The

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figure 2.11 shows the schematic diagram of UV-Vis. The expression of Beer-
Lambert law can be expressed by:

A = log (I0/I) = Ecl

Where,

A = absorbance

I0 = intensity of light incident upon sample cell

I = intensity of light leaving sample cell

C = molar concentration of solute

L = length of sample cell (cm.)

E = molar absorptivity

Figure 2.11 Schematic Diagram of UV-Vis

There are a few applications for UV-Vis such as detection of functional


groups, it is used to detect the presence or absence of chromophore in the
compound. Next, detection of extent of conjugation which the extent of
conjugation in the polyenes can be detected with the help of UV spectroscopy
by the increase in double bonds the absorption shifts towards the longer
wavelength. Moreover, identification of an unknown compound also can be

36 | P a g e
detected by the spectrum of unknown compound is compared with the spectrum
of a reference compound and if both the spectrums coincide then it confirms the
identification of the unknown substance. Determination of configurations of
geometrical isomers, it is observed that cis-alkenes absorb at different
wavelength than the trans-alkenes. The two isomers can be distinguished with
each other when one of the isomers has non-coplanar structure due to steric
hindrances. Lastly, UV-Vis can determine the purity of a substance which the
absorption of the sample solution is compared with the absorption of the
reference solution. The intensity of the absorption can be used for the relative
calculation of the purity of the sample substance.

2.19 Fourier Transform InfraRed FT-IR

Fourier Transform InfraRed stand for FT-IR and it is the preferred method of
infrared spectroscopy. In infrared spectroscopy, IR radiation is across through a
sample. Some of the infrared radiation is absorbed by the sample and some of it
is transmitted. The resulting spectrum represents the molecular absorption and
transmission, producing a molecular fingerprint of the sample. Like a fingerprint
no two unique molecular structures produce the same infrared spectrum. This
makes infrared spectroscopy useful for several types of analysis. The
information of FT-IR will provide identify unknown materials. Moreover, it can
determine the quality or consistency of a sample and also the amount of
components in a mixture. For the theory of FT-IR, it represents a fingerprint of a
sample with absorption peaks which correspond to the frequencies of vibrations
between the bonds of the atoms building up the material. There is no two
compounds produce the exact same infrared spectrum due to each different
materials is a unique combination of atoms. Therefore, infrared spectroscopy
can result in a positive identification (qualitative analysis) of every different kind
of material. In addition, the size of the peaks in the spectrum is a direct
indication of the amount of material present. With modern software algorithms,

37 | P a g e
infrared is an excellent tool for quantitative analysis. Figure 2.12 shows the
schematic diagram of FT-IR.

Figure 2.12 Schematic Diagram of FT-IR

2.20 Solvent

A solvent is a liquid that act as the medium for a reaction. It can acts with two
major purposes which are first Non-participatory that is to dissolve the reactants.
For example polar solvents are best for dissolving polar reactants such as ions
while nonpolar solvents are best for dissolving nonpolar reactants such as
hydrocarbons. Secondly is participatory, as a source of acid (proton), base
(removing protons), or as a nucleophile (donating a lone pair of electrons). Polar
solvents have large dipole moments as known as partial charges which they
contain bonds between atoms with very different electronegativities, such as
oxygen and hydrogen. Non polar solvents contain bonds between atoms with
similar electronegativities, such as carbon and hydrogen. Bonds between atoms
with similar electronegativities will lack partial charges and due for absence of
charge which makes these molecules as non-polar. Moreover, some solvents
are called “protic” and some are called “aprotic. Protic solvents have O-H or N-H
bonds. It is important because protic solvents can participate in hydrogen
bonding, which is a powerful intermolecular force.

38 | P a g e
Additionally, these O-H or N-H bonds can serve as a source of protons
(H+). Meanwhile, Aprotic solvents may have hydrogen on them somewhere, but
they lack O-H or N-H bonds, and therefore cannot make hydrogen bond with
themselves. The most suitable solvents to extract flavonoid are polar solvent
such as ethanol, methanol, ethyl acetate and more. (Yu et al. 2010). The figure
2.13 shows the solvent polarity strength of chart.

Figure 2.13 Solvent Polarity Chart

2.20.1 Petroleum Ether

Petroleum ether or it can be called as petroleum spirits. It is highly


flammable liquid distillate of petroleum heavy compare to the naphtha
and lighter than kerosene. It is known variously as benzine, varnish
makers and painters naphtha (VMandP), petroleum naphtha, naphtha
ASTM. Moreover, it is a lightweight hydrocarbon used chiefly as a
nonpolar solvent. Benzole (benzoline), Pether, ligroin, and X4 are related
hydrocarbon mixtures. Ligroin was used as a fuel by the first motor car,
which the brand of Benz Patent-Motorwagen. Petroleum ether is a
mixture of alkanes such as pentane, hexane, and heptane, whereas

39 | P a g e
benzene is a cyclic, aromatic hydrocarbon, C6H6. Likewise, petroleum
ether should not be confused with the class of organic compounds called
ethers, which contain the R-O-R' functional group.

