SCREENING FOR HIGH KAEMPFEROL CONTENT FROM DIFFERENT SPECIES
OF MALAYSIAN MEDICINAL PLANTS
KHAIRUNNISA BINTI ZAINOL ABIDIN
UNIVERSITI TEKNOLOGI MALAYSIA
SCREENING FOR HIGH KAEMPFEROL CONTENT FROM DIFFERENT SPECIES
OF MALAYSIAN MEDICINAL PLANTS
KHAIRUNNISA BINTI ZAINOL ABIDIN
A dissertation submitted in partial fulfillment of the
requirements for the award of the degree of
Master of Science (Biotechnology)
Faculty of Biosciences and Bioengineering
Universiti Teknologi Malaysia
DECEMBER 2010
iii
To my beloved parents, brothers and sister
iv
ACKNOWLEDGEMENTS
In preparing this thesis, I was in contact with many people that have contributed
to my understanding and thoughts. In particular I wish to express my sincere
appreciation to my supervisor, Dr. Azman Bin Abd. Samad for his advice and guidance.
I am also very thankful to Mr Ali Bin Mokhti and his wife for sharing their knowledge
in traditional medicine.
I also would like to thank research assistant Miss Nor’ain Binti Abdul Rahman,
senior postgraduate students Miss Aslamiah Binti Sarju and Miss Fadhilah Binti Mis for
their help and assistance during my research in Plant Tissue Culture Laboratory. Not to
forget all the staff at Universiti Teknologi Malaysia that has been helping me during this
research period.
Last but not least, I would like to extend my sincere appreciations to my family
and all my colleagues who have provided assistance at various occasions.
v
ABSTRACT
The flavonoids such as kaempferol that have anti-cancer properties are widely
distributed in plants kingdom. The purpose of this study was to screen high kaempferol
content in the selected medicinal plants and determine the kaempferol distribution in
different plant organ. Kaempferol content in mature leaf of twenty two randomly
selected Malaysian medicinal plants were determined using gas chromatography (GCFID). A commercial kaempferol was used as a standard (positive control). It was
discovered that the highest kaempferol content was detected in Tibouchina
semidecandra (4689.75 654.83 mg/kg) while the mature leaf contained higher
kaempferol than young leaf and shoot. However, no kaempferol was detected in young
stem of T. semidecandra. Therefore, this study suggests T. semidecandra as an
alternative source for kaempferol.
vi
ABSTRAK
Flavonoid seperti kaempferol bersifat anti-kanser serta mempunyai taburan yang
luas di alam tumbuhan. Kajian ini bertujuaan menyaring kandungan kaempferol tertinggi
didalam tumbuhan ubatan terpilih dan menentukan taburan kaempferol didalam organ
tumbuhan ubatan yang berbeza. Kandungan kaempferol didalam daun matang daripada
sejumlah dua puluh dua tumbuhan ubatan Malaysia yang dipilih secara rawak telah
disaring menggunakan kromatografi gas bersama pengesan nyalaan ion (GC-FID).
Kaempferol komersil dijadikan piawaian (penujuk positif). Hasil kajian menunjukkan
kandungan
kaempferol
tertinggi
dikesan
didalam
Tibouchina
semidecandra
(4689.75 654.83 mg/kg) manakala daun matang mengandungi kaempferol tertinggi
berbanding daun muda dan pucuk. Walaubagaimanapun, kaemperol tidak dapat dikesan
didalam batang. Oleh itu, kajian ini mengesyorkan T. semidecandra sebagai sumber
alternatif bagi kaempferol.
vii
TABLE OF CONTENTS
CHAPTER
TITLE
DECLARATION
ii
DEDICATION
iii
ACKNOWLEDGEMENTS
iv
ABSTRACT
v
ABSTRAK
vi
TABLE OF CONTENTS
vii
LIST OF TABLES
x
LIST OF FIGURES
xi
LIST OF ABBREVIATIONS
1
PAGE
xiii
INTRODUCTION
1
1.1 General Introduction
1
1.2 Statement of Problem
4
1.3 Objective of Research
4
1.4
5
Scope of Research
viii
2
3
LITERATURE REVIEW
6
2.1 Medicinal Plants
6
2.2
Flavonoids
21
2.2.1 Kaempferol
25
2.3 Gas Chromatography-Flame Ionized Detector
26
METHODOLOGY
28
3.1 Material
28
3.1.1 Plant sources
28
3.1.2 Chemicals
31
3.2 Method
31
3.2.1 Preparation of Standard
31
3.2.2 Medicinal plants extraction
32
3.2.3 GC-FID analysis
32
3.2.4
33
Quantification of kaempferol
3.2.5 Statistical analysis
34
ix
4
RESULT AND DISCUSSION
35
4.1
35
Screening of medicinal plants with highest
kaempferol content
4.2
5
Distribution of kaempferol in plant organ
CONCLUSION
42
44
FUTURE RESEARCH
45
REFERENCES
46
APPENDIX
51
x
LIST OF TABLES
TABLE NO.
TITLE
PAGE
2.1
The major flavonoids classes (Cook & Samman, 1996)
23
3.1
Selected Malaysian Medicinal Plants
29
4.1
Kaempferol distribution within in plant families
37
4.2
Kaempferol content in different organ of
43
Tibouchina semidecandra
xi
LIST OF FIGURES
FIGURE NO.
TITLE
PAGE
2.1
The generic structure of flavanoids (Coultate, 1990)
21
2.2
Phenylpropanoid biosynthesis leading to flavanones,
22
flavonols, isoflavones, anthocyanins, proanthocyanidins,
lignin and chlorogenic acid (Dixon, 2005)
2.3
Chemical structure of kaempferol
26
2.4
Diagram of Gas Chromatography-Flame Ionized Detector
27
(Zainab, 2009)
4.1
Kaempferol (mg/kg) concentration detected in selected
36
Malaysian medicinal plants
4.2
GC-FID chromatogram of
(a)
kaempferol commercial standard
(b)
T. Semidecandra
(c)
R. tomentosa
(d)
A. hispida
(e)
J. podagrica
39
xii
FIGURE NO.
TITLE
4.2
GC-FID chromatogram of
(f)
A. conchigera
(g)
E. anguliger
4.3
Distribution of kaempferol in organ of Tibouchina
semidecandra.
PAGE
39
43
xiii
LIST OF ABBREVIATIONS
GC-FID
-
Gas Chromatography-Flame Ionized Detector
GC-MS
-
Gas Chromatography-Mass Spectrophotometer
HPLC
-
High Performance Liquid Chromatography
SEM
SPSS
-
Standard Error Mean
Statistical Package Social Sciences
CHAPTER 1
INTRODUCTION
1.1
General Introduction
Malaysia is among the most biologically diverse countries in the world. There
are approximately 12,500 species of flowering plants, including species of valuable and
marketable timber and fruit. Plants that produce natural products such as secondary
metabolites confer adaptive benefits to plants and human. Important secondary
metabolites such as anthocyanin could serve as natural food colorant and shelf life
indicator for acidic food (Janna et al., 2006).
