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Physiological and Antioxidant Properties of Centella Asiatica in
Response to Phosphate Deficiency
Halgoord Nasraden Hassan
DISSERTATION.COM
Boca Raton
Physiological and Antioxidant Properties of Centella Asiatica in Response to Phosphate Deficiency
Copyright © 2014 Halgoord Nasraden Hassan
All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or by any
information storage and retrieval system, without written permission from the publisher.
Dissertation.com Boca Raton, Florida
USA • 2015
ISBN-10: 1-61233-436-9 ISBN-13: 978-1-61233-436-3
iv
Dedicated to my beloved father, mother, and to my beloved wife Darya
and my sweet daughter Lyan.
v
ACKNOWLEDGMENT
Alhamdulillah, first and foremost, I praise and acknowledge Allah, the most
beneficent and the most merciful. Secondly, my humblest gratitude to the Holy
Prophet Muhammad (Peace be upon him) whose way of life has been a continuous
guidance.
I am deeply indebted to my supervisor Dr. Fazilah Binti Abd Manan whose
continuous help, stimulating suggestions, encouragement and constructive criticism
throughout my work helped me in all the time of research and have greatly improved
the content of this paper.
I would like to express my appreciation to Kurdistan regional government
that provided me the opportunity to study and complete my master research.
Finally yet importantly, my deepest gratitude goes to my beloved parents
who have been so patient with me over the years. My deepest gratitude further goes
to my wife for being with me in any situation, which is the source of my strength.
Finally, I would like to thank all my friends who helped me during this research
project.
vi
ABSTRACT
Centella asiatica L. Urban (Umbelliferea) locally known as ‗Pegaga‘ is a
type of herbal plant that had been used in traditional medicine in Asia for many
centuries. This species has high medicinal value derived from the phenolic and
flavonoid compounds. This research was conducted to understand the physiological
and antioxidant properties of Centella asiatica L. Urban in response to different
phosphate concentrations. Experiment was arranged in a Completely Randomized
Design (CRD) consisted of five treatments with three replications. The treatments
was 0, 0.05, 0.1, 0.15, and 0.2 g of Phosphorus pentoxide (P2O5 )/kg. The results of
physical characteristics of Centella asiatica showed that P2O5 fertilizer significantly
increased the fresh and dry weight of plants at p <0.05. The highest production of
dry weight was 0.44±0.15 g for plant supplied with 0.1g P2O5. While the lowest dry
weight was recorded for plants that were not supplied with phosphate, which was
0.18 ±0.05 g . Accordingly, the highest production of fresh weight was 1.71±0.61 g
for 0.1g P2O5 treatment, and the lowest was 0.82±0.06 g for non-treated plants.
Results also showed that P2O5 fertilizer significantly affects total flavonoid and total
phenolic content of Centella asiatica. The flavonoid constituents detected was
43.7±4.6 mg quercetin equivalents /100g DW) for non-treated plants and 28.9± 0.8
mg quercetin equivalents/100g DW) for 0.1g P2O5 treated plants. The total phenolic
was (5.91±0.38 g gallic acid equivalents /100g DW) for non-treated and 4.05±0.61 g
gallic acid equivalents /100g DW) for 0.2 g P2O5 plants. Phosphate in plant tissues
has strong positive linear correlation phosphate fertilizer. Results show that different
concentrations of phosphate affects the physiological and biochemical properties of
plants.
vii
ABSTRAK
Centella asiatica L. Urban (Umbelliferea) dikenali sebagai ‗Pegaga‘ adalah
sejenis tumbuhan herba tempatan yang telah digunakan dalam perubatan tradisional
di Asia untuk beberapa abad. Spesis ini mempunyai nilai perubatan yang tinggi
diperolehi daripada fenolik dan sebatian flavonoid. Kajian ini dijalankan untuk
memahami ciri-ciri fisiologi dan biokimia Centella asiatica sebagai tindak balas
kepada tahap fosforus yang berbeza. Eksperimen telah disusun dalam ―Rekabentuk
Rawak Lengkap (RRL)‖ terdiri daripada lima rawatan dengan tiga replikasi.
Rawatan adalah 0, 0.05, 0.1, 0.15, dan 0.2 g daripada fosforus pentoksida (P2O5)/kg.