2.20.2 Methanol

Methanol is the simplest alcohol that is a light, volatile, colorless,


flammable liquid with a distinctive odor very similar to that of ethanol. At
room temperature, it is a polar liquid, and is used as an antifreeze,
solvent, fuel, and as a denaturant for ethanol. It is also used for producing
biodiesel via transesterification reaction. Methanol is produced naturally
in the anaerobic metabolism of many varieties of bacteria, and is
commonly present in small amounts in the environment. As a result, there
is a small fraction of methanol vapor in the atmosphere. Methanol can
burns in oxygen and producing carbon dioxide and water.

2.20.3 Ethanol

Ethanol also commonly called ethyl alcohol, drinking alcohol, or simply


alcohol is the principal type of alcohol found in alcoholic beverages,
produced by the fermentation of sugars by yeasts. It is a neurotoxic.
Ethanol is used as a solvent, an antiseptic, a fuel and the active fluid. It is
a volatile, flammable, colorless liquid with a strong chemical odor. Its
structural formula CH3CH2OH, is often abbreviated as C2H5OH, C2H6O
or EtOH. Ethanol is a 2-carbon alcohol. Its molecular formula is
CH3CH2OH. An alternative notation is CH3–CH2–OH, which indicates that
the carbon of a methyl group (CH3–) is attached to the carbon of a
methylene group (–CH2–), which is attached to the oxygen of a hydroxyl
group (–OH). It is a constitutional isomer of dimethyl ether. Ethanol is
sometimes abbreviated as EtOH, using the common organic chemistry
notation of representing the ethyl group (C2H5-) with Et.

40 | P a g e
2.21 Studying TFC Content on Different Parameters

There are different TFC content on different parameters. The experiment was
conducted to study the effect of different mass and time taken on the yield of
TFC, to compare the effect of yield produce between three solvents which is
petroleum ether, methanol, and ethanol to determine the yield for each solvents,
and lastly to compare the effect of yield produce by using two method which are
soxhlet extraction method and ultrasonic bath method.

2.21.1 Effect of Solvents on the Yield of TFC

As the flavonoid is the polar solvent, so the suitable solvent must be in


polar to extract the polar compound. Moreover, the highest the polarity of
solvent, the greater polar compound will be extracted. (Stankovi 2011)

2.21.2 Effect of Extraction Method on the Extraction Yield of TFC

Based from the journals that had been made, the soxhlet can be
considered better option compare to Ultrasonic bath method because of
longer extraction time to produce high efficiency number of yields of TFC.
(Bimakr et al., 2011)

2.21.3 Effect of Solid-to-Liquid Ratio on the Yield of TFC

The TFC is significantly increased when the mass of sample is increasing


due to the increase of the driving force for the mass transfer on the TFC.
(Lu et al. 2013)

2.21.4 Effect of the Time on the Extraction Yield of TFC

The yield of TFC will be increase if the time of extraction is getting


increase due to produce more efficiency of the yield. However, if the time
of extraction is exceed certain the limits, the yield of TFC will be reduced
cause of too much heat exposed and led to the degradation of flavonoid
compounds. (Vangalapati et al., 2014)

41 | P a g e
2.22 Identifying of TFC in the Hibiscus Leaves Using FT-IR

Based from the journals that had been made, all the polar solvent will extract the
polar flavonoid compound. Hence the TFC will present only when use the
solvent of methanol and ethanol as the solvent is polar. The TFC will not present
when use the solvent of petroleum ether as it non-polar solvent. (Finney,
Pomeranz, and Hoseney 1976).

42 | P a g e
CHAPTER 3 :METHODOLOGY

3.1 Materials

The solvent used for this research are methanol, ethanol, and petroleum ether
that came from same company which is Hmbg chemicals. The grade of solvents
is analytical grade. (Sebah 2007).

The ingredients for formulation of shampoo are Cocamidea DEA,


Cocamidoproply Betaine, Formaldehyde, Ethylenediaminetetraacetic acid
(EDTA) Disodium, Sodium Lauryl Sulfate (SLS), and Sodium Hydroxide. These
ingredients come from same company which is Take It Global Sdn. Bhd.