2
Medicinal plant is defined as a plant which has been proven or claimed or
thought to have medicinal remedies (Majid et al., 1995). In Peninsular Malaysia, there
are about 1,200 species of higher plants which have been reported to have medicinal
properties for treatment of various diseases and ailments (Jantan, 1998). However, so far
only about hundred have been investigated fully for their medicinal potential (Jamal et
al., 2010; Ismail, 2001).
The Orthosiphon stamineus extract is widely used in Malaysia as drug for the
treatment of diabetes and kidney stones. Traditionally, it was consumed as herbal tea to
facilitate body detoxification. More than 50 components including flavonoids, terpinoids
and caffiec acid derivatives with different biological activity were successfully isolated.
Majority of the flavonoid were obtained from the leaves (Hossain and Ismail, 2011) and
the major bioactive compound was rosmarinic acid that is caffiec acid derivatives
(Akowuah et al., 2005).
An enormous variety of secondary metabolites such as alkaloid, terpenes,
saponines, quinones and polyphenols are synthesized by plants to serve as a chemical
defense against herbivores and microbial attack (Wink, 1988), toxicant and repellent for
insects, and attractants for pollinators (Close and McArthur, 2002). For example, the
Radopholus similis (nematode) resistance in Musa cultivars (banana) was caused by
flavan-3,4-diols and condensed tannin ( Collingborn et al., 2000).
3
Flavonoids are potent antioxidants, free radical scavengers, and metal chelators
and lipid peroxidation inhibitor. They have been reported to exhibit a wide range of
biological effects, including antibacterial, antiviral, (Hanasaki et al., 1994) antiinflammatory, antiallergic, (Middleton, and Kandaswami, 1993; Hanasaki et al., 1994;
Hope et al., 1983) and vasodilatory actions (Duarte et al., 1993). In addition, flavonoids
inhibit lipid peroxidation (LPO) (Salvayre et al., 1988) platelet aggregation, (Tzeng et
al., 1991; Robak et al., 1988) capillary permeability and fragility (Torel et al., 1986;
Budavari et al., 1989) and the activity of enzyme systems including cyclo-oxygenase
and lipoxygenase (Hope et al., 1983; Middleton, and Kandaswami, 1993; Hodnick et al.,
1988). For example, baicalin isolated from Scutellaria baicalensis that have been used in
Chinese and Japenese medicine inhibit HIV-1 replication (Li et al., 1993).
Kaempferol is a flavonoid classified as flavonol that widely distributed group of
polyphenolic compounds characterized by a common benzo-pyrone structure. Over
4,000 different flavonoids have been identified and distributed in the leaves, seeds, bark
and flowers of plants. For example, the leaves of Centella asiatica which belong to
Umbelliferrae family also known as “Pegaga” in Malay has been reported to contain
high kaempferol content besides other flavonoids such as naringin, rutin, quercetin,
catechin, apigenin and luteolin (Mohd Zainol et al., 2009). Therefore, this study was
initiated to screen high kaempferol producing plants in mature leaves because previous
study shows that flavonol act as antioxidant in chloroplast of spinach (Hernandez et al.,
2009; Takahama, 1984).
4
1.2
Statement of problem
The potential of high flavonoids producer in Malaysian medicinal plants was not
fully explored although extensive studies of bioactive compounds and their total
phenolic content in many species have been carried out. Besides that, there was lack of
research that focus on identification of kaempferol despite diverse plant secondary
metabolites and its wide distribution in plants. In addition, the plant secondary
metabolites such as flavonoids vary in type and quantity due to duration of stress
exposure and plant species. Therefore, screening of medicinal plants with high flavonoid
content particularly kaempferol and its distribution in plant organs will elucidate the
biochemical nature of bioactive compounds.
1.3
Objectives of research
The objectives of this study are to:

screen plant with high kaempferol content from selected Malaysian medicinal
plants

determine distribution of kaempferol in different plant organs of T. semidecandra
5
1.4
Scope of research
The scope of my research includes screening of kaempferol in twenty two
randomly selected Malaysian medicinal plants mature leaves using Gas Chromatography
with Flame ionized detector (GC-FID). The analysis was done in triplicate and repeated
three times. The mature leaf was selected because kaempferol is a flavonoid classified in
flavonol group that act as antioxidant in chloroplast (Hernandez et al., 2009) and
preliminary study showed that the mature leaf of Justicia gendarussa exhibit highest
antioxidant activity (Yani, 2009). The other plant organ such as shoot, young leaf and
young stem of identified plant with highest kaempferol content was screen for highest
organ producer and all the data were analyzed using Statistical Package for the Social
Sciences (SPSS).
CHAPTER 2
LITERATURE REVIEW
2.1
Medicinal Plants
Traditional knowledge of medicinal plants has always guided the search for new
cures. In spite of the advent of modern high throughput drug discovery and screening
techniques, traditional knowledge systems have given clues to the discovery of valuable
drugs (Buenz et al., 2004). The traditional medicinal plants include forest plants, weed,
fruit plants, vegetables, spices, ornamental plants and other class of plants. Although
many of these are known as medicinal plants it may also use as non-medicinal purpose.
Even among the known medicinal species there are variations and differences in the
method of utilization and ailments treated (Ong and Nordiana, 1999).
7
Traditional medicinal plants are often cheaper, locally available and consumable,
raw or as simple medicinal preparations. Nowadays, traditional medicinal practices form
an integral part of complementary or alternative medicine. Although their efficacy and
mechanisms of action have not been tested scientifically in most cases, these simple
medicinal preparations often mediate beneficial responses due to their active chemical
constituents (Park and Pezzutto, 2002; Survesvaran et al., 2006).
2.1.1
Strobilanthes crispus
This medicinal plant (Appendix-Figure 1a) is commonly known as “Pecah Kaca”
in Malay due to the crushed leaves sound that resembles breaking of the glass. It belongs
to family Acanthaceae which native to tropical countries like Madagascar and Malay
Archipelago. Strobilanthes crispus can be found on riverbanks and abandoned fields.
The leaves are oblong-lancelet, rather abtuse and shallowly crenate-crispate (Liza et al.,
2007). Traditionally, the leaves of Strobilanthes crispus were used as antidiabetic,
diuretic, antilytic, laxative and have been proven scientifically to poses high antioxidant
activity, anti-AIDS, and anti-cancer properties. It is commonly consumed in the form of
herbal tea (Mohd Fadzelly et al., 2006). The Strobilanthes crispus leaf extract contains
several flavonoids such as quercetin, naringenin, and kaempferol (Liza et al., 2007).
However, the flavonoids detected in Strobilanthes crispus leaf extract vary due to
extraction method (Liza et al., 2007).
