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OPTIMIZATION OF SUPERCRITICAL FLUID EXTRACTION OF BIOACTIVE

COMPOUND FROM STROBILANTHES CRISPUS

MUHAMMAD AFIQ SYAMIM BIN RAHIMI


55201113563

FINAL YEAR PROJECT 2


PROPOSAL

Malaysian Institute of Chemical & Bio Engineering Technology


University Kuala Lumpur

JANUARY 2016
Table of Contents

CHAPTER 1 ....................................................................................................... 10
INTRODUCTION ................................................................................................ 10
1.1 Background of studies............................................................................... 11
1.2 Problems Statement.................................................................................. 14
1.3 Significant of Study ................................................................................... 16
1.4 Scope of Study .......................................................................................... 17
1.5 Objectives ................................................................................................. 18

CHAPTER 2 ....................................................................................................... 19
LITERATURE REVIEW ...................................................................................... 19
2.1 Bioactive Compounds ............................................................................... 19
2.2 Strobilanthes Crispus ................................................................................ 22
2.2.1 Strobilanthes Crispus characteristic ................................................... 22
2.2.2 Uses of Strobilanthes Crispus ............................................................ 23
2.2.3 Chemical and Physical Properties of Strobilanthes Crispus ............... 26
2.3 Diabetes Mellitus Disease ......................................................................... 27
2.4 Extraction Method ..................................................................................... 29
2.4.1 Conventional Extraction Method ......................................................... 29
2.4.2 Non-Conventional Extraction Method ................................................. 31

CHAPTER 3 ....................................................................................................... 33
METHODOLOGY ............................................................................................... 33
3.1 Outline of the methodology ......................... Error! Bookmark not defined.
3.2 Methodology ............................................................................................. 35
3.2.1 Sample Collection and Preparation .................................................... 35
3.2.2 Solvent preparation ............................................................................ 36
3.2.3 Sample Extraction .............................................................................. 37
3.2.4 Sample Analysis ................................................................................. 38
3.2.5 Sample Data ....................................................................................... 39

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CHAPTER 4 ....................................................................................................... 42
EXPECTED RESULT ......................................................................................... 42

CHAPTER 5
CONCLUSION ................................................................................................... 43

RESEARCH PLAN ............................................................................................. 44

REFERENCES ................................................................................................... 45

APPENDICES .................................................................................................... 48
Chemicals and Materials ................................................................................. 49
Solvent Preparations ....................................................................................... 50
Example of Calculation ................................................................................... 50

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List of Figures

Figure Tittle Page

1.1 The Prevalence of diabetes in adult by the age in Malaysia (IDF, 2015) 15
2.1 Strobilanthes Crispus plants in Malaysia (Lin, 2013) 23
2.2 Strobilanthes Crispus leaved sold in Malaysia (Lin, 2013) 24
2.3 The islets contain beta cell cells in human organ (NIH, 2014) 28
2.4 The ultrasonic-assisted extraction
(University of British Columbia, 2012 30
2.5 The maceration method of plant extraction (Rahul, 2013) 30
2.6 The supercritical fluid extraction method (Suzan Mahdi & Altikriti, 2010) 31
2.7 The Pressurized liquid extraction
(Department of Pharmaceuticals Sciences, 2015) 32
3.1 The outline of the methodology in this studies 34

3.2 The ideal gas law formula (Corbett, 2014) 36

List of Tables

Tables Tittle Page

2.1 The Medical uses of Strobilanthes Crispus plants (GlobinMed, 2010) 25


2.2 The chemical and physical properties of the Strobilanthes Crispu
(Ghasemzadeh, Jaafar, & Rahmat, 2015) 27
1 Project cost estimation for Final Year Project 1 48
2 Table of chemicals/materials and apparatus 49
3 Solvent Preparation for Ethanol 50

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ABSTRACT

Supercritical Fluid Extraction (SFE) has become one of the most popular
green extraction techniques nowadays since it has demonstrated many
advantages compared to traditional or conventional extraction process. Aspects
such as improved selectivity, higher selection yields, better fractionation
capabilities and lower environmental impacts have been crucial to the important
growth of SFE. In this study, non-conventional Supercritical Fluid Extraction by
using Tubular Batch Reactor was used to extract the bioactive compound from
Strobilanthes Crispus besides using Supercritical Fluid Extractor.

Strobilanthes Crispus is an herbal medicine plant which is native to country


from Madagascar to Indonesia. This plant are used in medicinal and to treat a
variety of ailments in the various traditional systems of medicine. Phytochemical
investigations have revealed that the plant contains polyphenols, flavonoids,
catechins, alkaloids, caffeine, tannis, and compound known to possess health
beneficial effects. Preclinical studies have shown that the plant possess
antioxidant, anti-cancer, anti-diabetic, and anti-microbial activities.

Hence, the aim of this study was to identify the bioactive compounds in
extracted Strobilanthes Crispus which could be used to treat diabetes mellitus
disease. The bioactive compound which is quercetin will be analyzed to cure the
diseases.
ABSTRAK

6
APPROVAL PAGE

We have supervised and examined this report and verify that it met the program
and the University’s requirements for the Bachelor in Chemical Engineering
Technology in Process.

Date: Signature: …………………………………


Supervisor Name: Mdm. Nor Aini Bt Burok
Official Stamp:

7
DECLARATION

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

Date: Signature:
…………………………………
Full Name: MUHD AFIQ SYAMIM
BIN RAHIMI
ID Number: 55201113563

8
ACKNOWLEDGEMENT

Alhamdulillah, all praises to Allah SWT, the Almighty and may Allah’s peace
and blessing be upon His Servant and Messenger Muhammad SAW and upon his
family and Companions. Thanks to Allah whom with His willing giving me the
opportunity to complete the final year project report.

I would like to express deepest gratitude to my advisor, Madam Nor Aini


Binti Burok for her full support, expert guidance, understanding, and
encouragement throughout my study and research. Without her incredible
patience and timely wisdom and counsel, my thesis work would have been a
frustrating and overwhelming pursuit. To my parents, no words that I could use to
express my genuine appreciation for their endless support and invaluable
expenditure of motivation. I would not have been able to complete this thesis
without their continuous love and encouragement.

