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
ii
CHAPTER 4 ....................................................................................................... 42
EXPECTED RESULT ......................................................................................... 42
CHAPTER 5
CONCLUSION ................................................................................................... 43
REFERENCES ................................................................................................... 45
APPENDICES .................................................................................................... 48
Chemicals and Materials ................................................................................. 49
Solvent Preparations ....................................................................................... 50
Example of Calculation ................................................................................... 50
iii
List of Figures
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
List of Tables
iv
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.
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.
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.
9
CHAPTER 1
INTRODUCTION
Overview
10
1.1 Background of studies
11
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.
12
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.
13
1.2 Problems Statement
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)
14
Figure 1.1: The Prevalence of diabetes in adult by the age in Malaysia (IDF,
2015)
15
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.
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.
17
1.5 Objectives
18
CHAPTER 2
LITERATURE REVIEW
2.1.1 Flavonoid
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.
20
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
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
21
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.
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
22
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.
2.2.2.1 Drinks
23
Figure 2.3: Strobilanthes Crispus leaved sold in Malaysia (Lin, 2013)
24
on to wounds caused by the bites of poisonous snakes or other animals. (Afrizal,
2008)
Table 2.1: The Medical uses of Strobilanthes Crispus plants (GlobinMed, 2010)
Requirement Uses
Renal activity To treat kidney stones and its ability to erode renal stones.
25
Both fermented and unfermented leaves of Strobilanthes
Anti-diabetic activity
Crispus leaves was able to lower blood glucose level.
26
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%
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.
27
Figure 2.4: The islets contain beta cell cells in human organ (NIH, 2014)
28
2.4 Extraction Method
29
Figure 2.5: The ultrasonic-assisted extraction (University of British Columbia,
2012)
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)
30
2.4.2 Non-Conventional Extraction Method
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.
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.
Ethanol
Temperature: 243oC
Pressure: 62.18atm
34
3.2 Methodology
The details of the experiment from this study were briefly explained in this
section.
35
3.2.2 Solvent preparation
36
3.2.3 Sample Extraction
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
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.
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.
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
𝑚 𝑒𝑥𝑡𝑟𝑎𝑐𝑡
𝑌 𝑒𝑥𝑡𝑟𝑎𝑐𝑡 (%) = ( ) 𝑥 100
𝑚 𝑓𝑒𝑒𝑑
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.
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:
http://www.idf.org/membership/wp/malaysia
Kalish, M. (2010). Similar Characteristics in Rats & Humans. Retrieved from
eHow: http://www.ehow.com/info_8413774_similar-characteristics-rats-
humans.html
45
Leo M, L Nollet, & Toldra, F. (2012). Handbook of Analysis of Active Compounds
in Functional Food. eBook, 415-416.
Leo, M., Nollet, L., & Toldra, F. (2012). Handbook of Analysis of Active
Compounds in Functional Foods. eBook, 415-416.
Lin, K. K. (2013). Strobilanthes crispus Tea - Teh Pecah Beling. Retrieved from
http://www.sabahsnakegrassherb.com/:
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
NPCR . (2016, September). Retrieved from http://www.omicsonline.org/:
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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
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70.htm
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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
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47
APPENDICES
Materials and
Quantity Price
Equipments
15 mL ¾” OD
Bulkhead 1 RM212.40
Union
¾” OD Cap 2 RM68.60
TOTAL RM668.00
48
Chemicals and Materials
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
49
Solvent Preparations
Ethanol
Example of Calculation
Ethanol
50
To find the volume of solvent
PV = nRT
n = 0.022 mol
𝑚𝑒𝑡ℎ𝑎𝑛𝑜𝑙
0.022 mol =
58.08 𝑔/𝑚𝑜𝑙
methanol = 1.27 g
1.27 𝑔
789 g/L =
𝑉𝑒𝑡ℎ𝑎𝑛𝑜𝑙
Vethanol = 1.61 L
51