8
2.1.2
Wrightia religiosa
This plant (Appendix-Figure 1b) is called “Anting Puteri” in Malay which
literally means “Princess’s Earrings” due to the flower morphology and “Water
Jasmine” in English. It is a very fragrant fruity scented, enchanting shrub, pendant and
born along its twiggy branches that native to tropical region. The foliage is thin and
slightly hairy and it can flower almost all year. Wrightia religiosa is a popular
ornamental plant which belongs to family Apocynaceae. The plants size and shape can
easily prune to create unique shape bonsai tree within short period of time due to its fast
growth rate. The root was used to cure skin disease in traditional medicine.
2.1.3
Polyscias scutellaria
Polyscias scutellaria (Appendix-Figure 1c) belongs to Araliaceae family that
grows in Pasific countries including Malaysia. It was known as “Puding Mangkuk” in
Malay due to the bowl shape of the leaves. Traditionally, the water from boiled roots
was drank to stimulate sweating and urination. Besides that, it was also used to cure
constipation by drinking of the leaves decoction three times per day (Ong and Nordiana,
1999). Other than that, Polyscias scutellaria also have been used in culinary such as in
“Kerabu” and “Botok-botok” and as an ornamental plant. Today, it has been used as
anti-inflammatory drug (Paphassarang et al., 1989). Previous studies identified seven
triterpenic glycosides including new triterpenic glycosides: saponin C (Paphassarang et
al., 1989) in the leaves.
9
2.1.4
Epiphyllum anguliger
This plant (Appendix-Figure 1d) is distributed geographically from Southern
America to Southeast Asia belong to Cactaceae family and was known as “Bakawali” in
Malay, “Ric Rac Cactus”, “Moon Cactus” and “Queen of the Night in English. It poses
an exotic flower with strong scent that only bloom during full moon for certain period of
time. The flower scent was the strongest when it fully blooms at 12.00 a.m but it only
stay until dawn. Epiphyllum anguliger release its pollen to the air when it blooms. It was
used in Malay traditional medicine as eye cleanser and refresher, to balance the facial
skin tone, mind and body refresh. Some people believe that the bloomed flower was
guard by mystical people, whereas some believe that the bloomed flower will bring
wealth and fortune to the owner, and there are some that believe the flower have close
relation with its owner which means if the owner dies it will also dies. The flower was
also claimed to have anti-cancer effect by consume as salad.
2.1.5
Gynura procumbens
Gynura procumbens (Appendix-Figure 1e) also known as “Sambung Nyawa”
and “Akar sembiak” belong to family Compositae is an annual evergreen shrub with
fleshy stem that is widely used in Southeast Asian particularly Indonesia, Malaysia, and
Thailand. In the traditional treatment, it was used to treat many ailments such eruptive
fevers, rash, kidney disease, diabetes mellitus and hyperlipidemia.
10
Pharmacological studies indicate that it has anti-herpes simplex virus (Nawawi et al.,
1999), anti-hyperglycaemic (Akowuah et al., 2001), anti-inflammatory (Iskander et al.,
2002), anti-hyperlipidaemic (Zhang and Tan, 2000) and blood hypertension reduction
capabilities (Lam et al., 1998; Kim et al., 2006). The benefits of the traditional use of
Gynura procumbens have also been supported by the isolation and identification of
several possible active chemical constituents from this plant, including flavonoids,
saponins, tannins, and terpenoids (Akowuah et al., 2002)
2.1.6
Acalypha hispida
Acalypha hispida (Appendix-Figure 1f) is one of plant that belongs in the family
Euphorbiaceae selected in this study. In Malaysia, Acalypha hispida is commonly
known as “Ekor Kucing”’ that literally means cat’s tail due to the furry flowers which
resembles a tail of a cat. Leaves and fruits of Acalypha hispida are green while the
flowers are red. The flowers are chewable fresh together with a mixture of curry leaves
and young betel nuts as remedy for blood vomiting, intestine inflammation and small
wounds, the water of boiled root can be drank to treat blood cough while the leaves are
effective to treat skin disease and leprosy. Previous studies indicate that Acalypha
hispida mostly composed of phenolic, saponin, and flavonoid compounds. However,
there are also glycoside, steroid, and phlobatannins presence in the plant. Acalypha
hispida is typically uses for treatment of arthrosclerosis, cancer, obesity, and anti-aging
(Iniaghe et al, 2009).
11
2.1.7
Acalypha indica
Acalypha indica (Appendix-Figure 1g) is famously known as “Kucing Galak” in
Malay and “Indian acalypha” or “Oliver” in English belongs in family Euphorbiaceae.
Acalypha indica is used in traditional Malay medicine to treat gastrohelcosis, lung
infection and scaly skin ailment. Besides that, it has been reported used for treatments of
bronchitis, asthma, pneumonia, and scabies (Azmahani et al, 2002). Previous research
indicates that it can act as anti-fertility agent in rats and has potential to be applied in
humans (Hiremath et al, 1998). Recent studies identified chemical constituents of
Acalypha indica were acalyphamide, aurantiamide, alkaloids, kaempferol, and tannins
(Rahman et al, 2010).
2.1.8
Jatropha podagarica
In Malaysia, this plant is known as “Jarak Tapak Gajah” (Appendix-Figure 1h)
and widely grown as an ornamental plant. Jatropha species which belong to family
Euphorbiaceae are well known for their medicinal value. Many species from this genus
have been reported to be widely used in traditional medicine especially in the treatment
of malaria (Baraguey et al., 2000), arthritis, gout, jaundice (Nengchomnong et al.,
1986), ulcers (Evans et al., 1983), and cancer (Manandhar, 1989). Previous study have
been successfully isolated 8-hydroxy-6,7-dimethoxycoumarin, acetylaleuritolic acid and
γ-sitosterol from Jatropha podagarica that were cytotoxic towards cervical carcinoma
cell line (Ee et al., 2005).
12
2.1.9
Pedilanthus tithymaloides
This plant (Appendix-Figure 1i) possess many common names including “Getah
lelipan” in Malay, “Devil’s backbone”, “Fiddle flower”, and “Slipper plant” in English.
Those names are given due to its distinctive erect leaves that are arranged oppositely on
stem, resembling a ladder or backbone or centipede. The leaves of Pedilanthus
tithymaloides are green, glabrous, and acuminate whilst its flowers are bright red. In
traditional medicine, latex sap was used as pain reliever (analgesic) from centipede and
scorpion venomous bites and also used to remove skin rashes. Previous studies provide
scientific support of Pedilanthus tithymaloides usage as anti-inflammatory agent (Abreu
et al, 2006), antimicrobial and antifungal in traditional medicine and successfully
identified constituents of Pedilanthus tithymaloides which was terpenoids and long
chain alcohols. Examples of chemical compound found in Pedilanthus tithymaloides are
pyrogallol, nicotinamide, and proline (Vidotti et al, 2006).