Last, but by no means least, my sincere appreciation goes to my sister Nur


Farthiah Bt Mohd Adanan and all my friends who patiently helped in revising and
correcting every single page of my thesis report. I also would like to thank The
technicians of UniKL MICET, En.Shukri for guiding, sharing advice and giving
strong cooperation to help me during handling equipment and apparatus in the lab.
I hope that with all the knowledge and experiences that I gained from this project
will be beneficial for me in the real working in the future.

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CHAPTER 1

INTRODUCTION

Overview

A bioactive compound is a substance present in natural source that having


an impact or cause a response to the living organism, tissue or cell. Nowadays, it
becomes a trend to used bioactive compound in geo-medicine, plant science,
modern pharmacology, agrochemicals, cosmetics, food industry and nano-
bioscience. Based on the problem statement below, there were over a million
cases of diabetes in Malaysia in 2015 and it getting increase year by year.
Quercetin exhibits a wide range of biological functions and gives a lot of benefits
to human health. Thus, the Strobilanthes Crispus leaves was used to extract
quercetin that can be used to treat diabetes disease. The plants contained high
amount of mineral content and vitamin C, B1, and B2. The extraction technique
used in this study was non-conventional Supercritical Fluid Extraction by using
Tubular Batch Reactor.

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1.1 Background of studies

Bioactive compound are extra nutritional constituents that typically occur in


small quantities in food and has an effect on living organism, tissue or cell. These
compounds utilized as a part of extensive variety of conventional and present day
applications. They are being intensively studied to evaluate their effects on health
widely. Nowadays, it becomes a trend to used bioactive compound in geo-
medicine, plant science, modern pharmacology, agrochemicals, cosmetics, food
industry and nano-bioscience. Bioactive compounds can have an influence on
health and they are being studied in the prevention of cancer, diabetes, and other
disease.

Strobilanthes Crispus (pecah kaca pecah beling leaves) is a member of the


Acanthaceae family and is an herbal medicine plant which is native to countries
throughout Madagascar region to Indonesia. Traditionally, it is known as “pecah
kaca” in Malay, “daun picah beling” in Jakarta, and “kejibeling” in Java. (Chong,
Koh, Kiong Ling, Chye, & Yew, 2014). The plants contained high amount of mineral
content and vitamin C, B1, and B2. The plants is a well-known her in Malaysia with
various pharmaceutical properties and contain several biologically active chemical
constituents which are responsible for its pharmaceutical quality .The plant part
will be used to extract the bioactive compound is the leaves.

Phytochemical investigations have revealed that the plant contains


polyphenols, flavonoids, catechins, alkaloids, caffeine, tannis, and compound
known to possess health beneficial effects. Preclinical studies have shown that the
plant possess antioxidant, anti-cancer, anti-diabetic, and anti-microbial activities
and can gives an advantage in order to prevent the disease. (Nurraihana,
Norfarizan Hanoon, & N.a, 2013)

Extraction in chemistry is a separation process consisting in the separation


of a substances from a matrix. There are two extraction methods can be used to
extract bioactive compound which is conventional and non-conventional method.

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The examples of conventional method are soxhlet extraction, macerations and
hydrodistillation that had been used in order to retrieve bioactive compounds.
Supercritical fluid extraction (SFE), and pressurized liquid extraction (PLE) are
non-convetional methods that offers a more economical, environmental friendly
and ozone-depleting emissions.

Scalia et al., (1999) in their studies state that the conventional techniques
to obtain plant extracts such as steam distillation and organic solvent extraction
usually requires several hours or even days causing the extraction process require
large amount of solvent. The solute or solvent separation may result in degradation
of the thermo labile components and traces of the solvent may be present in the
product, which it may reduce quality assessment of the extraction yield.

Process optimization and antioxidant activity supercritical fluid extraction


studies by (Yuefei, Le, Da, Shikang, Yuejin, & Ping, 2011) in their journal
supercritical carbon dioxide extraction (SC-CO2) of bioactive compounds from
Ampelopsis grossedentata stems was to investigate extraction parameters such
as pressure, temperature, dynamic time and modifier. The best conditions
obtained for SC-CO2extraction of flavonoids was 250 bar, 40 °C, 50 min, and with
a modifier of methanol/ethanol (1:3, v/v), and that for phenolics extraction was 250
bar, 40 °C, 50 min, and with a modifier of methanol/ethanol (1:1, v/v). This shows
that methanol and ethanol are the best solvent to extract flavonoids and phenolics
compounds.

A comprehensive study on different methods of extraction was done by


Vibha, Pallavi, & Devendra, 2012 by using Guajava Leaves for curing various
health problems. This study was to introduce the different extraction processes
with different solvent such as ethanol, methanol, ethyl acetate and water. The
method used was Supercritical Fluid Extraction (SFE), Soxhlet Extraction, Steam
Distillation and Ultrasound Extraction. The yield shows Soxhlet process gives
highest global yield but separation of compounds is difficult in this method but in
SFE gives better recovery of functional compounds.

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Supercritical Fluid extraction (SFE) has been documented as an effective
method for preparing bioactive products from plant materials. The combined liquid-
like solvating capabilities and gas-like transport properties of supercritical fluids
make them particularly suitable for the extraction of diffusion-controlled matrices
such as plant tissues. The remarkably high selectivity can be achieve when the
solvent strength of supercritical fluid can manipulated by changing pressure and
temperature.

Subsequently, based on previous studies, this study was conducted to


extract bioactive compounds from Strobilanthes Crispus by using non-
conventional extraction method which is supercritical fluid extraction (SFE)
method. Ethanol was used to identify bioactive compounds content from
Strobilanthes Crispus and acts as a supercritical fluid. Extraction parameter
studied was solid loading, temperature, and extraction time to identify their effect
on extracted Strobilanthes Crispus.