Keputusan ciri-ciri fizikal Centella asiatica menunjukkan bahawa baja P2O5
meningkat dengan ketara bagi berat segar dan kering tumbuh-tumbuhan pada p
<0.05. Pengeluaran paling tinggi daripada berat kering adalah 0.44 ±0.15 g bagi
tumbuhan yang dibekalkan dengan 0.1 g P2O5. Berat kering yang paling rendah
dicatatkan bagi tumbuh-tumbuhan yang tidak dibekalkan dengan fosforus iaitu 0.18
±0.05 g. Oleh itu, pengeluaran tertinggi berat segar adalah 1.71 ±0.61 g untuk
rawatan 0.1 g P2O5, dan yang paling rendah adalah 0.82 ±0.06 g untuk tumbuh-
tumbuhan tidak dirawat. Keputusan juga menunjukkan P2O5 dengan ketara memberi
kesan kepada jumlah flavonoid dan jumlah kandungan fenolik daripada Centella
asiatica. Juzuk flavonoid dikesan adalah 43.7 ± 4.6 mg setara quercetin /100g DW)
untuk tumbuhan tidak dirawat dan 28.9±0.8 mg setara quercetin/100g DW) untuk
0.1 g P2O5 tumbuhan dirawat. Jumlah fenolik adalah 5.91±0.38 g setara asid Gallic
/100g DW untuk bukan dirawat dan 4.05±0.61 g setara asid Gallic / 100g DW) untuk
rawatan P2O5 sebanyak 0.2 g. Fosfat dalam tisu tumbuhan mempunyai korelasi linear
positif terhadap fosforus. Keputusan menunjukkan bahawa kepekatan fosfat yang
berbeza memberi kesan kepada fisiologi dan biokimia tumbuhan.
viii
TABLE OF CONTENTS
CHAPTER TITLE PAGE
DECLARATION ii
DEDICATION iv
ACKNOWLEDGMENT v
ABSTRACT vi
ABSTRAK vii
TABLE OF CONTENTS viii
LIST OF TABLES xii
LIST OF FIGURES xiii
LIST OF ABBREVIATION/SYMBOLS xiv
LIST OF APPENDICES
xvi
1 INTRODUCTION 1
1.1 Research Background 1
1.2 Significance of Study 3
1.3 Research Objectives 3
1.4 Problem Statement 4
ix
1.5 Scope of the Research 4
2 Literature Review 5
2.1 Description of Centella asiatica 5
2.2 Antioxidant Compounds in Centella asiatica 7
2.2.1 Flavonoid 8
2.2.1.1 Important Functions of
Flavonoids
9
2.3 Phenolic 10
2.3.1 Important Functions of Phenolic Acid 11
2.4. Phosphate 11
2.5 Phosphate Deficiency and the Level of
Antioxidants in Plants in Plants
12
2.6 Phosphate Level in Malaysian Soil 13
2.7 Significance of Phosphate for Plants 14
2.8 Availability of Phosphate 15
2.8.1 Phosphate Uptake by Plants 16
2. 8.2 Phosphate Deficiency: Effect on Plant
Growth
16
2.8.3 Improving Phosphate Efficiency in the
Soil
18
2.8.3.1 Plant Adaptations to Low
Phosphate
18
2.9 Conclusion 20
x
3 MATERIALS AND METHOD 21
3.1 Plant Cultivation 22
3.2 Physiological Characteristics of Centella
asiatica
23
3.2.1 Plants Fresh Weight and Dry Weight 23
3.2.1.1 Measuring Fresh Weight 23
3.2.1.2 Measuring Dry Weight 23
3.2.2 Root: Shoot Ratio 23
3.3 Antioxidant Characterization 24
3.3.1 Sample Preparation for Determination
of Total Phenolic and Total Flavonoid
24
3.3.2 Determination of Total Phenolic
Content
24
3.3.3 Determination of Total Flavonoid
Content
25
3.3.4 Determination of Phosphate
Concentration in Plant Tissues
25
3.4 Statistical Analysis
26
4 RESULT AND DISCUSSION 27
4.1 Effect of Different Phosphate Fertilizer on Plant
Fresh Weight.