3.2 Equipment

The equipment that had been used in the experiment is analytical balance which
B204-S Mettler Toledo, USA to measure the different weight of samples. Next,
Ultrasonic bath from brand D-78224 Singan/Htw Elma to extracts the sample by
using types of solvent using water as heating medium. Moreover, Soxhlet
extractor which from brand of Buchi of B-811 LSV to extract the sample with
long time which is above from 60 minutes.

The equipment that had been used for testing the Total Flavonoid
Content (TFC) from the experiment is Ultraviolet Visible Spectroscopy which
brand of Lambda EZZ10 Perkin Elmer to determine the concentration of TFC
present in the sample. Moreover, Fourier Transform InfraRed (FT-IR) from
Thermo scientific company that brand of Nicolet iS10.

43 | P a g e
3.3 Procedure

It is include the pre-treatment process. Next, include preparation of extraction


process by using different method which are ultrasonic assistant extraction
method and soxhlet extractor method. Moreover, include the way to conducted
the testing of Total Flavonoid Content (TFC) by using FT-iR and UV-Vis. Lastly,
it also include the formulation of herbal shampoo.

3.4 Pre-treatment Process

Pre-treatment process is the process to clean sample before it can be used.


Moreover, after it been done for cleaning, the sample must be dry to remove
moisture content and make the sample become powdered to increase the
surface and hence the components of the sample will easier to extracts.

Fresh Hibiscus rosa-sinensis leaves were pluck and put into a basin. The
Hibiscus rosa-sinensis were rinse with tap water to remove any rubber that
attached to the leaves and also insects. (Mak, 2013). Then, the Hibiscus rosa-
sinensis leaves were left for a several days for drying process. (Divya et al.,
2013) After a day, the 50 gram of leaves was put into a blender to blend to
increase surface area. The blender was left to blend leaves for 1 minutes.
(Mgaya Kilimia et al., 2014) The steps were repeated until the mass of leaves
were enough to conduct the experiment. Powdered dried hibiscus leaves are
produced. Figure 2.14 shows pre-treatment process of hibiscus leaves.

44 | P a g e
Figure 3.1 Pre-treatment Process of Hibiscus Leaves

3.5 Preparation for Extraction Process

Extraction process is the process to extracts the powdered dried hibiscus leaves
as the sample. The extraction process has two methods for this experiment
which is Ultrasonic assistant extraction method and Soxhlet extraction method.

3.5.1 Soxhlet Extraction Method

Soxhlet extraction is only required where the desired compound has not
very great in amount of solubility in a solvent, and the impurity is insoluble
in that solvent. If the desired compound has a significant solubility in a
solvent then a simple filtration can be used to separate the compound
from the insoluble substance.

Approximately 4050 ml plastic Tupperware were used for the


extraction product. The powdered Hibiscus rosa-sinensis leaves was
weighted and then put in a thimble and placed into soxhlet apparatus.
The powdered leaves extracted using 150 ml each of solvent which is
petroleum ether, methanol, and ethanol. Approximately 1350 ml on every

45 | P a g e
each of solvents were used to extract the product. The samples were
running with 3 different mass of samples which are 10 gram, 20 gram,
and 30 gram with 3 different times of extraction such as 120 minutes, 240
minutes, and 360 minutes. The extracted product was labeled on certain
parameter before done through extraction and filled in the plastic
Tupperware. Lastly, the plastic Tupperware filled with extracted product
were going to be analysis. Figure 3.2 shows the flow of process Soxhlet
extraction method.

Figure 3.2 Process of Soxhlet Extraction Method

3.5.2 Ultrasonic Assistance Extraction Method

Ultrasonic assistant extraction is effectiveness in increasing extraction


yield is proven as the ultrasonic wave generates supersonic liquid micro
jet that has the ability to disrupt plant cells when the cavitating bubbles
collapse at close proximity.

Approximately 4050 ml plastic Tupperware were used for the


extraction product. Three solvents were prepared that are methanol,
ethanol, and petroleum ether. 9 beakers were being poured with 150 ml
petroleum ether, 9 beakers being poured with 150 ml ethanol, and 9
beakers were being poured with 150 ml methanol. The Hibiscus rosa-
46 | P a g e
sinensis powdered leaves were weighted into 3 different mass which is
10 gram, 20 gram, and 30 gram and mixed with petroleum ether in the
250 ml beaker. The weighing processes were repeated for solvents of
methanol and ethanol. Every beakers was labeled before being left in the
ultrasonic bath. The ultrasonic cleaner was switching on before using it.
Approximately 3800 ml of water was poured in the ultrasonic to act as
medium. The times were set for 15 min, 20 min, and 30 min by rotating
the knob. For every 15 minutes, the label with certain mass were taken
out from the ultrasonic bath for filtration.