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1.2 Problems Statement

Cancer is a major public health problem worldwide with millions of new


cancer patients diagnosed each year and many deaths resulting from this disease.
Many scientist had been trying to develop the cure for cancer, unfortunately their
attempt had failed. Chemotherapy remains the principal mode of treatment for
various cancers and the patients need to spend a lot of their money for the
treatment. Tamoxifen, non-steroidal, and anti-estrogen drug is used in the
treatment of estrogen receptor that positive in breast cancer patients. This
statement is supported by (Yaacob, et al., 2010) in his titled “Anticancer activity of
a sub-fraction of dichloromethane extract of Strobilanthes crispus on human breast
and prostate cancer cells in vitro”

There is no treatment that can completely cure diabetes mellitus until today.
Presently, insulin is used to treat diabetes mellitus type 1 and the pharmacological
agents currently used for the treatment of type 2 include sulphnonylureas,
biguanide, and acarbose. These agents however have restricted usage due to
several undesirable side effect and failure to significantly the course of diabetic
complications. (Bakar, 2005)

According to the International Diabetes Federation (IDF) reports, there were


3.3 million cases of diabetes in Malaysia in 2015. The number of deaths in adults
due to diabetes is 34,576. Based on the figure 1.1 below, the figure describes
which are groups in population have the highest proportions of diabetes. The
dotted lines is the distributions of diabetes prevalence by age of the world, the
black line is the distribution for the region and the country region is plotted in the
red line. Many middle and low income countries have more people under the age
of 60 with diabetes compared to the world average.

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Figure 1.1: The Prevalence of diabetes in adult by the age in Malaysia (IDF,
2015)

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1.3 Significant of Study

The bioactive compound extracted from this study will be used for diabetes
disease. The bioactive compound existed in the leaves was able to lower blood
glucose level in strptozocin induced diabetic rats by extract of both fermented and
unfermented leaves using hot water. It was found that the content of vitamins C,
B1 and B2 with catechin, caffeine, tannin were the major contributors to the
antioxidant activity. In order to enhance the defense system especially the
incidence of degenerative diseases, the daily consumption of the tea could
contribute towards it.

The extraction technique use in this study was Supercritical Fluid Extraction
(SFE). This techniques was widely used in industrial extraction operations because
of regulatory and environmental pressures on hydrocarbon and ozone-depleting
emission. Nowadays, the most currently available solvent free extraction system
will utilize carbon dioxide (CO2) which is generally considered as safe for solvent
free extraction processes. In this study new technique were applied, SFE will be
conducted by using tubular batch reactor. This reactor was design by using 15 mL
bulkhead union (3/4” OD) with two steel caps (3/4” OD).

In addition, this study will build up the people about the benefits of the leaves
thus it can consume the leaves in their daily life since it can make as a tea.
Traditionally, it is widely used to treat gastrointestinal and kidney diseases at a
certain place around Malaysia.
1.4 Scope of Study

The Strobilanthes Crispus leaves was used in this study because it has been
of much interest due to traditional claims of its anti-cancer properties and other
diseases in this country. The leaves was collected from the area around Alor Gajah
and will be followed with a few step until it is ready to use.

In this study, supercritical fluids extraction method by using tubular batch


reactor design was applied in order to extract the bioactive compound of
Strobilanthes Crispus leaves. Ethanol was used as a supercritical fluid because it
is a very polar molecule due to its hydroxyl (OH) group with high electronegativity
of oxygen allowing hydrogen bonding to take place with other molecules, thus it
can attract polar and ionic molecules. Ethanol can dissolve both polar and non-
polar substances because it can attract non-polar molecules.

The parameter studied in this project were types of solid loading, temperature
and extraction time. Temperature use is 300oC and 400oC, solid loading 30% and
50%, and retention time 15min and 25min. The critical state temperature and
pressure for ethanol are 243oC and 63.18atm respectively.

In order to identify the existence of bioactive compound in Strobilanthes


Crispus leaves, Ultraviolet-Visible Spectrophotometer (UV-Vis) was used to
analyze the extractant. To determine the optimal yield bioactive compound in
Strobilanthes Crispus, Response Surface Methodology (RSM) was used by using
statistical software which is Minitab 17.

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1.5 Objectives

1. To extract the bioactive compound in Strobilanthes Crisus leaves.


2. To identify the bioactive compound found in Strobilanthes Crispus
leaves.
3. To optimize the process condition of supercritical fluid extraction to
obtain better yield using Response Surface Methodology (RSM).

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CHAPTER 2

LITERATURE REVIEW

2.1 Bioactive Compounds

Bioactive compounds by definition is a substance has an organic activity if


it consumes direct effects on a living organism. These effects can both be
advantageous or inconsistency on the substance and the bioavailability. It is
usually linked with positive effects on an organism. (NPCR , 2016) Bioactive
compounds have actions in order to promote good health in the body.

Bioactive substances present as natural essential in food to provide health


benefits beyond the basic nutritional value of the product. Many bioactive
compounds have been discovered and have been widely in chemical structure,
function, and are grouped accordingly. Phenolic compounds including flavonoids
are present in all plants and have been studied extensively in tea, vegetables, fruits
and others. Many phenolic compound have antioxidant properties, and it have
been proved favorable effects of some disease such as thrombosis and
tumorogenesis.
Bioactive compound in plants are compound produced by plants having
pharmacological or toxicological effects in human and animals. Typically, bioactive
compounds in plants are produced as secondary metabolites. Hence, the definition
of bioactive compounds in plants in known as secondary plant metabolites. The
secondary metabolites compounds in plants come out to be randomly synthesized.
Most of the bioactive compound found have different characteristic and functions
for example, flavonoids can protect against free radical generated during
photosynthesis. It is slightly different with primary metabolites that course of
compounds aimed at plant growth and development such as carbohydrates, amino
acid, proteins, and lipids. It can be observe as the side track of biochemical
products in plant cell and not needed for the plant daily functioning.

2.1.1 Flavonoid

Flavonoids or bio-flavonoids from the Latin word meaning yellow based on


their color in nature. Chemically, it have general structure of a 15-carbon skeleton
which consists of two phenyl rings A and B. According to the IUPAC nomenclature,
it can be classified into flavonoids or bio-flavonoids, Iso-flavonoids, and Neo-
flavonoids. (McNaught, Wilkinson A., & Andrew, 1997)

Flavonoids are abundant in plants, in which they perform several functions.