2.1.10 Phyllanthus amarus
In Malaysia, this plant (Appendix-Figure 1j) which belongs to family
Euphorbiaceae was known as “Dukung Anak”. All part of the plant is used in traditional
medicine. The water mixture from the boiled plant used to relieve diarrhea (Ong and
Nordiana, 1999) and reduce high blood pressure while the sap liquid obtained from
pressing the leaves is used to heal the wound.
13
Besides that, the water mixture was also effective to cure diabetes. Modern research has
found that it can be used to cure hepatitis B by suppressing the growth and replication of
the viruses (Yeh et al., 1993: Jayaram and Thyagarajan, 1996: Lee et al., 1996).
2.1.11 Coleus atropupureus
It (Appendix-Figure 1k) has many binomial names including Coleus blumei,
Solenostemon scutellariodes, and Ocimum scutellariodes. Besides, Coleus atropupureus
also has several common names such as “Painted nettle”, “Flame nettle”, and “Ati-ati
Merah”. This plant produces colorful leaves with small flowers and can grow up to one
meter. Leaves of Coleus atropupureus are oval-shaped with rounded tooth edges while
the colors varied from maroon, dark-red, brown, and blue. It was used in Malaysian
traditional medicine to reduce high blood pressure and stop bleeding (Ong and Nordiana,
1999). Other than that, this plant is widely used as ornamental plants for houses and
buildings due to the attractive colors of its leaves. Previous study discovered the major
constituent of Coleus atropupureus was caffeic acid called rosmarinic acid (Bauer et al,
2004).
14
2.1.12 Hibiscus rosa-sinensis (white variety)
The red variety of Hibiscus rosa-sinensis is an official flower of Malaysia
belongs to family Malvaceae. It is a woody shrub with large varieties of flower that
distributed geographically in a pan-Pacific tropical area. All varieties are known to have
special medicinal properties. The white variety (Appendix-Figure 1l) which is known as
“Bunga Raya Putih” was usually used in Malay traditional medicine for example to
reduce high blood pressure by drinking the decoction of the flower (Ong and Nordiana,
1999). Besides that, the inflorescence was also been used to cure stomachache. The
ethanol flower extract of Hibiscus rosa-sinensis show anti-diabetic effect (Sachdewa and
Khemani, 2003). It was reported to contain flavonoid that associated with antioxidant,
antipyretic, analgesic and spasmolytic and also saponin that enable reduction of blood
pressure and cholesterol (Krishnaiah et al., 2009).
2.1.13 Tibouchina semidecandra
The local name of Tibouchina semidecandra (Appendix-Figure 1m) is
“Senduduk Biru” or “Senduduk Baldu” and its belong to family Melastomataceae which
comprise approximatelay 4,500 species in less than 200 genera distributed in tropical
and subtropical region (Abdullah and Yong, 2007). This plant was used for both
medicinal and food purpose (Janna et al., 2006; Zakaria and Ali Mohd, 1994). The
flower pigment consist of anthocynin which is water soluble and non-toxic and reported
to be safe as dietary supplements (Bride et al., 1997).
15
Besides that, the leaves extract of the plant contain flavonoids such as quercetin,
quercetin 3-О-α-ʟ (2”-О-acetyl) arabinofuranoside, avicularin, and quercitrin (Sirat et
al., 2010).
2.1.14 Rhodomyrtus tomentosa
This plant (Appendix-Figure 1n) which native in Southeast Asia was known as
“Kemunting” in Peninsular Malaysia and “Rose Myrtle” in English belong to family
Myrtaceae. It leaves were leathery glossy green above and densely grey or rarely
yellowish-hairy beneath. The raw ripe fruits are consumed to treat diarrhea (Ong and
Nordiana, 1999) while the roots and leaves were used as ingredient in boiling stock to
reduce high blood pressure and as treatment for women after childbirth. The leaves paste
is used to treat sores and headache. Recently, an antibacterial drug called rhodomyrtone
from this plant was reported to have powerful in vitro activity against a broad range of
Gram-positive bacteria including antibiotic resistant strains (Kayser et al., 2009).
16
2.1.15 Cynbopogon nardus
Cymbopogon nardus (Appendix-Figure 1o) is commonly known as “Citronella
grass” in Taiwan, “Naid grass” in India, and “Serai Wangi” in Malaysia. This is another
variety of grass from family Poaceae. Cymbopogon nardus is not being consumed
because it is unpalatable to human and some animals such as cow. Hence, it is known
more as unwanted grass since it can cause loss of animal population wherever it grows.
Nevertheless, a few other animals like buffalo and elephant did consume it but only for
desperate times where there is lack of food especially during dry seasons. Therefore,
Cymbopogon nardus is more famous as an ingredient of aromatic oils rather than as
cooking element. Cymbopogon nardus grows easily throughout the world including
south and north-east Africa and Asia. It was used to make anti-dandruff shampoo and
crushed leaved were place in bath water to invigorate the body and also to get rid of
body odours (Ong and Nordiana, 1999). Since it is unpalatable, there is not much
research and analysis about its phytochemicals and medicinal uses.
2.1.16 Imperata cylindrica
Imperata cylindrica (Appendix-Figure 1p) is a grass type that belongs in the
grass family Poaceae, together with other grasses like lemongrass and citronella grass.
This grass is commonly called “Kunai grass” or “Congo grass” in English while “Lalang
Hitam” in Malay. It has similar morphology to both lemongrass and citronella grass
except the different color of its leaves, which is purple.
17
Imperata cylindrica is distributed throughout Southeast Asia, India, Australia, and
Africa. It is a weed that can easily grows on disturbed grounds such as near roadsides,
building sites, and timber harvesting sites. However, Imperata cylindrica is not just a
useless weed because it can be used to treat several diseases like febrifuge, diuretic,
diabetes (Villasenor et al., 2006) and headache by drinking the rizom decoction with
sugar (Ong and Nordiana, 1999). These medicinal properties of Imperata cylindrica
existed because phytochemical studies indicate that this plant possesses active
compounds including tannins, saponins, and flavonoids (Krishnaiah et al., 2009).
2.1.17 Ruta angustifolia
This plant (Appendix-Figure 1q) which belongs to family Rutaceae is called
“Geruda” in Malay. The crushed leaves produces unpleasant aroma. It was used to cure
high fever in traditional medicine. A new natural coumarin, angustifolin was isolated
from the aerial parts of Ruta angustifolia besides the other two coumarins (scoparone
and 6,7,8-trimethoxycoumarin) and the alkaloid graveoline (Del Castillo et al., 1984)
while dictamnine and γ-fagarine were isolated from the root (Visudevan and Luckner,
1968, Kong et al., 1984). In addition, graveoline was also contained in the root and
whole plant extract (Visudevan and Luckner, 1968; Ulubelen and Terem, 1988). The
graveoline show antibacterial effect on Gram-negative bacteria.