3.5.2.1 Filtration Process

Filtration process is the process of separation of solids from a suspension


in a liquid by means of a porous medium or screen which retains the
solids and allows the liquid to pass.

Conical flask, filter funnel, and filter paper were prepared for the
filtration process. The filter paper was fold into triangular shape and
placed in the filter funnel. The beaker filled with sample submerge with
solvent was poured into the filter funnel contained filter paper for filtration.
After third quarter an hour, the extracted product was collected inside the
conical flask. Next, the extracted products were being transferred into
plastic Tupperware before going for the analysis. Figure 3.3 shows the
flow of process Ultrasonic assistant extraction method in simples way to
understand.

47 | P a g e
Figure 3.3 Process Ultrasonic Assistant Extraction Method

3.6 Fourier Transform infrared radiation (FTIR) analysis.

In infrared spectroscopy, IR radiation is across through a sample. Some of the


infrared radiation is absorbed by the sample and some of it is transmitted. The
resulting spectrum represents the molecular absorption and transmission,
producing a molecular fingerprint of the sample.

FTIR spectra were obtained from extracted product with every solvents
that is petroleum ether, methanol, and ethanol. The samples were analyzed in
the absorption mode of FTIR and all spectra were recorded from 4000 to 500
cm-1 at a data acquisition rate of 2 cm-1 using FTIR spectrophotometer. (Mak et
al. 2013)

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3.7 Analysis Using Uv Vis

UV spectroscopy is type of absorption spectroscopy in which light of ultra-violet


region from 200-400 nm. is absorbed by the molecule. Absorption of the ultra-
violet radiations results in the excitation of the electrons from the ground state to
higher energy state.

The extracted samples of 2.5 ml were added with 12.5 ml Distilled Water
and mixed with 5% NaNO3 solution of 1 ml. After 6 minutes, 1.5 ml of 10% AlCl3
solution was added. After 5 minutes, 5 ml of 1M Sodium Hydroxide is added
sequentially. The absorbance of the solution at a wavelength is 510 nm. Then,
the FeSO4.7H2O solution was prepared for 1Mm solution as standard solution
for Uv Vis. (Settharaksa et al., 2014)

3.8 Rotary Evaporator

The best three of sample were chosen that contained optimum flavonoids were
going under rotary evaporator to separate between oil contained in the sample
and the solvent which is placed in the water bath under the reduced pressure,
and heating with the rotation of the flask to make the solvent in the bottle spread
and evaporate. The solvent will vaporize because of low boiling point compare
to the sample and had been suck through vacuum system by reduced pressure
and transfer to the condenser with coil passing coolant into which coolant
mixtures such as dry ice and acetone are placed. Solvent after it re-condenses
is placed at the condensate-collecting flask at the bottom of the condenser. The
sample that contained oil at standard evaporating flask is transferred to the
formulating herbal shampoo.

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3.9 Formulation Herbal Shampoo

The definition of Herbal shampoo is the problem solver that is used to removed
dirt, oil, skin particles and dandruff from hair which had been build up in hair
frequently and need to be discharge to maintain the practice of keeping yourself
and your living and working areas clean in order to prevent illness and disease
using extracted herbs that containing a lot of beneficial and reduced the
synthetic ingredient that can cause harmful in our body. Table 3.1 shows
chemical use for the formulation of herbal shampoo.

Table 3.1 Chemical Formulation of Herbal Shampoo

Chemical names Weight % Function

Deionized water 57.95 Diluent

Sodium Laureth Sulfate (28 wt. %) 20.00 Surfactant

Cocamidopropyl Betaine (30 wt. %) 5.00 Surfactant

Cocamide DEA (88 wt. %) 0.60 Surfactant

Oil Hibiscus extracted 15.00 Hair growth promoter agent

Disodium EDTA 0.05 Chelating agent

Formaldehyde 0.40 Preservative

Sodium Hydroxide (18%) 1.00 Neutralizer

For references making the herbal shampoo was based on the formula
economic herbal shampoo that had been created by The Lubrizol Corporation.
Firstly, Add 60 gram deionized water and mixed with 20 gram of Sodium Laureth
Sulfate (28 wt %), SLS to diluent the surfactants until uniform in the main batch.
After that, mixed with all 5 gram of Cocamidoropyl Betaine (30 wt %), 0.6 gram

50 | P a g e
of Cocamide DEA (88 wt %), and 1.5 gram of sample that contained Hibiscus
rosa-sinensis until uniform one at a time into the main batch to homogenize the
surfactants with oil plant extracts that act as conditioner. In separated vessel,
dissolve Disodium EDTA in Deionized Water to dilute the chelating agents first
and then add to the main batch and mixed until uniform. Next, add 0.4 gram of
formaldehyde in the main batch as preservatives. After formaldehyde has been
added, add 1 gram of Sodium Hydroxide (18 wt %) to neutralize batch, adjusting
pH to 6.2-6.5 and mix until uniform. Lastly, after all ingredients were mixed well
in the main batch, transfer it to the bottle and became product herbal shampoo.
Figure 3.4 shows flow of formulation herbal shampoo.