They are essential pigments for producing the colors needed to attract pollinating
insects. In higher order plants, flavonoids are also required for nitrogen fixation,
UV filtration, cell cycle inhibition and can act as chemical messengers. Some
flavonoids also inhibits certain spores to protect against certain plant disease.
Flavonoids are plentiful in plants and are the most common type of polyphenolic
compound found in human diet.

The abundance of flavonoids coupled with their low toxicity relative to other
plant compounds means they can be ingested in large quantities by animals
including humans. The examples of foods that are rice in flavonoids include onions,
blueberries, red wine, dark chocolate, and bananas.

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Flavonoids, including quercetin are important in anti-inflammatories
because they act as anti-oxidants which mean the literally fight the natural process
of oxidation that takes place over time of the age. Quercetin can help stop
damaging particles in the body that known as free radical which negatively impact
on how the cells works.

2.1.2 Quercetin

Quercetin exhibits a wide range of biological function. Quercetin is a type of


flavonoids anti-oxidants that’s found in plant foods including leafy greens,
tomatoes, broccoli and others. Technically, it is considered as a plant pigment
which is exactly found in deep colored, nutrient packed fruits and vegetables.

They scavenge particles in the body known as free radicals which damage
cell membranes, tamper with DNA, and even cause cell death. Anti-oxidants can
neutralize the free radicals and it may reduce or even help to prevent some of the
damage free radical causes. In test tubes, quercetin has strong anti-oxidant
properties and the researches are not sure whether taking quercetin and many
other anti-oxidants has the same effect on the body. (Ehrlich, 2015)

Many of the benefits often hear attributed to anti-oxidants that refer to the
effects associated with quercetin, some of them are supports normal respiratory.
For example when respiratory system is irritated, redness, and swelling can result
from the release of histamines and quercetin has been reported to have an anti-
histamine effect. From the previous research that have been done by Chirumbolo
(2010), the lab test shown quercetin influences intracellular enzymes and may help
inhibit histamine release.

The study conducted by Perez Vizcaino and Duarte (2010) shown that the
consumptions of flavonoids, specifically quercetin offer a dual benefits in promoting
overall cardiovascular health and it encourages the blood flow very well. Next,
researchers have witnessed that quercetin anti-oxidant action pretect against LDL

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cholesterol oxidation and this may be useful because oxidation causes LDL
cholesterol to stick to artery walls. Figure 2.1 below shows the structure of
quercetin that is believe can give a lot of benefits to human body.

Figure 2.1: The structure of quercetin

2.2 Strobilanthes Crispus

Strobilanthes Crispus is a member of the Acanthaceae family and is known


by the following names “Pecah Kaca” or “Pecah Beling” in Malaysia and
“Kecibeling” or “Kejibeling” in Indonesia. It is a Plant that originated from
Madagascar and all the way to Indonesia and it grows rapidly in Malaysia. The
leaves are used traditionally for anticancer treatment and diabetes mellitus
disease. (Wikipedia, 2009)

2.2.1 Strobilanthes Crispus characteristic

The Strobilanthes Crispus is a woody spreading shrub that can easily reach
more than a meter in height of the cultivation. The stem has a diameter of between
0.2-0.7cm with the external bark being purplish in color when young and brown

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when matured. The leaves are elliptical in shape and have an attractive color which
is glossy sheen. The surface of the leaves are also rough to the touch. The
Strobilanthes Crispus leaves rarely produces flowers. It has rough in texture and
measures 5-8cm and 2-5cm wide. The buds are borne in leafy sheaths and the
yellow flowers that emerge are said to be able reach up to 2cm in diameter. Figure
2.2 below shows the Strobilanthes Crispus plants were found in many places in
Malaysia as it is easily to grow.

Figure 2.2: Strobilanthes Crispus plants in Malaysia (Lin, 2013)

2.2.2 Uses of Strobilanthes Crispus

2.2.2.1 Drinks

In Malaysia, Strobilanthes Crispus plants were extracted in certain process


in order to make it as healthy tea or herbal tea. The people believe especially
Chinese race that this tea can relief cough, removal of kidney stones and urinary
bladder problems. It is slightly bitter taste and has the sweet aroma. Figure 2.3
shows the example of Strobilanthes Crispus tea packet were sold in Malaysia

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Figure 2.3: Strobilanthes Crispus leaved sold in Malaysia (Lin, 2013)

2.2.2.2 Traditionally Uses

Strobilanthes Crispus plants been used traditionally to treat kidney stones.


The responsible for this action occurs because the phenolic compound in the leaf
that was extracted. The phenolic compounds has inhibitory actions on calcium
oxalate crystal growth. The most effective and the highest in phenolic compound
content was found in the extraction by using methanol that acts as a solvent. This
statement is supported by (Afrizal Itam, Ismail, & Abdul Majid, 2007) in his article
titled “In vitro studies of calcium oxalate crystal growth inhibition of Strobilanthes
Crispus extracts”. To further enhance its ability to erode renal stones, the aqueous
extract of the leaves of Strobilanthes Crispus also has it diuretic properties.

In addition, Strobilanthes Crispus is used to treat constipation because it


considered as a laxative. It is also used in the treatment of liver problems, hepatitis,
and liver problems. The leaves part has always been used in a decoction process
to produce the healthy water.

Strobilanthes Crispus had been encouraged in the medication of poisonous


bites including snakebites. The poultice of the fresh leaves can be directly applied

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on to wounds caused by the bites of poisonous snakes or other animals. (Afrizal,
2008)

2.2.2.3 Medical Uses

The use of Strobilanthes Crispus plants as medicine is not a new thing in


Malaysia. This plants used by the local people to season food also yield medicinal
compounds. As part of response to the threat of the disease in Malaysia, the use
of the Strobilanthes Crispus leaves in cuisine is developed. Table 2.1 below shows
the medical uses of Strobilanthes Crispus leaves.