18
2.1.18 Cestrum nocturnum
This evergreen woody shrub belongs to family Solanaceae that is native to
tropical and sub tropical region. The leaves are simple, smooth and glossy, narrow
lanceolate with an entire margin. The flowers are greenish-white with a slender tubular
corolla that contains five acute lobes produced in cymose inflorescences at night. It
(Appendix-Figure 1r) was grown as ornamental due to the strong-scented flower. It was
known as “Sedap Malam”, “Harum Sundal Malam” and “Seri Pekan” in Malay while it
is commonly known as “Water Jasmine” and “Lady of the Night” in English because the
fragrance becomes intoxicating at night. This plant was used as one of the ingredients in
Malay traditional talc called “Bedak Rambai” that have been used to remove the scar
and make the skin look finer. It was and also used as ingredient in perfume due to its
strong scent. The plants extract of Cestrum nocturnum studied shows larvacidal activity
on Aedes aegypti (Jawale et al., 2010) and antitumor effect (Zhong et al., 2008). It also
contain steroidal glycosides in its leaves (Mimaki et al., 2002)
2.1.19 Alpinia conchigera
Alpinia conchigera (Appendix-Figure 1s) is herbaceous perennial 2-5 ft tall,
found in Eastern Bengal and southward towards Peninsular Malaysia and Sumatera
(Smith, 1990; Larsen et al., 1999). It is known as “Lengkuas Padi”, “Lengkuas Padang”,
Lengkuas Ranting”, “Lengkuas Geting” and etc in Malay.
19
It has been used as a condiment in northern state of Peninsular Malaysia and
occasionally in traditional medicine in east coast to treat antifungal infections (Ibrahim
et al., 2000). Previous study show that the 1’-(S)- 1’-Acetoxychavicol acetate (ACA)
isolated from Alpinia conchigera induce cytotoxicity on tumor cell lines but no adverse
cytotoxic effect on normal cell (Awang et al., 2010). Besides that it shows
antinociceptive and anti-inflammatory effect (Sulaiman et al., 2010).
2.1.20 Alpinia purpurata
This plant (Appendix-Figure 1t) is also classified under Zingiberaceae family
and it is native to Southeast Asia. The local called it “Halia Merah” which means “Red
Ginger in English because it poses a red colour flower that suitable to be use as an
ornament. It is used in Malay traditional medicine as one of the ingredient in mixture of
herb for the treatment of after childbirth. Its rizom contain unstable labdane diterpenes,
labda-8(17), 12-diene-15, 16-dial and alkaloid piperine (Sirat and Rizal Liamen, 1995).
20
2.1.21 Kaempferia pulchra
This plant (Appendix-Figure 1u) is another Zingiberaceae family member that
has been selected known as “Cekur Hutan” in Malay and “Peacock Ginger” in English it
was geographically distributed from India to Southeast Asia. In Malay traditional
medicine, it was used to cure skin diseases and to stabilize the body temperature after
childbirth. Recent study showed that crotepoxide (a substituted cyclohexane diepoxide)
isolated from Kaempferia pulchra linked to antitumor and anti-inflammatory activities
synthesized tumor cell to cytokines and chemotherapeutic agent (Prasad et al., 2010).
Besides that, pimarane (Tuchinda et al., 1994) and diterpenes (Sematong et al., 1998)
have been isolated from this plant.
2.1.22 Zingiber zerumbet
Zingiber zerumbet (Appendix-Figure 1v) is locally known as “Halia Hutan” in
Malay which literally mean wild ginger or also known as “Lempoyang”. It is the most
common ingredient in Malaysian traditional medicine such as health supplement and
tonic. Zingiber zerumbet belong to family Zingiberaceae which among the most prolific
plants in tropical rainforests. In Southeast Asian countries, several species are commonly
used as spices, medicines, flavouring agents as well as the source of certain dyes
(Burkill, 1996). This plant was eaten as an appetizer or to cure stomachache (Ruslay et
al., 2007). Its rhizome contains zerumbone that can inhibit proliferation of cervical, skin
and colon cancer cells lines (Taha et al., 2010).
21
2.2
Flavonoids
Flavonoids are a widely distributed group of polyphenolic compounds
characterized by a common benzo-pyrone structure as show in the Figure 2.1 Over 4,000
different flavonoids have been identified were widely distributed in the leaves, seeds,
bark and flowers of plants. The flavonoids diversity was due to complex and diverse
secondary metabolic processes as shown in Figure 2.2. They occur naturally in fruits and
vegetables, mainly as flavonoid glycosides, and are thus important constituents of the
human diet (Semalty et al., 2010). In plants, these compounds afford protection against
ultraviolet radiation, pathogens, and herbivores (Harborne and Williams, 1992). The
major flavonoid classes include flavonols, flavones, flavanones, catechins (or flavanol),
anthocyanidins, isoflavones, dihydroflavonols, and chalcones as shown in Table 2.1.
Figure 2.1: The generic structure of flavanoids
(Coultate, 1990)
22
Figure 2.2: Phenylpropanoid biosynthesis leading to flavanones, flavonols, isoflavones,
anthocyanins, proanthocyanidins, lignin and chlorogenic acid (Dixon, 2005).
23
Table 2.1: The major flavonoids classes (Cook & Samman, 1996)
Class and General Structure
Flavonoids
Substitution pattern
Flavonol
Kaempferol
3,5,7,4’-OH
Quercertin
3,5,7,3’,4’-OH
Myricetin
3,5,7,3’,4’,5’-OH
Tamarixetin
3,5,7,3’-OH,4’-OMe
Chrysin
5,7-OH
Apigenin
5,7,4’-OH
Rutin
5,7,3’,4’-OH,3-rutinose
Luteolin
5,7,3’,4’-OH
Luteolin
5,7,3’-OH,4’-glucose
glucosides
5,4’-OH,4’,7-glucose
Naringin
5,4’-OH,7-rhmnoglucose
Naringenin
5,7,4’-OH
Taxifolin
3,5,7,3’,4’,-OH
Eriodictyol
5,7,3’,4’-OH
Hesperidin
3,5,3’-OH,4’-OMe,7-rutinose
Leucocyanidol
3,4,5,7,3’,4’-OH
(+)-Catechin
3,5,7,3’,4’-OH
(+)-Epicatechin
3,5,7,3’,4’-OH
Flavone
Flavanone
Catechin
24
Anthocyanidin
Isoflavone
Dihydroflavonol
Chalcone
Apigenidin
5,7,4’-OH
Cyanidin
3,5,7,4’-OH,3,5-OMe
Genistin
5,4’-OH,7-glucose
Genistein
5,7,4’-OH
Daidzin
4’-OH,7-glucose
Daidzein
7,4’-OH
Taxifolin
3,5,7,3’,4’-OH
Fustin
3,7,3’,4’-OH
Butein
3,4,4’,6-OH
Phloretin
4,2’,4’,6’-OH
Phloridzin
4’,2’,4-Oglucose
25
2.2.1
Kaempferol
Kaempferol is synonyms as campherol, indigo yellow, nimbecetin and 3,4’,5,7tetrahydroxyflavone. Its IUPAC name is 3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1benzopyran-4-one with molecular formula C15H10O6 and chemical structure as shown in
Figure 2.3. Other than that it is also known as robigenin, pelargidenolon, rhamnolutein,
rhamnolutin, populnetin, trifolitin, kempferol and swartziol. It can be isolated from
various natural sources including apples, onions, leeks, citrus fruits, grapes, red wines,
green tea seeds (Park et al., 2006) and saffaron petals (Hadizadeh et al., 2003).