Figure 3.4 Flow of Formulation of Herbal Shampoo

51 | P a g e
CHAPTER 4 :RESULT AND DISCUSSION

4.1 Introduction

The results for this research were recorded in table 1. The volumes of extracted
products were stored by using Tupperware. The standard absorbance obtain
from the analyzing using uv vis, yield and rate of extraction were calculated
while to confirm the flavonoid compound, the sample was tested using FTIR.

The objectives of this experiment are to determine the active ingredient in


Hibiscus rosa-sinensis that can improve blood circulation to nourish the hair
follicle and thereby promoting hair growth, to study the effect of different mass
and time taken on the yield of Total Flavonoid Content (TFC), to compare the
effect of yield produce between three solvents which is petroleum ether,
methanol, and ethanol to determine the yield for each solvents, and lastly to
compare the effect of yield produce by using two method which are soxhlet
extraction method and ultrasonic bath method. Research had been made and it
is proved that Hibiscus leaves contain active ingredient called flavonoid that had
numerous benefit to human health. Flavonoid content had been used in food
industry and mostly in cosmetic products.

Page | 52
4.2 Parameters of the Experiments

The experiment on extraction of Hibiscus leaves had been conducted to obtain


the yield of TFC by using two types of method which are soxhlet extraction
method and ultrasonic bath method, three solvents such as petroleum ether,
methanol and ethanol as medium. Times taken for every experiment are 10
minutes, 20 minutes, and 30 minutes for ultrasonic bath method and 120
minutes, 240 minutes, and 360 minutes for soxhlet extraction method. For the
mass are 10 gram, 20 gram, and 30 gram as dry hibiscus powdered.

4.3 Effect of Solvents on the Yield of TFC

The type solvent influences the efficiency of TFC. Relating to the whole of a
thing, ethanol is better for extraction of polar flavonoid compounds in the
Hibiscus leaves. The effect of solvents on extraction yield of TFC is shown in
figure 4.1. Extraction was carried out with 3 different solvents such as petroleum
ether (40 until 60 0C), ethanol, and methanol while other extraction parameters
were constant which are soxhlet extraction method, 30 per 150 solid-to-liquid
ratio, and 120 minutes extraction of time.

The TFC became the highest which is 65.155 mg FeSO4 per 100 gram of
sample when the solvent extraction is ethanol among the 2 other solvents. The
reason may be because the ethanol is the highest polar solvent compare to
methanol and petroleum ether is non-polar solvent. The ethanol extract more
polar flavonoids in the Hibiscus leaves which are the major compound compare
to the methanol. (Fatiha et al., 2012) Meanwhile, the petroleum ether only
extracts the non-polar and very least flavonoid compound in the Hibiscus leaves.
(Finney et al., 1976) The examples of non-polar compound are carotenoids and
chlorophyll. (Shui et al., 2004)

Page | 53
250

Flavonoids Contents (mg FeSO4 per 100


200

gram of sample)
150

100

50

0
Petroleum Ether Methanol Ethanol
Solvent (Types)

Figure 4.1 Flavonoids Contents (mg FeSO4 per 100 gram of sample) against
Type of Solvents

4.4 Effect of Extraction Methods on the Extraction Yield of TFC

Method of extraction is a factor that would significantly influence the extraction


efficiency of TFC from Hibiscus leaves or edible vegetables. The effect of
different methods on extraction yield of TFC is shown in figure 4.2. Extraction
was carried out at different methods which are soxhlet extraction method and
ultrasonic bath method while other extraction parameters were kept constant (30
per 150 solid-to-liquid ratios, and types of solvent) except for the times of
extraction. For the soxhlet extraction method, the samples was taken at the
second minute which is 120 minutes while for the ultrasonic bath method, the
sample was taken at the second minute too which is 10 minutes.