Table 2.1: The Medical uses of Strobilanthes Crispus plants (GlobinMed, 2010)

Requirement Uses

To induce the apoptosis in the liver cells through the


Cytotoxic activity
mechanism action of the extraction process.

Renal activity To treat kidney stones and its ability to erode renal stones.

To enhance the defense system especially towards the


Anti-oxidant activity
incidence of degenerative disease.

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Both fermented and unfermented leaves of Strobilanthes
Anti-diabetic activity
Crispus leaves was able to lower blood glucose level.

It can induced the human body weight from the study of


Anti-obesity activity
Strobilanthes Crispus extract on lipolysis.

Anti-viral activity It is found to be an efficiently in virucidal agent.

2.2.3 Chemical and Physical Properties of Strobilanthes Crispus

The different types of matter can be distinguished through two components


which are physical and chemical properties. The physical properties of the
Strobilanthes Crispus plants can be observed or measured without the changing
of composition while for chemical properties is the potential of the plants to
undergo some chemical change or reaction by purity of its composition. Table 2.2
below shows the chemical and physical properties of the plants, it is very extensive
to study the properties of the Strobilanthes Crispus plant in order to achieve the
quality samples during the extraction process.

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Table 2.2: The chemical and physical properties of the Strobilanthes Crispus
(Ghasemzadeh, Jaafar, & Rahmat, 2015)

Properties Value
Low Temperature (oC) 21oC-23oC
High Temperature (oC) 32oC-33oC
Height above sea level (m) 3m-56m
Humidity (%) 76%-81%
Light intensity (umol/m2/s) 54.6
Moisture 66.3%
Potassium (%) 51%
Calcium (%) 24%
Sodium (%) 13%
Iron (%) 1%
Phosphorus 1%

2.3 Diabetes Mellitus Disease

Diabetes mellitus is a group of metabolic diseases in which there are high


blood sugar levels over a continuous period and commonly referred to as diabetes.
Frequent urination, increased thirst and increased hunger are the symptoms of
high blood sugar. Diabetes can cause many complication to human health if left
untreated. The long-term problems include stroke, cardiovascular disease, chronic
kidney failure, and damage to the eyes. (Wikipedia, 2016)

Diabetes is a disorder of metabolism that the body use to digest food for
energy and it will breaks down to carbohydrates, sugars and starches found in
many meals into glucose. Diabetes evolves when the body does not make enough
insulin or is not capable to use insulin efficiently. Insulin is made in the pancreas
and contains clusters of cells called islets. Beta cells within the islets make insulin
and release it into the bloodstream.

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Figure 2.4: The islets contain beta cell cells in human organ (NIH, 2014)

The assessment of the anti-hyperglycemic properties were investigated and


the etnopharmacological value of the Strobilanthes Crispus plant such as the
development of nutraceutical Strobilanthes Crispus herbal tea with condition
fermented and unfermented. The hot water extract from the fermented and
unfermented reduced blood glucose and also improve lipid profile shown by the
result of the experiment which is tested on rats. The antihyperglycemic and
antilipidemic properties are contributed by the presence of antioxidant and
polyphenol content in the extracts. (Abu Bakar, Othman, & R Asmah, 2006)

In addition, rats are used in lab research because of their physical


similarities to human. Rats seem to analyze consequences in a similar way that
humans and other primates do and this phenomenon is called metacognition.
These similarities include physical, psychological, behavioral and social
characteristics. (Kalish, 2010)

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2.4 Extraction Method

Extracting the bioactive compound from plant materials is the most


appropriate and standard method used in different sectors such as
pharmaceutical, food and chemical industries. Eventhough a lot of new methods
have been established along with conventional method, there is no method that
can be classified as standard for extracting bioactive compounds from plants.
There are two extraction methods which are conventional and non-conventional.
The methods depend on the critical input parameters, nature of plant matrix,
chemistry of bioactive compounds, and scientific expertise.

2.4.1 Conventional Extraction Method

Conventional extraction method is synthesized in small quantities in nature


that had been used several years ago which classified as the classical extraction
technique to obtain bioactive compounds. It requires labor intensive and time
consuming purification procedures which present as conjugate or mixture in
extract.

2.4.1.1 Ultrasonic-Assisted Extraction (UAE)

In ultrasonic assisted extraction, the use of ultrasound is expediting the


extraction which is a modified from maceration method. A vial is used to place the
plant powder which later placed in an ultrasonic bath. Ultrasound is used to induce
a mechanical stress on the cells through the production of cavitation in the sample.
The solubilization of metabolites in the solvent is increased by the cellular
breakdown and improves the extraction yields. The initial extraction of a small
amount of material commonly use this method.

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Figure 2.5: The ultrasonic-assisted extraction (University of British Columbia,
2012)

2.4.1.2 Maceration Processes

The plant material cut into small pieces and moderately coarse powder. A
closed vessels is used to place the substances and the selected solvent is added
in a suitable ratio. Allowed to stand for seven days shaking occasionally and the
liquid is strained off. The process is repeated for once or twice with fresh solvent.
Finally, by using a mechanical press or a centrifuge, the last residue of extract is
pressed out of the plant particles. Both initial and bulk extraction is suitable in this
method. The main disadvantage of maceration is that the process can be quite
time-consuming, taking from a few hours up to several weeks. (Singh, 2012)

Figure 2.6: The maceration method of plant extraction (Rahul, 2013)

30
2.4.2 Non-Conventional Extraction Method

The non-conventional method was introduced since the conventional


method is no longer suitable because of the long extraction time, costly, lot of
solvent amount used, low selectivity and thermal decomposition. The production,
detection, separation and characterization of bioactive compounds are able to be
improved by using this method.

2.4.2.1 Supercritical-Fluid Extraction

Supercritical fluids extraction is increasingly demanded in industry replacing


organic solvents which is used in industrial extraction because of regulatory and
environmental pressures on hydrocarbon and ozone-depleting emissions. Carbon
dioxide (CO2) which is considered as safe for solvent free extraction processes is
utilized by most of the available solvent free extraction. By regulating its
temperature and pressure, the liquid CO2 is forced into supercritical state. Next,
Supercritical CO2 has solvent power and extracts predominantly lipophilic and
volatile compounds. Gaseous CO2 returns to CO2 tank. The new extraction starts
with circulating CO2 after a full round.