Kaempferol is a strong antioxidant and help to prevent oxidative damage to our
cell, lipids and DNA. It seems to prevent arteriosclerosis by inhibiting the oxidation of
low density lipoprotein and the formation of platelets in the blood. Studies have also
confirmed that kaempferol acts as a chemopreventive agent, which means that it inhibit
the formation of cancer cells. Besides that, it can reduce the risk of pancreatic cancer
(Nöthling et al., 2007) and enhances the effect of cisplatin through down regulation of
cMyc in promoting apoptosis of ovarian cancer cells (Luo et al., 2010).
26
Figure 2.3: Chemical structure of kaempferol
2.3
Gas Chromatography-Flame Ionized Detector (GC-FID)
Chromatography is a powerful analytical method suitable for the separation and
quantitative determination of considerable number of compounds even from complicated
matrix. The flame ionization detector is the most widely used GC detector and it respond
to all organic compounds that burn in the oxy-hydrogen flame. Analytical method used
for identification and quantification of kaempferol was Gas chromatography-flame
ionized detector (GC-FID) as shown in Figure 2.4.
27
Gas chromatography with flame ionized detector (GC-FID) is one of the most
efficient chromatographic techniques for separating volatile mixtures. Identification is
based on direct comparison of retention time with standard. The previous study indicates
that GC-FID assay was found to be the simplest and most efficient and facilitated the
simultaneous detection and determination of flavonoids within reasonable time
compared to High Performance Liquid Chromatography (HPLC) because HPLC
analysis exhibit low resolution of separation and low detection sensitivity. In addition,
either ethanol or methanol can be used in GC-FID due to identical effectiveness. Both
solvent exhibit equivalent solubilizing abilities and detection responses and yield similar
retention times and separation resolution (Sutthanut et al., 2007).
Figure 2.4: Diagram of Gas Chromatography-Flame Ionized Detector
(Zainab, 2009)
CHAPTER 3
METHODOLOGY
3.1
Materials
3.1.1
Plant sources
A total of twenty two Malaysian medicinal plants from fourteen plants family
age ranged in between three to eleven month were purchased from local nursery at
Skudai, Johor. The medicinal plants were selected randomly. The plant purchased were
grown and maintained in the green house. The list of twenty two selected medicinal
plants was shown in Table 3.1.
29
Table 3.1: Selected Malaysian Medicinal plants
Number
Family
Species
Vernacular names
1
Acanthaceae
Strobilanthes crispus
Pecah kaca, pecah beling
2
Apocynaceae
Wrightia religiosa
Anting puteri, jeliti
3
Araliaceae
Polycias scutellaria
Puding mangkuk
4
Cactaceae
Epiphyllum anguliger
Bakawali
5
Compositae
Gynura procumbens
Sambung nyawa
6
Euphorbiaceae
Acalypha hispida
Ekor kucing
Acalypha indica
Kucing galak
Jatropha podagarica
Jarak tapak gajah
Pedilanthus tithymaloides
Getah lelipan
Phyllanthus amarus
Dukung anak
7
Lamiaceae
Coleus atropupureus
Ati-ati merah
8
Malvaceae
Hibiscus rosa-sinensis
Bunga raya putih
9
Melastomataceae Tibouchina semidecandra
10
Myrtaceae
Rhodomyrtus tomentosa
Kemunting
11
Poaceae
Cymbopogon nardus
Serai wangi
Imperata cylindrica
Lalang hitam
Senduduk baldu/biru
12
Rutaceae
Ruta angustifolia
Geruda
13
Solanaceae
Cestrum nocturnum
Harum sundal malam
30
Continued
14
Zingiberaceae
Alpinia conchigera
Lengkuas padi/ranting
Alpinia purpurata
Halia merah
Kaempferia pulchra
Cekur
hutan/perang/merah
Halia hutan,lempoyang
Zingiber zerumbet
31
3.1.2
Chemicals
Kaempferol standard was purchased from Sigma Aldrich Corporation.
3.2
Method
3.2.1
Preparation of Standard
A total of 2.0 mg kaempferol standard was added with 1000 µL methanol
(MeOH) and vortexes until it completely dissolved. The standard solution was filtered
with 0.2 µm nylon filter and then 1 mL standard solution was pipette from the vial to the
vial agilent and the lid was sealed with parafilm before injected into GC-FID
chromatography analysis.
32
3.2.2
Medicinal Plants Extraction
The sample was prepared in triplicate for each plant by using methanol (MeOH)
extraction method. Firstly, the mature leaf was cleaned from debris and powdered by
crushing with liquid nitrogen using mortar and pastel. Then, the extraction solvent
(MeOH) with ratio 3 mLg-1 was added in the 50 mL Erlenmeyer flask containing the
sample before shaken for 30 minutes at 150 rpm in room temperature. After that, the
plant extract was filtered using 0.2 µm nylon filter. Finally, a total of 1000 μL plant
extract was pipette from the vial to the vial agilent before sealing the lid with parafilm
All the glassware used were soaked in 10% sulphuric acid (H2SO4) overnight and
washed with deionized water before usage and rinsed with methanol (MeOH) after
washing with soap prior to usage.
3.2.3
GC-FID analysis
The plant extracts were diluted with methanol and 1 μL was sampled for the gas
chromatography with flame ionization detector. The kaempferol standard and medicinal
plants samples were injected into a HP-5 capillary column (30 m length x 0.32 mm i.d.,
0.25 µm film thickness), Agilent Technologies, USA GCMS model, consisting of 6890
N Gas Chromatograph.
33
Operating conditions: oven temperature program from initial temperature at 100°C for 1
minutes to 325°C maximum temperature at 1 minute equilibration time and the final
temperature at 325°C was kept for 17 min; "split mode" ratio 50:1; carrier gas Helium
(He) with 20 mL/min flow rate; the temperature of flame ionization detector (FID) were
fixed at 270°C, respectively. The total run time was of 35.50 min.
