Physiological and Antioxidant Properties of Centella ... · Physiological and Antioxidant Properties of Centella Asiatica in Response to Phosphate Deficiency Halgoord Nasraden Hassan

Physiological and Antioxidant Properties of Centella Asiatica in

Response to Phosphate Deficiency

Halgoord Nasraden Hassan

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Physiological and Antioxidant Properties of Centella Asiatica in Response to Phosphate Deficiency

Copyright © 2014 Halgoord Nasraden Hassan

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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.

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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

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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

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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