Figure 2.7: The supercritical fluid extraction method (Suzan Mahdi & Altikriti,
2010)

31
2.4.2.2 Pressurized Extraction

The extraction cell is loaded with the powdered plant materials, which is
placed in an oven. In order to fill the cell, the solvent is then pumped from a
reservoir which is heated and pressurized at programmed levels for a set of period
time. The cell is flushed with nitrogen gas, and the extract which is automatically
filtered, is collected in a flask. To rinse the cell and solubilize the remaining
components, a fresh solvents is used. To dry the material, a final purge with
nitrogen gas is performed. This method offers a few advantages which are more
economical and environmental friendly.

Figure 2.8: The Pressurized liquid extraction (Department of Pharmaceuticals


Sciences, 2015)

32
CHAPTER 3

METHODOLOGY

Overview

This chapter discusses about the methodology that used in this study. The
extraction process were done in this study was Supercritical Fluid Extraction (SFE)
by using Tubular Batch Reactor. The preparation sample of the Strobilanthes
Crispus, extraction process, and the sample analysis were discussed in this
chapter. The bioactive compound of the leaves were analyzed by using Ultraviolet-
Visible spectrophotometer (UV-Vis) while Response Surface Methodology (RSM)
was used to get the optimal yield value of the leaves.
The outline of the methodology was plotted as shown in figure 3.1 before
run the experiment to make sure the effectiveness of the study.

Sample collection of Strobilanthes Crispus leaves

The leaves were cleaned and dried at 40oC

The cleaned dried leaves were grounded and stored

Preparation the volume of solvent (ethanol) by using Ideal-Gas Law


formula PV=nRT

Ethanol
Temperature: 243oC
Pressure: 62.18atm

Supercritical Fluid Extraction (SFE) of bioactive compounds using


Tubular Batch Reactor

Tested with different parameters

Temperature: Extraction Solid


Type of Loading
time:
solvents:  300oC and
 15min and  30% and
 Ethanol 400oC
25min 50%

Analysis of bioactive compounds exist by using Ultraviolet-Visible


Spectrophotometer (UV-Vis)

The experimental data obtained by using Response Surface Methodology


(RSM)
Figure 3.1: The outline of the methodology in this studies

34
3.2 Methodology

The details of the experiment from this study were briefly explained in this
section.

3.2.1 Sample Collection and Preparation

The Strobilanthes Crispus leaves can be found on riverbanks or abandoned


fields. The leaves of the plants collected from the area around Alor Gajah, Melaka.
The leaves were separated from the stalks and then completely washed with tap
water. The leaves were rinsed with distilled water that had been supplied and dried
in ventilated drying oven at 40oC for 24 hours. The dried leaves were grounded in
a dry mill blender to form a powder in order to increase the surface area of sample.
The grinded fresh leaves were directly stored in air-tight bottles and dark place
prior to extraction process. Figure 3.2 below shows the powder of the Strobilanthes
Crispus leaves.

Figure 3.2: The powder form of the Strobilanthes Crispus leaves

35
3.2.2 Solvent preparation

In preparation of supercritical fluid, the volume of the solvent used in the


reactor to extract Strobilanthes Crispus must be considered Ethanol was widely
used in industrial and it is the second most important solvent after water. In this
extraction process, ethanol was used as supercritical fluid solvent. By referring
figure 3.3, the volume of the solvent used was calculated by using Ideal-Gas Law
formula. In this case, the temperature and pressure of the solvent calculated must
be above their supercritical state. Where the critical state temperature and
pressure for ethanol are 243oC and 62.18atm respectively. (Material Safety Data
Sheet Ethanol, 2013)

Figure 3.3: The ideal gas law formula (Corbett, 2014)

36
3.2.3 Sample Extraction

These extraction methods are based on Supercritical Fluid Extraction (SFE)


merthod. Since SFE equipment is not available at the laboratory, so Tubular Batch
Reactor was designed to replace SFE equipment to extract anti-oxidants
compound which is bioactive compound in Strobilanthes Crispus. Tubular batch
reactor was designed by using 15mL bulkhead union (3/4” OD) with two steel caps
(3/4” OD). This reactor was made of stainless steel 316 because it can adapt with
high temperature and pressure. One end of the bulkhead union was closed with
one steel cap. Then, Strobilanthes Crispus sample of 30% and 50% solid loading
and the solvent was placed in the bulkhead union and closed with another steel
cap at the other end of bulkhead union. Both end of bulkhead union must be closed
tightly to avoid the reactor exploded while heating in the furnace, due to its high
temperature and pressure.

Once the reactor filled with sample and solvent, the tubular batch reactor
was placed in the furnace with different extraction time 15 and 25 minutes. The
temperature of the furnace was set to 300 OC and 400OC for each extraction time
samples to convert the solvent to its supercritical fluid. The extracted sample was
cooled in water bath immediately to avoid another reaction take placed in the
reactor that can lead to the error of the data. After the reactor was cooled, the
extractant was transfer to a universal bottle. The cap of the reactor does not open
until the reactor cooled to avoid the sample burst out due to high pressure. Figure
3.4 and 3.5 below shows the tubular batch reactor in order to extract the bioactive
compound from the leaves.

37
Figure 3.4: The 15ml bulkhead union of the tubular batch reactor

Figure 3.5: The steel cap of the tubular batch reactor

3.2.4 Sample Analysis

The extracted leaves were analyzed by using Ultraviolet-Visible


Spectrophotometer (UV-Vis) to identify the bioactive compound in Strobilanthes
Crispus. This equipment was left ON for 30 to 60 minutes before proceeding. At
first, a cuvette is filled with quercetin and another cuvette is filled with absolute
ethanol solvent. Before cuvette contain quercetin is placed in sample holder and
cuvette contain ethanol is placed into reference holder compartment to run, the
sides of the cuvette was wiped with soft tissues. This wavelength scan is run in
order to determine lambdamax for the extracts. Then, both cuvettes filled with

38
blanks ethanol is scanned in order to get zero value of the instrument. To run the
photometric scan, the cuvette contain chloroform placed in sample holder is took
out and replace with the cuvette filled with standard solution which are 1.00ppm,
50.00ppm, 100.00ppm, 250.00ppm, and 500.00ppm respectively. The other blank
is left in the reference holder for subsequent runs.