3.2.4
Quantification of kaempferol
The concentration of the kaempferol was expressed by peak area (pA) obtained
from gas chromatography chromatogram. The F factor had been calculated by known
concentration and peak area of positive standard (kaempferol) and external standard (a
series n-alkane). Kaempferol content of various plants extract was calculated by
concentration, using the formula below:
Concentration and area of positive standard
Known
Concentration and area of external standard
Concentration positive standard = F Concentration external standard
Area positive standard
Area external standard
34
F = factor for positive standard
F = Concentration positive standard in sample
Area positive in sample
F X Area positive standard in sample = Concentration positive standard in sample
3.2.5
Statistical Analysis
Results were express as mean values with
standard error of mean (SEM). All
experiments triplicate data were analyzed using statistical software, SPSS for Window
software (SPSS 17.0 for Windows Evaluation Version software, SPSS Inc., USA). Oneway ANOVA was used to determine the statistical significance of kaempferol content in
selected medicinal plants. The means were compared using one-way ANOVA with Post
Hoc Multiple Comparison of Bonferroni analysis in order to compare all of plants
extract samples with control. The mean differences were considered significant at p ≤
0.05.
CHAPTER 4
RESULTS AND DISCUSSION
4.1
Screening of medicinal plants with highest kaempferol content
Phytochemical analysis of twenty two randomly selected Malaysian medicinal
plants mature leaf indicates that twelve plants contain kaempferol. This study focused
on mature leaf because leaf is subject to illumination and consequently has an efficient
antioxidant system (Masuda et al., 1999). The twelve medicinal plant species that were
identified to contain kaempferol included Wrightia religiosa, Polyscias scutellaria,
Epiphyllum
anguliger,
Acalypha
hispida,
Jatropha
podagrica,
Pedilanthus
tithymaloides, Tibouchina semidecandra, Rhodomyrtus tomentosa, Cynbopogon nardus,
Imperata cylindrica, Cestrum nocturnum, and Alpinia conchigera as shown in Figure
4.1.
36
Figure 4.1: Kaempferol (mg/kg) concentration detected in selected Malaysian medicinal
plants
37
Table 4.1: Kaempferol distribution within plant families
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Family
Species
Flavonoids
content (mg/kg)
Acanthaceae
Strobilanthes crispus Not detected
0.00 0.00
Apocynaceae
Wrightia religiosa
Kaempferol 1853.19 104.66
Araliaceae
Polycias scutellaria Kaempferol
532.90 532.90
Cactaceae
Epiphyllum
Kaempferol
345.79 345.79
anguliger
Compositae
Gynura procumbens Not detected
0.00 0.00
Euphorbiaceae
Acalypha hispida
Kaempferol 3696.28 206.52
Acalypha indica
Not detected
0.00 0.00
Jatropha podagarica Kaempferol
767.52 386.01
Pedilanthus
Kaempferol 1582.27 313.85
tithymaloides
Phyllanthus amarus Not detected
0.00 0.00
Lamiaceae
Coleus atropupureus Not detected
0.00 0.00
Malvaceae
Hibiscus
Not detected
0.00 0.00
rosa-sinensis
Melastomataceae Tibouchina
Kaempferol 4689.75 654.83
semidecandra
Myrtaceae
Rhodomyrtus
Kaempferol 3182.66 295.05
tomentosa
Poaceae
Cymbopogon nardus Kaempferol
350.13 350.13
Imperata cylindrica Kaempferol
757.33 757.33
Rutaceae
Ruta angustifolia
Not detected
0.00 0.00
Solanaceae
Cestrum nocturnum Kaempferol
2590.79 161.12
Zingiberaceae
Alpinia conchigera
Kaempferol
2355.53 462.57
Alpinia purpurata
Not detected
0.00 0.00
Kaempferia pulchra Not detected
0.00 0.00
Zingiber zerumbet
Not detected
0.00 0.00
*The value of kaempferol content are means
is significant at the 0.05 level
Flavonoids
SEM (n = 3) and the mean difference, p
38
The highest kaempferol content was identified in Tibouchina semidecandra
(4689.75 654.83 mg/kg) which locally known as “Senduduk Biru” or “Senduduk
Baldu” as shown in Table 4.1. Existence of kaempferol in T. semidecandra was
determined by comparing the plant extract chromatogram peak to the commercial
kaempferol standard chromatogram peak. The compounds were separated according to
their molecular weight. Whereby, compound with lower molecular weight was separated
earlier than the higher ones. The commercial kaempferol standard (4689.75 0.00
mg/kg) was detected at retention time 28.926 minutes while kaempferol in T.
semidecandra was detected at retention time 28.531 minutes as shown in Figure 4.2 (a)
and Figure 4.2 (b). Therefore, the retention time for kaempferol was estimated at the 28 th
minutes. The chromatogram peak of kaempferol detected indicates that it was the most
synthesized flavonoid by T. semidecandra which belong to family Melastomataceae
from five peaks in the chromatogram. The first peak that appear in the chromatogram
indicate the presence of methanol in the commercial kaempferol standard and plant
extract. The peak was high because methanol was used as solvent to extract the
flavonoid and it was detected earlier than kaempferol due to its lower molecular weight.
Another medicinal plant that synthesizes kaempferol as its major flavonoids was
Rhodomyrtus tomentosa that belong to family Myrtaceae and classified in the Myrtales
order same as Tibouchina semidecandra (Yoshida et al., 2010). As shown in Figure 4.2
(c), a mean of 3182.66 295.05 mg/kg kaempferol was detected in this medicinal plant.
Besides this two species, another species in the same order that have been identified to
contain kaempferol was Melastoma malabatricum. However, it was identified in the
flower but not in the leaves (Susanti, 2007).
39
(a)
(b)
(c)
(d)
(e)
(f)
(g)
Figure 4.2: GC-FID chromatogram of (a) kaempferol commercial standard (b) T.
semidecandra (c) R.tomentosa (d) A.hispida (e) J. podagrica (f) A. conchigera (g) E.
anguliger
40
A maximum number of medicinal plants were selected from the family
Euphorbiaceae which total up five plants. Among the five plants selected three of which
positive contained kaempferol. The highest was detected in Acalypha hispida (3696.28
206.52 mg/kg) while the lowest was detected in Jatropha podagrica (767.52
386.01
mg/kg) as shown in Figure 4.2 (d) and (e). However kaempferol was not the major
flavonoid synthesized by this family member. Besides selected plants another plant from
this family that have been reported containing kaempferol was Euphorbia retusa that
grows wild in the Middle East region of Egypt and Saudi Arabia used to treat tumor and
warts (Fathalla et al., 2009).
The second among the plant family selected besides family Euphorbiaceae, was
family Zingiberaceae. However, despite the high number of selected plant from
Zingiberaceae which total up five plants only one species contain kaempferol. The plant
that contain kaempferol was Alpinia conchigera (2355.53
462.57 mg/kg) as shown in
Figure 4.2 (f). Although the kaempferol content in Alpinia conchigera was high, it was
not the major flavonoid synthesize in this species from a total of nine compounds
separated. The unknown flavonoid can be identified by comparing to other flavonoids
standard or analyse using GC-MS (Guliyev, 2004).
