ABSTRACT Tacca integrifolia Ker-Gawl is belongs to the family of

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ABSTRACT

Tacca integrifolia Ker-Gawl is belongs to the family of Taccaceae and locally known as “Belimbing Tanah”. It has been used traditionally for the treatment of hypertension,

hemorrhoids, heart failure and kidney disease. The thin layer

chromatography (TLC) of hexane, petroleum ether, chloroform, methanol and water extract of leaves and rhizome showed the presence of phenols, flavonoids, terpenoids, essential oil and alkaloid compounds. The analysis of the leaves and rhizome extract with LCMS/MS showed that it contained proanthocyanidin, proanthocyanidin

trimer,

p-hydroxybenzoic

acid,

phenolic

acid

conjugate,

protocatechuic acid, quinic acid and dicaffeolquinic acid conjugate. The total phenol contents w e re highest in leaves and rhizomes extract of water at 792.7mgGAE/g and 350.8mgGAE/g respectively. Whereas, the highest total flavonoids contents were highest in petroleum ether leaves extract and in chloroform rhizome extract at 376.7mgQE/g and 193.4mgQE/g respectively. The ACE inhibition activity at 100mg/ml was highest in leaves extract of water (45.5%) and in rhizome extract of methanol (53.6%). The isolated compounds label as A5, A7, A8, A9, B3, B5, B8, C3, C9, E1, E3, E4, F1, G1, G3 and G6 showed ACE inhibition more than 50%. Since the water extract showed highest ACE inhibition and because it is often used in traditional medicine for hypertension treatment, the water extracts were analyzed in vivo with SHR rats. The sub-acute toxicity test has shown that there were no mortality occurs under experimental conditions. Brine Shrimp Lethality Assay (BSLA) also has shown low toxicity at LC50 22981µg/ml for leaves extract and LC50 4378µg/ml of rhizome extract. The blood pressure of spontaneously hypertensive rats were reduced significantly (p<0.05) at 50mg/kg and 100mg/kg of water leaves extract and water rhizome extract respectively. The liver function test has indicated significant

ii

difference in AST except when compared 100 mg/kg water rhizome extract SHR group with control group, and significance also showed in total protein and ALT except when compared 50 mg/kg water leaves extract and 50 mg/kg Captopril compared to control normal SD rat, respectively. While sodium test in renal function test showed significance in difference. In the antioxidant DPPH assay the IC50 of methanol leaves, chloroform leaves and water leaves extract was 88µg/ml, 350µg/ml and 480µg/ml respectively. The ferric reducing power assay has showed that both hexane extract from leaves and rhizome gave high reducing activity. The metal chelating activity of the chloroform leaves extract showed the highest metal chelating activity of IC50 at 1.98mg/ml. In conclusion, water extract from leaves and rhizome of Tacca integrifolia contained active phytochemical compounds as detected

in

LCMS/MS

that

responsible

in

reducing

blood

pressure

of

spontaneously hypertensive rats significantly at 50mg/kg and 100mg/kg and possessed high antioxidant activity that could scavenged free radicals and prevented oxidative stress that related to hypertension. Thus, these results support and provide scientific evidence to the claimed made by traditional practitioner that used Tacca integrifolia for antihypertension treatment.

iii

ABSTRAK

Tacca integrifolia Ker-Gawl daripada keluarga Taccaceae juga dikenali sebagai "Belimbing Tanah". Ia telah digunakan secara tradisional untuk rawatan hipertensi, buasir, kegagalan jantung dan penyakit buah pinggang. Kromatografi lapisan nipis (TLC) pada ekstrak heksana, petroleum eter, klorofom, metanol dan air daripada daun dan rizom menunjukkan kehadiran sebatian fenol, flavonoid, terpenoid, minyak pati dan alkaloid. Analisis LCMS/MS ekstrak daun dan rizom menunjukkan bahawa ia mengandungi proantosianidin, trimer proantosianidin, asid p-hydroxybenzoic, konjugat asid fenolik, asid protocatechuic, asid quinic dan konjugat asid dicaffeolquinic. Jumlah kandungan fenol paling tertinggi dikesan dalam ekstrak air pada daun dan rizom iaitu sebanyak 792.7mgGAE/g dan 350.8mgGAE/g masing-masing. Manakala, jumlah kandungan flavonoid tertinggi dikesan di dalam ekstrak petroleum eter daun dan ekstrak kloroform rizom sebanyak 376.68mgQE/g dan 193.4mgQE/g masing-masing. Aktiviti perencatan ACE pada kepekatan 100mg/ml adalah tertinggi dalam ekstrak daun air (45.5%) dan dalam ekstrak rizom metanol (53.6%). Sebatian terpencil berlabel A5, A7, A8, A9, B3, B5, B8. C3, C9, E1, E3, E4, F1, G1, G3 dan G6 menunjukkan perencatan ACE melebihi 50%. Oleh kerana ekstrak air menunjukkan perencatan ACE yang tertinggi dan sering digunakan oleh pengamal tradisional untuk rawatan hipertensi, maka ekstrak air dianalisis secara in vivo dengan menggunakan tikus SHR. Ujian ketoksikan sub-akut telah menunjukkan bahawa kematian tidak berlaku sepanjang eksperimen. Asei kematian anak udang (BSLA) juga telah menunjukkan ketoksikan adalah rendah pada LC50 22981.46µg/ml bagi ekstrak daun dan LC50 4378.011µg/ml bagi ekstrak rizom. Tekanan darah tikus hipertensi yang diberi dos ekstrak air daun dan rizom sebanyak 50mg/kg dan 100mg/kg telah berkurang dengan ketara (p <0.05). Ujian fungsi hati telah menunjukkan tiada perbezaan yang signifikan dalam jumlah protein,