Page | 54
Soxhlet extraction method can get the higher TFC which are 111.032,
293.145, and 317.793 (mg FeSO4 per 100 gram of sample) by using solvents of
petroleum ether, methanol and ethanol respectively compare with ultrasonic
bath method which can get 2.986, 132.059, and 170.663 (mg FeSO4 per 100
gram of sample) by using solvents of petroleum ether, methanol and ethanol
respectively. It is proved that soxhlet can be considered better option compare
to Ultrasonic bath method because of longer extraction time to produce high
efficiency number of yields of TFC. (Bimakr et al., 2011) Soxhlet also proved
better for extraction of antioxidant compounds on more number of occasions
than Ultrasonic Bath Method. Moreover, Soxhlet was found to be superior
for getting high extraction efficiency from all the plant seeds. (Kothari etal.,
2012)

350
Flavonoids Contents (mg FeSO4 per

300

250
100 gram of sample)

200

150 Ultrasonic Bath


100 Soxhlet Extractor
50

0
Petroleum Methanol Ethanol
Ether
Types of Solvents

Figure 4.2 Flavonoids Contents (mg FeSO4 per 100 gram of sample) against
Type of Methods

Page | 55
4.5 Effect of Solid-to-Liquid Ratio on the Yield of TFC

The effect of solid-to-liquid ratio on the extraction yield of TFC is shown in figure
4.3. Extraction was carried out at different solid-to-liquid ratios which are 10 per
150, 20 per 150, and 30 per 150 (gram per ml) while other extraction parameters
were constant (types of solvents, soxhlet extraction method and 120 min
extraction time). The extraction yields of TFC significantly increased from 6.112
to 111.032 (mg FeSO4 per 100 gram of sample) for petroleum ether, 24.663 to
293.145 (mg FeSO4 per 100 gram of sample) for methanol, and 80.968 to
317.793 (mg FeSO4 per 100 gram of sample) for ethanol, due to the increase of
the driving force for the mass transfer of the TFC. (Lu et al., 2013)

350.000
Flavonoids Contents (mg FeSO4 per 100

300.000

250.000
gram of sample)

200.000
Petroleum Ether
150.000
Methanol
100.000 Ethanol

50.000

0.000
10 / 150 20 / 150 30 / 150
Material : Solvent (gram / ml)

Figure 4.3 Flavonoids Contents (mg FeSO4 per 100 gram of sample)
against Material per Solvent (gram per ml)

Page | 56
4.6 Effect of Time on the Extraction Yield of TFC

Extraction time is a factor that would important enough to have effect on the
extraction efficiency of TFC from Hibiscus leaves which is medicine plant. (Yu et
al. 2010). The effect of different times on extraction yield of TFC is shown in
figure 4.4. Extraction was carried out at different times which are 120, 240, and
360 (minutes) while other extraction parameters were kept constant (types of
solvents, 30 per 150 solid-to-liquid ratio and soxhlet extraction method).

When extraction time varied from 120 minutes to 240 minutes, the yield of
TFC was significantly increased from 24.524 to 111.032 (mg FeSO 4 per 100
gram of sample) for petroleum ether, 26.628 to 259.353 (mg FeSO 4 per 100
gram of sample) for methanol, and 309.206 to 317.793 (mg FeSO 4 per 100
gram of sample) for ethanol while when time was increased to 360 minutes the
yield of TFC was decreased to 26.628 (mg FeSO 4 per 100 gram of sample) for
petroleum ether, 27.386 (mg FeSO4 per 100 gram of sample) for methanol and
151.075 (mg FeSO4 per 100 gram of sample) for ethanol.

It may be that the excess of increasing time can cause too much heat
exposed and led to the degradation of flavonoid compounds since it is one of the
antioxidant activity compound in the Hibiscus leave that can’t expose too much
heat or light. Thus 240 minutes was picked as the favorable extraction time to
extract the yield of TFC in the Hibiscus leaves. (Vangalapati et al., 2014)

Page | 57
350

Flavonoids Contents (mg FeSO4 per 100


300

250

gram of sample)
200
Petroleum Ether
150
Methanol
100 Ethanol

50

0
2 4 6
Time (hour)

Figure 4.4 Flavonoids Contents (mg FeSO4 per 100 gram of sample)
against Time (hour)

4.7 Confirmation of TFC in the Hibiscus Leaves Using FTIR

FT-IR spectrum was analyzed to fine the most important Functional groups of
flavonoid extract of the sample by KBr disk technique using FT-IR. (Wightianum
et al., 2013). Table 4.1 shows the functional groups of the purified flavonoid
compound from IR-spectrum.