27
4.2 Effect of Different Phosphate Fertilizer on
Plant Dry weight
28
xi
4.3 Effect of Different Phosphate Fertilizer on Root-
Shoot Ratio of Plant
29
4.4 Effect of Different Phosphate Fertilizer on Root
Length
30
4.5 Effect of Different Phosphate Fertilizer on Total
Phenolic Acid
32
4.6 Effect of Different Phosphate Fertilizer on Total
Flavonoid
33
4.7 Effect of Different Phosphate Fertilizer on
Phosphate Content
34
4.8 Correlations Between Variables in the Study 35
4.8.1 Fresh Weight Correlation with Phenolic
and Flavonoid Compound
35
4.8.2 Phosphate Fertilizer Correlation with
Phosphate Content
37
4.8.3 Correlation between Total Flavonoid and
Total Phenolic
38
5 CONCLUSION AND RECOMMENDATION 40
5.1 Conclusions 40
5.2 Recommendation 41
REFERENCES 42
APPENDICES 53
xii
LIST OF TABLES
TABLE NO. TITLE PAGE
1.1 Taxonomy of Pegaga 7
xiii
LIST OF FIGURES
FIGURE NO. TITLE PAGE
3.1 Different Treatments for Centella asiatica 21
3.2 General Framework of Study 22
4.1 Effect of Different Phosphate Fertilizer on Plant Fresh
Weigh
27
4.2 Effect of Different Phosphate Fertilizer on Plant Dry
Weight
28
4.3 Effect of Different Phosphate Fertilizer on Root-Shoot
Ratio
29
4.4 Effect of Different Phosphate Fertilizer on Root Length
31
4.5 Centella asiatica Roots Length From Different
Treatments
31
4.6 Effect of Different Phosphate Fertilizer on Total
Phenolics
32
4.7 Effect of Different Phosphate Fertilizer on Total
Flavonoid
33
4.8 Purple Colored Ctem in 0.05 g/kg P2O5
34
4.9 Effect of Different of Phosphate Fertilizer on Centella
asiatica Phosphate Content
35
4.10 Fresh Weights and Total Phenolic Correlation
36
4.11 Fresh Weight and Total Flavonoid Correlation
37
4.12 Phosphate Fertilizer and Phosphate Content Correlation
38
4.13 Total Flavonoid and Total Phenolic Correlation 39
xiv
LIST OF ABBREVIATION/SYMBOLS
% Percentage
≤ Greater than or equal
≥ Less than or equal
°C Degree celcius
µg microgram
AE Agronomic Effectiveness
AL3+
Aluminum ion
ANOVA Analysis of Variance
Ca Calcium
CEC Cation Exchange Capacity
cm Centimetre
DNA Deoxyribonucleic Acid
g gram
H2PO4 2-
Dihydrogen phosphate
H3PO4 Phosphoric acid
HPO4 2-
hydrogen phosphate
xv
Hz Hertz
LSD Least Significant Difference
Mg milligram
mg/L milligram per liter
min minutes
mL milliliter
mM millimolar
N Normality
Na2CO3 Sodium carbonate
nm Nanometre
P Phosphate
P2O5 Phosphorus pentoxide
pH Degree of acidity and alkalinity
PR Phosphate Rocks
ROS Reactive Oxygen Species
SE Standard Error
v/v Percent ―volume in volume‖
λ Wavelength
μL microliter
xvi
LIST OF APPENDICES
APPENDIX TITLE PAGE
A Anova Tests 53
B Fisher Least Significant Difference(LSD)Tests 57
C Pearson Correlation 61
D Antioxidants and Phosphate Standard Curves
62
1
CHAPTER 1
INTRODUCTION
1. 1 Research Background
About 1200 species of higher plants in Malaysian forests are reported to have
medicinal uses. Plant-based products such as herbal medicines and health foods gained
better are popularity among Malaysians, and to date about 200 species have been used
for these purposes. According to the data obtained from 4000 Chinese herbal stores, the
annual sales value in Malaysia reached 500 million$ in 1994 and the estimated market
value of traditional medicine was between 1 to 2 billion$ in 1995 (Joy et al., 1998).
Centella asiatica is one of the most important medicinal plants that have been
used as traditional medicine in Malaysia. The potential of Centella asiatica as an
alternative natural antioxidant and the use of this plant for protection against age-related
changes in brain antioxidant defense system, have notably increased in recent years
(Subathra et al., 2005). A variety of biochemical components such as different
secondary metabolites have been found in Centella asiatica. The chemical constituents
of Centella asiatica have important roles in medicinal and nutraceutical applications
derived from its biologically active components (Hashim, 2011). Due to their
importance, they have been used as a biomarker component for quality assessment of
Centella (Dash et al., 2011).
2
In general, plant growth and development depends on environmental variants
such as temperature, water availability, light intensity, and base metals. Therefore,
careful inspection of plant growth can help to identify a specific nutrient stress. If a plant
is lacking in a particular nutrient, visible symptoms may appear (Burdon, 1987).
Symptoms of deficiency of nutrients do not appear directly. Rather, the normal plant
processes are thrown out of balance by accumulation of some organic compounds and a
shortage of others leading to abnormal conditions. Visual evaluation of nutrient stress is
not enough and should be used only as a supplement to other diagnostic techniques such
as soil and plant analysis (Crawford, 2008).