41
Other than that, other plant families studied that contain kaempferol were
Apocynaceae (Wrightia religiosa-1853.19 104.66 mg/kg), Araliaceae (Polycias
scutellaria-532.90 532.90 mg/kg), Poaceae (Cymbopogon nardus-350.13 350.13
mg/kg, Imperata cylindrica-757.33 757.33 mg/kg), and Solanaceae (Cestrum
nocturnum-2590.79 161.12 mg/kg).
As shown in Figure 4.2 (g), the lowest kaempferol content was detected in
Epiphyllum anguliger (345.79
345.79 mg/kg) that belong to the Cactaceae family. To
my knowledge, there was no publication that reveals the presence of kaempferol in
Epiphyllum anguliger to date although kaempferol and other flavonoid such as quercetin
and isorhamnetin had been identified in other species in Cactacea family (Burret et al,
1982).
Although there was study that had been identified kaempferol in Strobilanthes
crispus (Liza et al., 2010) and Acalypha indica (Rahman et al., 2010), it was not exist in
the selected plant. Besides that, Gynura procumbens, Phyllanthus amarus, Coleus
atropupureus, Hibiscus rosa-sinensis, Ruta angustifolia, Kaempferia pulchra, Alpinia
purpurata, and Zingiber zerumbet also does not contain kaempferol. The negative result
might be caused by distribution of kaempferol in other part of plants organ and lost
during sample preparation or analysis. Other than that, the kaempferol might not contain
in the selected leaves but it presence in other leaves. In addition, the absence of
kaempferol in these medicinal plants maybe due insufficient exposure to sunlight.
42
4.2
Distribution of kaempferol in plant organ
The flavonol kaempferol was widely distributed in Tibouchina semidecandra
species. From a total of twelve samples taken from various part of the plant, eight
samples were detected containing kaempferol. All nine samples were taken from shoot,
young leaf and mature leaf exhibit positive result except one sample from young leaf.
Whereas, all three samples taken from young stem exhibit negative result that indicate
absence of kaempferol. The leaves contain kaempferol because flavonol act as
antioxidant in chloroplast (Hernandez et al., 2008). The kaempferol content increases as
the plant become mature. This can be prove by the result that show highest kaempferol
content in mature leaves (4689.75
654.83 mg/kg) compared to other selected organs
such as young leaves (1945.04 138.81 mg/kg) and shoot (727.51 371.79 mg/kg). The
mature leaf contain highest kaempferol because the duration of exposure to sunlight was
longer than young leaves and shoot due to longer lifespan (Chew et al., 2009).The same
goes to the kaempferol content in the young leaves because it was exposed longer than
the shoot. The leaves provide an oxidative protection from reactive chemical and
harmful by product formed when excess light energy was received during
photosynthesis. This result supported T. semidecandra as the best kaempferol producer
and mature leaf as the best organ producer because it can produce the flavonoid in
almost all part of its organ although at early growth phase.
43
Figure 4.3: Distribution of kaempferol in organ of Tibouchina semidecandra.
Table 4.2 : Kaempferol content in different organs of Tibouchina semidecandra
Medicinal plants
Kaempferol content mean (mg/kg)
Shoot
727.51 371.79
Young leaves
1945.04 138.81
Mature leaves
4689.75 654.83
Young stem
*The value of kaempferol content are means
is significant at the 0.05 level
0.00 0.00
SEM (n = 3) and the mean difference, p
CHAPTER 5
CONCLUSION
In this study, Malaysian medicinal plant that produces the highest kaempferol
content among twenty two selected Malaysian medicinal plants was Tibouchina
semidecandra. Kaempferol was distributed in shoot, young leaf and mature leaf of
Tibouchina semidecandra. Maximum kaempferol content was obtained in mature leaf
while no kaempferol consist in the young stem. The kaempferol content in Tibouchina
semidecandra was higher than Allium fistulosum (onion leaves), Carica papaya (papaya
shoot), Cucurbita maxima (pumpkin), Daucus carota (carrot), Camellia chinensis (black
tea), and Solanum melongena (brinjal). However, kaempferol was not present in ten
plants species selected namely Strobilanthes crispus, Gynura procumbens, Acalypha
indica, Phyllanthus amarus, Coleus atropupureus, Hibiscus rosa-sinensis, Ruta
angustifolia, Kaempferia pulchra, Alpinia purpurata and Zingiber zerumbet.
FUTURE RESEARCH
The prospect areas to further this research that I suggest include screening
process of the selected medicinal plants that show negative results because kaempferol
might be distributed in other leaves in the same plant but not the leaves that had been
used as sample. Besides kaempferol, the highest flavonoid that was still unknown can
also be identified in the selected plants.
Other than that, I also suggest micropropagation of T. semidecandra to
regenerate plant with higher kaempferol content. After that, the kaempferol content
between the wild type and regenerant were analyzed and compared using GC-FID. The
regenerant that produce highest kaempferol were exposed to various stress condition to
enhance kaempferol production. In addition, the kaempferol content can also be
improved by Agrobacterium mediated transformation. The kaempferol produced from
the mature leaf will then isolated and tested on cancer cell to evaluate the anti-cancer
activity.
46
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51
APPENDIX
a
b
c
d
Figure 2.1 a) Strobilanthes crispus. Bar line = 6.0 cm, b) Wrightia religiosa. Bar line =
6.0 cm c) Polyscias scutellaria. Bar line = 6.0 cm, d) Epiphyllum anguliger. Bar line =
12.0 cm
52
e
f
g
h
Figure 2.1 e) Gynura procumbens. Bar line = 3.0 cm, f) Acalypha hispida. Bar line =
12.0 cm, g) Acalypha indica. Bar line = 5.0 cm, h) Jatropha podagarica. Bar line = 4.0
cm
53
j
i
k
l
Figure 2.1 i) Pedilanthus tithymaloides. Bar line = 2.5 cm, j) Phyllanthus amarus. Bar
line = 2.5 cm, k) Coleus atropupureus. Bar line = 6.0 cm, l) Hibiscus rosa-sinensis. Bar
line = 8.0 cm
54
m
o
n
p
Figure 2.1 m) Tibouchina semidecandra. Bar line = 5.0 cm, n) Rhodomyrtus tomentosa.
Bar line = 6.0 cm, o) Cynbopogon nardus. Bar line = 2.8 cm, p) Imperata cylindrica.
Bar line = 2.8 cm
55
r
q
s
t
Figure 2.1 q) Ruta agustifolia. Bar line = 4.0 cm, r) Cestrum nocturnum. Bar line = 7.5
cm, s) Alpinia conchigera. Bar line = 8.0 cm, t) Alpinia purpurata. Bar line = 10.5 cm
56
v
u
Figure 2.1 u) Kaempferia pulchra. Bar line = 5.5 cm, v) Zingiber zerumbet. Bar line =
12.0 cm