Table 4.1 the functional groups of the purified flavonoid compound from IR-
spectrum

Wave number Band shape Band Functional


(cm-1) group

3500-3200 Broad O-H Stretching of


phenolic-OH

3300-2500 Broad O-H Bending of


phenolic -OH

Page | 58
1760-1665 Medium, broad C=O Stretching of
ketone
Carbonyl

1320-1000 sharp C-O-C Stretching of


ether

4.7.1 Confirmation TFC Using Solvent Extraction of Petroleum Ether

The confirmation of TFC of Hibiscus leaves by using petroleum ether as solvent


extraction is present but the yield of TFC is the least compare to the methanol
and ethanol as solvents extraction because of the sample did not contain
phenolic compound (3500-3200 cm-1) that indicates the presence of high
content flavonoids. (Rani et al., 2013). Moreover, the petroleum ether is a non-
polar solvent that cannot extract the polar compound which is flavonoids
compound. (Finney et al., 1976).

4.7.2 Confirmation TFC Using Solvent Extraction of Methanol

The confirmation of TFC of Hibiscus leaves by using methanol as solvent


extraction is present and the yield of TFC is the higher than petroleum ether as
solvents extraction because of the sample contain carbonyl compound which is
at the wavelength 1649.99 cm-1 that indicates the presence of high content
flavonoids. (Rani et al., 2013). Moreover, the methanol is a polar solvent that
can be extracting with the polar compound which is flavonoids compound.
(Hrdlovic et al., 2010).

Page | 59
4.7.3 Confirmation TFC Using Solvent Extraction of Ethanol

The confirmation of TFC of Hibiscus leaves by using ethanol as solvent


extraction is present and the yield of TFC is the highest compare to the
petroleum ether and methanol as solvents extraction because of the sample
contain carbonyl compound which is at the wavelength 1649.53 cm -1 that
indicates the presence of high content flavonoids. (Rani et al., 2013). In addition,
the ethanol is a high polar solvent, so it can be extract high amount polar
compound which is flavonoids compound. (Fatiha et al., 2012). The type of
flavonoid that can be finding in Hibiscus leaves using ethanol as solvent
extraction is anthocyanin. (Mak et al., 2013)

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CHAPTER 5 :CONCLUSION AND RECOMMENDATION

5.1 Conclusion

Alopecia areata is a disease that affects the hair follicles, which are part of the
skin from which hairs grow and there is no treatment done for this disease. A
few researchers had been done related to this disease and conclude that
Hibiscus leaves can cure the Alopecia disease by its numerous benefits to
health. Knowing that, the Hibiscus leaves have valuable active substances that
can be extracted and give profit, it is the best opportunity to create wealth from
the waste and the most highlight opportunity is to create health from the waste
so an experiment based on this Hibiscus leaves were done to obtain the active
substance by extracting the Hibiscus leaves. The active substance called
flavonoid is widely used in food industry but mostly in cosmetic products. In the
experiment, two types of method and three solvents are used as a medium of
extraction time and mass constant for each and every sample. The objectives of
this experiment to determine the active ingredient in Hibiscus rosa-sinensis that
can improve blood circulation to nourish the hair follicle and thereby promoting
hair growth and to determine the yield of TFC in different parameters. Yield of
TFC was calculated to obtain the actual amount in every sample. The result was
showed that the highest yield of TFC is 317.793 (mg FeSO4 per 100 gram of
sample) for 30 gram of sample at 240 minutes of extraction process using
ethanol as solvent and soxhlet extraction method. This proved that the
extraction process using soxhlet extraction method is the best method due to
longer extraction time to produce high efficiency number of yields of TFC. Hence
from the result obtained, it can be conclude that the best solvent for extraction is
ethanol as it can produce TFC at the highest amount.as for the best extraction
time is 240 minutes. However if the extraction time more than 240 minutes,

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the yield of TFC will be reduced due to too long exposed to the heat that can
cause degradation of flavonoids. Lastly, the best solid-to-liquid ratio for
extraction highest amount of TFC is 30 per 150.

5.2 Recommendations

This researcher should be continuing for pill production to treat Alopecia patient
since the Hibiscus leaves is edible. In the future, other method of extraction of
Hibiscus leaves to produce flavonoid should be tested in producing higher yield.
Moreover, after extract the TFC from Hibiscus leaves, it must be stored in
cooling temperature which below than 150C to keep the yield from being
reduced. Next, use suitable method for analyzing data of TFC content to get
precise of yield. Besides that, food grade solvent as medium of extraction also
should be consider in the research to produce pills that are safe to consume for
the patient.