Nutrient shortage symptoms cause the plant not to function properly. In this case,
it would have been useful to have applied fertilizer before appearance of symptoms.
Since the objective is to get the limiting nutrient into the plant as quickly as possible,
nutrients may be supplied with foliar applications or side dressings (O'Sullivan et al.,
1997).
Phosphorus (P) is one of the most important elements among many inorganic
nutrients that significantly affect plant growth and metabolism. Phosphorus is an
important pillar in the metabolism and vital energy of nucleic acids and membrane
development, Phosphorus also plays important roles in the process of photosynthesis
and respiration, and in the organization of a number of enzymes (Schaffert et al., 1999).
Plants take up phosphorus the form of orthophosphate . In phosphate deficiency, plant
growth will be slowed and stunted, and the older leaves will have a purple coloration,
particularly on the underside.
Lack of phosphate is the main obstacle to plants, and this issue has been
observed, especially in agricultural fields of many countries all over the world. This
affects plants in many circumstances, including the growth, development, and
production of many important metabolites. These conditions will also affect the
3
production of antioxidants in plants. To survive under various environmental conditions,
plants have to have certain physical and chemical adaptation mechanisms.
The nutritional and mineral component is also an important factor in determining
the quality of the herbs. Study by Caris-Veyrat et al. (2004) indicated that nutrient
composition of the plants during maturation was influenced by factors such as genetics,
agronomic practices, region, and rate of growth, variety, and climatic conditions.
Phosphate excess will not have a direct effect on the plant. High phosphate may also
interfere with the normal calcium nutrition (Munson and Kalra, 1998).
This study aims to characterize the physiological and biochemical aspects of
Centella asiatica in different phosphate concentrations and study the relationship of
phosphate concentrations and the production of antioxidants.
1.2 Significance of Study
This study attempts to determine the physiological and biochemical responses of
Centella asiatica to different levels of phosphate. Although this species is exposed to
phosphate stress, lack of studies has been conducted to look in detail into this issue.
1.3 Research Objectives
1) To characterize the physiological aspects of Centella asiatica under different
phosphate concentration.
4
2) To analyze the antioxidant properties of Centella asiatica under different phosphate
concentrations.
3) To determine phosphate concentration in plant tissues.
1.4 Problem Statement
Phosphate deficiency is a major limiting factor in many Malaysian acidic soils.
The amount of phosphate in these soils are either inherently low or are fixed in the forms
that are unavailable to plants (Pushparajah et al., 1990). Low level of phosphate in the
soil normally affect the growth and development of plants. The production of secondary
metabolites, also the antioxidant level of plants will be altered under phosphate stress.
1.5 Scope of the Research
In this research, the ability of Centella asiatica to cope with phosphate
deficiency, and the changes that occurred during phosphate stress were studied through
investigation of physiological characteristics (fresh weight, dry weight and the root-
shoot ratio). Antioxidant properties represented by determination of total phenolic, total
flavonoid and phosphate level in plant tissues were characteriazed .
5
CHAPTER 2
LITERATURE REVIEW
2.1 Description of Centella asiatica
Centella asiatica L. belongs to the family Apiaceae or Umbelliferae, the plants
that grow on the edge of agricultural land and river banks. Plant flourishes in wet areas
of Malaysia, Indonesia, India, and other parts of Asia including China. The herb is also
known as ―Pegaga‖ in Malaysia, ―Indian pennywort‖ and ―Gotu Kola‖ in Europe and
America, ―Mandookaparni‖ in India, ―Pegagan or Kaki Kuda‖ in Indonesia, and ―Luei
Gong Gen‖ or ―Tung Chain‖ in China (Tolkah, 1999).
There are several types of Centella asiatica that can be found in Malaysia such
as Pegaga Cina or Nyonya, Pegaga Daun Lebar, Pegaga Salad and Pegaga Renek. The
use of Centella (whole plant parts) in food and beverages has increased over the years,
basically due to its health benefits such as antioxidant, anti-inflammatory, wound
healing, memory enhancing property and many others (Hashim, 2011). Free radicals
have been claimed to play an important role in the ageing process and capable of
damaging many cellular components (Gülcin, 2006). Natural antioxidant from Centella
asiatica protects age-related changes in brain (Subathra et al., 2005).
Centella asiatica L. is important herbal medicinal plant used for various
applications (James and Dubery, 2009). In Malaysia and Indonesia, Centella is
commonly eaten fresh as vegetable (ulam or salad) especially among the locals Malay