Next, each sample of 30 extracted was diluted with absolute ethanol with
0.1g sample in 10ml absolute ethanol. Photometric scan was conducted to get the
absorbance results and the standard calibration graph was generated
automatically by using the equipment. The purpose of photometric scan was to
determine the concentration (single component) of the extract from the standard
calibration graph.

3.2.5 Sample Data

Before the experimental procedure was run, basically Response Surface


Methodology (RSM) was designated for the whole process. RSM is a collection of
statistical and mathematical methods that are useful for the modelling and
analyzing engineering problems. In this technique, the main objective is to optimize
the response surface that is influenced by various parameters such as solid
loading, temperature, and extraction time. Response surface also quantifies the
relationship between the controllable input parameters and the obtained response
surface.

In this study, The Box Behnken Design (BBD) was employed for the
experimental design chosen with a fraction factorial for three independent
variables at three level or for finding out the relationship between the yield of
extraction and the three variables. This design will be a first option because
relatively few experimental combinations of the variables are acceptable to
estimate complex response function.

The various process parameters involved in total extraction yield of


Strobilanthes Crispus solid loading 30% and 500%, temperature 300oC and 400oC,

39
extraction time 15 and 25 minutes with the level code as -1 (low) and +1 (high)
respectively. Table 3.1 below shows the actual experimental parameters and the
coded experimental BBD level used.

Table 3.1: The actual experimental and the coded level used.

Coded Level
Parameters
Factor Low (-1) High (+1)
Solid Loading (%) A 30 50
Temperature (C) B 300 400
Extraction Time (min) C 15 25

RSM was applied to the experimental data using statistical software,


Minitab17. The statistical terms and their definitions used in the software are well
defined elsewhere. The linear and second order polynomials were fitted to the
experimental data to obtain the regression equations. The sequential F-test, lack
of fit test and other measures were used in selecting the best model.

The total extraction yield of Strobilanthes Crispus was determined by


using the equation below.

𝑚 𝑒𝑥𝑡𝑟𝑎𝑐𝑡
𝑌 𝑒𝑥𝑡𝑟𝑎𝑐𝑡 (%) = ( ) 𝑥 100
𝑚 𝑓𝑒𝑒𝑑

Where Yextract is percentage of extraction yield, mextract is the crude extract


mass (g) and m feed is the feed mass (g). The quadratic polynomial equation will
be as the response surface with the equation below.

Yield = A0 + A1X1 + A2X2 + A3X3 + A4X1X2 +A5X2X3 + A6X1X3 + A7X12


+ A8X22 + A9X32

Where, A0 is constant, A1, A2, and A3 is linear coefficients, A4, A5, and A6 is
cross product coefficients, A7, A8, and A9 is quadratic coefficients respectively. For
this three-level or three-factorial Box-Behnken experimental design, a total of 30

40
experimental runs are needed. The RSM is carried out from the design of
experiment (DOE). The parameters selected are inserted in Box-Behnken Design.
Then, the number of replicates was changed to two in order to get a better
spherical design and the value of the factors are inserted based on the table above.
After that, the different value of each factors will appeared on the new sheet along
with 30 standard runs. Figure below indicates the range of parameters that was
produced by Box-Behnken design using Minitab17.