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APPENDICES

Appendix A Table for Raw Material

Table Result of Flavonoid Content Using Soxhlet Extraction Method

Soxhlet Extraction
Abs Flavonoids Contents (mg
No. Material / (510 FeSO4 per 100 gram of
Time
Solvent Solvent (Types) nm) sample)
(mins)
(gram / ml)

1 120 10 / 150 Petroleum Ether 0.022 3.110

2 120 10 / 150 Methanol 0.152 23.209

3 120 10 / 150 Ethanol 1.523 235.386

4 120 20 / 150 Petroleum Ether 0.034 4.967

5 120 20 / 150 Methanol 0.364 56.011

6 120 20 / 150 Ethanol 2.000 309.206

7 120 30 / 150 Petroleum Ether 0.160 24.524

8 120 30 / 150 Methanol 1.678 259.353

9 120 30 / 150 Ethanol 2.000 309.206

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10 240 10 / 150 Petroleum Ether 0.041 6.112

11 240 10 / 150 Methanol 0.161 24.663

12 240 10 / 150 Ethanol 0.525 80.968

13 240 20 / 150 Petroleum Ether 0.236 36.268

14 240 20 / 150 Methanol 0.508 78.276

15 240 20 / 150 Ethanol 1.622 250.657

16 240 30 / 150 Petroleum Ether 0.719 111.032

17 240 30 / 150 Methanol 1.896 293.145

18 240 30 / 150 Ethanol 2.055 317.793

19 360 10 / 150 Petroleum Ether 0.092 13.925

20 360 10 / 150 Methanol 0.099 15.070

21 360 10 / 150 Ethanol 1.750 270.462

22 360 20 / 150 Petroleum Ether 0.073 11.109

23 360 20 / 150 Methanol 0.267 41.095

24 360 20 / 150 Ethanol 0.315 48.507

25 360 30 / 150 Petroleum Ether 0.174 26.628

26 360 30 / 150 Methanol 0.179 27.386

27 360 30 / 150 Ethanol 0.978 151.075

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Table Result of Flavonoid Content Using Ultrasonic Assistant Extraction Method

No. Ultrasonic Bath Abs Flavonoids Contents


(510 (mg FeSO4/100 gram of
Time Material / Solvent (Types)
nm) sample)
(min) Solvent
(gram / ml)

1 10 10 / 150 Petroleum Ether 0.052 7.7623

2 10 10 / 150 Methanol 0.093 13.4596

3 10 10 / 150 Ethanol 0.462 65.1554

4 10 20 / 150 Petroleum Ether 0.021 3.4089

5 10 20 / 150 Methanol 0.196 27.9619

6 10 20 / 150 Ethanol 0.736 103.4968

7 10 30 / 150 Petroleum Ether 0.554 85.4553

8 10 30 / 150 Methanol 0.925 142.8435

9 10 30 / 150 Ethanol 1.176 181.6954

10 20 10 / 150 Petroleum Ether 0.034 4.9665

11 20 10 / 150 Methanol 0.552 85.0995

12 20 10 / 150 Ethanol 0.703 108.5715

13 20 20 / 150 Petroleum Ether 0.109 16.5710

14 20 20 / 150 Methanol 0.483 74.4697

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15 20 20 / 150 Ethanol 0.558 86.1052

16 20 30 / 150 Petroleum Ether 0.021 2.9860

17 20 30 / 150 Methanol 0.855 132.0591

18 20 30 / 150 Ethanol 1.105 170.6634

19 30 10 / 150 Petroleum Ether 0.018 2.5373

20 30 10 / 150 Methanol 0.221 33.9932

21 30 10 / 150 Ethanol 0.392 60.3587

22 30 20 / 150 Petroleum Ether 0.026 3.8215

23 30 20 / 150 Methanol 0.262 40.2751

24 30 20 / 150 Ethanol 0.455 70.1838

25 30 30 / 150 Petroleum Ether 0.048 7.1481

26 30 30 / 150 Methanol 0.550 84.9293

27 30 30 / 150 Ethanol 1.370 211.6660

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Appendix B Figure of Functional Groups of Compound Inside the
Sample

Figure Functional Groups of Compound inside the Sample Extracted using


Ethanol Solvents

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Figure Functional Groups of Compound inside the Sample Extracted using
Methanol Solvents

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Figure Functional Groups of Compound inside the Sample Extracted using
Methanol Solvents

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Appendix C Figure of Standard Calibration of Ferrous Sulphate
(FeSO4)

Figure of Standard Calibration 1 of FeSO4

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Figure of Concentration of Flavonoid (ppm) in the Samples Based on Standard
Calibration 1
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Figure of Standard Calibration 2 of FeSO4

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Figure of Concentration of Flavonoid (ppm) in the Samples Based on Standard
Calibration 2

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