Figure 3.6: The range of parameters By Boh-Behnken design using Minitab17

41
CHAPTER 4

EXPECTED RESULT

42
CHAPTER 5

CONCLUSION

43
RESEARCH PLAN

44
REFERENCES

Abu Bakar, M., Othman, F., & R Asmah. (2006). Effects of Strobilanthes crispus
Tea Aqueous Extracts on Glucose and Lipid Profile in Normal and
Streptozotocin-Induced Hyperglycemic Rats. ResearchGate, 7-12.
Afrizal. (2008). ANALYTICAL, BIOACTIVITY AND STABILITY STUDIES ON
STROBILANTHES CRISPUS L. BREMEK AND SONCHUS ARVENSIS L.
EXTRACTS. ANALYTICAL, BIOACTIVITY AND STABILITY STUDIES ON
STROBILANTHES CRISPUS L. BREMEK AND SONCHUS ARVENSIS L.
EXTRACTS AFRIZAL UNIVERSITI SAINS MALAYSIA, 34-35.
Afrizal Itam, Ismail, Z., & Abdul Majid, A. (2007). In Vitro studies of calcium
oxalate crystal growth inhibition of Strobilanthes Crispus extract.
Malaysian Journal of Pharmaceutical Science, 96-97.
Asmah Rahmat, & Mandana, B. (2009). IChemE. Supercritical carbon dioxide
extraction of bioactive flavonoid from Strobilanthes crispus (Pecah Kaca),
3.
Bakar, M. F. (2005). Effects of Strobzlanthes Crispus crued and tea in
streptozotocin included hyperglycemic rats. Universiti Putra Malaysia, 24-
25.
Corbett, A. (2014). Chapter 12 Molecular Composition of gases. Retrieved from
slideplayer.com: http://slideplayer.com/slide/266930/
Department of Pharmaceuticals Sciences. (2015). Retrieved from Universiti
Basel: https://pharma.unibas.ch/home/
Ghasemzadeh, A., Jaafar, h., & Rahmat, A. (2015). Phtochemical constituents
and biological activities of different extracts of Strobilanthes Crispus
Bremek Leaves grown in different locations of Malaysia. Research Article,
3-4.
GlobinMed. (2010). Retrieved from www.globinmed.com:
http://www.globinmed.com/index.php?option=com_content&view=article&i
d=83483:strobilanthes-crispus&catid=721:s
IDF. (2015). Retrieved from International Diabeted Federation Wstern PAaific:
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Kalish, M. (2010). Similar Characteristics in Rats & Humans. Retrieved from
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Leo M, L Nollet, & Toldra, F. (2012). Handbook of Analysis of Active Compounds
in Functional Food. eBook, 415-416.
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Compounds in Functional Foods. eBook, 415-416.
Lin, K. K. (2013). Strobilanthes crispus Tea - Teh Pecah Beling. Retrieved from
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http://www.sabahsnakegrassherb.com/2013/03/strobilanthes-crispus-tea-
teh-pecah.html
Liza Md Salleh, Russly Abdul Rahman, Jinap Selamat, Azizah Hamid, & Zaidul,
M. (n.d.). Optimization Of Extraction Condition for Supercritical Carbon
Dioxide (SC-CO2) Extraction of Strobilanthes Crispus (PEcah Kaca)
leaves by Response Surface Methodology. Food Processing and
Technology, 3-6.
Material Safety Data Sheet Ethanol. (2013, May). Retrieved from
www.sciencelab.com:
http://www.sciencelab.com/msds.php?msdsId=9923955
NIH. (2014, June). National Institute of Diabetes and Digestive and Kidney
Disease. Retrieved from www.niddk.nih.gov:
http://www.niddk.nih.gov/health-information/health-
topics/Diabetes/causes-diabetes/Pages/index.aspx
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articles-ppts-list.php
Rahul, B. (2013, January). Extraction of plant constituents. Retrieved from
Slideshare: http://www.slideshare.net/rahulbs89/extraction-of-plant-
contituents
Singh, J. (2012). Maceration, Percolation and Infusion techniques of extraction of
medicinal and aromatic plants. Central Institute of Medicinal and Aromatic
Plants (CIMAP), 7-8.
Suzan Mahdi, & Altikriti, Y. (2010). Extraction of Natural Products. Retrieved from
Biologiskt Aktiva Naturprodukter:
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70.htm
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http://wiki.ubc.ca/Course:FNH200/2011w_Team20_Vanilla

46
Wikipedia. (2009, February). Retrieved from
https://en.wikipedia.org/wiki/Strobilanthes_crispa
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https://en.wikipedia.org/wiki/Diabetes_mellitus
Yaacob, N. S., Hamzah, N., Nik Mohamed Kamal, N., Zainal Abidin, S., Choon
Sheen Lai, Viswwswaran Navaratnam, & Norazmi, M. (2010). Anticancer
activity of a sub-fraction of dichloromethane extract of Strobilanthes
Crispus on human breast and prostare cancer cells in vitro. BioMed
Central, 10-42.

47
APPENDICES

Table 1: Project cost estimation for Final Year Project 1

Materials and
Quantity Price
Equipments

Strobilanthes 1kg RM10


Crispus leaves

Ethanol 1500mL RM99

0.1% Formic 3500mL Rm139


acid

Acetonitrile 3500mL RM139

15 mL ¾” OD
Bulkhead 1 RM212.40
Union

¾” OD Cap 2 RM68.60

TOTAL RM668.00

48
Chemicals and Materials

Table 2: Table of chemicals/materials and apparatus

Method Chemicals / Materials Apparatus

 Scissors
Sample  Strobilanthes Crispus
 Glove
Collections leaves
and  Grinder
Preparations  Tap Water
 Air tight Bottles

 10 mL of measuring
Solvent  Ethanol cylinder
Preparations
 Beaker

 15 mL ¾” OD Bulkhead
 Ethanol Union (Swagelok)
 Strobilanthes Crispus  ¾” OD Cap (Swagelok)
Sample
Extraction samples  Furnace
 Tap Water  Thongs
 Universal Bottles

 Extracted Strobilanthes  High Performance Liquid


Crispus leaves Chromatography (HPLC)
Sample  0.1% formic acid  Syringe
Analysis
 Acetonitrile  HPLC Waste Bottles
 Distilled water  Gloves

49
Solvent Preparations

Ethanol

Molecular Weight, Mr = 58.08 g/mol

Critical Temperature, Tcritical = 516.15 K

Critical Pressure, Pcritical = 6.30 MPa (62.18 atm)

Density, ρ = 789 g/L

Gas Constants, R = 0.082057 L atm/mol.K

Volume of Reactor, Vreactor = 0.015 L

Table 3: Solvent Preparation for Ethanol


Temperature, oC Volume of Solvent, mL Solid Loading, % Mass of Sample, g
50 0.64
300 1.61
100 1.27
50 0.35
700 1.61
100 0.70

Example of Calculation

Ethanol

Molecular Weight, Mr = 58.08 g/mol

Critical Temperature, Tcritical = 516.15 K

Critical Pressure, Pcritical = 6.30 MPa (62.18 atm)

Density, ρ = 789 g/L

Gas Constants, R = 0.082057 L atm/mol.K

Volume of Reactor, Vreactor = 0.015 L

50
To find the volume of solvent

1. Find the number of mole of ethanol.

PV = nRT

(62.18 atm) (0.015L) = n (0.082057 L.atm/mol.K) (516.15 K)

n = 0.022 mol

By formula no. of mole, find the mass of ethanol.


𝑚𝑒𝑡ℎ𝑎𝑛𝑜𝑙
n=
𝑀𝑟𝑒𝑡ℎ𝑎𝑛𝑜𝑙

𝑚𝑒𝑡ℎ𝑎𝑛𝑜𝑙
0.022 mol =
58.08 𝑔/𝑚𝑜𝑙
methanol = 1.27 g

From density formula,


𝑚𝑒𝑡ℎ𝑎𝑛𝑜𝑙
ρ=
𝑉𝑒𝑡ℎ𝑎𝑛𝑜𝑙

1.27 𝑔
789 g/L =
𝑉𝑒𝑡ℎ𝑎𝑛𝑜𝑙

Vethanol = 1.61 L

2. To find mass of sample

At 50% Solid Loading


50
× 1.27 g ethanol = 0.64 g of sample.
100

51

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