iv

ALT dan AST. Begitu juga, dalam ujian fungsi renal, tiada perbezaan yang signifikan telah diperhatikan dalam kandungan natrium, kalium dan kreanitin apabila dibandingkan antara kumpulan tikus SHR kawalan dengan kumpulan tikus SHR yang diberi rawatan dengan ekstrak air daun dan ekstrak air rizom. Dalam asei antioksidan DPPH, IC50 ekstrak daun metanol, klorofom dan air adalah 88µg/ml, 350µg/ml dan 480µg/ml masing-masing. Asei pengurangan kuasa ferric telah menunjukkan bahawa kedua-dua ekstrak heksana daripada daun dan rizom memberikan aktiviti perencatan yang tinggi. Aktiviti pengkelat logam bagi ekstrak kloroform daun menunjukkan aktiviti perencatan tertinggi pada IC50 di 1.98mg/ml. Kesimpulannya, ekstrak air dari daun dan rizom Tacca integrifolia mengandungi sebatian fitokimia aktif yang dikesan dalam analisis LCMS/MS yang bertanggungjawab dalam mengurangkan tekanan darah tikus hipertensi secara ketara pada dos 50mg/kg dan 100mg/kg dan memiliki aktiviti antioksidan yang tinggi yang boleh menyingkirkan radikal bebas dan menghalang tekanan oksidatif yang berkaitan dengan tekanan darah tinggi. Oleh itu, hasil kajian ini menyokong dan menyediakan bukti saintifik seperti yang didakwa oleh pengamal tradisional yang menggunakan Tacca integrifolia sebagai rawatan hipertensi.

v

ACKNOWLEDGEMENT

In the name of Allah, the Most Gracious and the Most Merciful.

Alhamdulillah, all praises to Allah for His blessing in completing of this thesis. First and foremost, I offer my sincere gratitude and special appreciation to my supervisor, Dr Jamaludin Bin Mohamad, for his supervision, patient, knowledge and constant support. His invaluable help of constructive comments and suggestion throughout the experiment and thesis works have contributed to the success of this research. Sincere thanks to all my friends and my colleagues especially Ashriya Azmil, Apiah Mohd Amin, Faezah Mohamad, Muhammad Fahrin Maskam, Nadzrul Aida Md Darus, Asmawati Abrul Rahim and Md Hoirol Azri for their kindness contribution and moral support during my postgraduate study. Thanks for the friendship and memories. I would like to express my deepest gratitude to all the laboratory technicians and staff of Institute of Science Biology, Faculty of Science, University of Malaya especially to Encik Roslan, Tuan Haji Elias, Syed Mohd Aliff, Mrs. Ruzaimah and Mrs.Marsha for their co-operations and help in using laboratory and its equipment. Last but not least, I am grateful for the endless love, prayers, support and encouragement from my beloved parents; Mr. Jamaludin Md Said and Mrs. Zaiton Mahmood, and my siblings; Zahrul, Afiq and Laila Nabila. Million thanks also to those who indirectly contributes in this research. Thank you very much.

vi

TABLE OF CONTENTS

DECLARATION

i

ABSTRACT

ii

ABSTRAK

iv

ACKNOWLEDGEMENT

vi

TABLE OF CONTENTS

vii

LIST OF FIGURES

xiii

LIST OF TABLES

xx

LIST OF SYMBOLS AND ABBREVIATIONS

xxviii

CHAPTER 1 INTRODUCTION

1

CHAPTER 2 LITERATURE REVIEW

6

2.1

Hypertension

6

2.1.1 Essential Hypertension

7

2.1.2 Secondary Hypertension

8

2.2

Renin Angiotensin Aldosterone System (RAAS)

10

2.3

Angiotensin Converting Enzyme (ACE)

11

2.3.1 Angiotensin Converting Enzyme (ACE) mechanism

12

2.3.2 Angiotensin Converting Enzyme inhibitors (ACEI)

14

Antioxidants

19

2.4.1 Reactive Oxygen Species (ROS)

24

2.4.2 Vitamin C

29

Medicinal Plant Studied - Tacca integrifolia (Belimbing Tanah)

31

2.5.1 Plant Descriptions

34

2.5.2 Medicinal uses

34

2.5.3 Chemical constituents

35

2.4

2.5

vii

2.6

Research objectives

36

CHAPTER 3 METHODOLOGY

37

3.1

Plant materials

37

3.2

Extraction of plant chemical compounds

38

3.3

Separation and isolation of chemical compounds

41

3.3.1 Thin Layer Chromatography (TLC)

42

3.3.2 Column Chromatography (CC)

45

3.3.3 High Performance Liquid Chromatography (HPLC)

46

3.3.3.1 Detection of standard phenol and flavonoid

49

3.3.3.2 Separation of chemical compounds in extracts of Tacca integrifolia 3.3.3.3 Determination of standard Hippuric acid

49 51

3.3.4 Determination of chemical compounds in extract of Tacca integrifolia using Liquid Chromatography Mass Spectrometry/ Mass Spectrometry (LCMS/MS) 3.4 Detection of chemical compounds

52 53

3.4.1 Visible light

53

3.4.2 UV light

54

3.4.3 Dragendorff reagents

54

3.4.4 Vanillin-Sulphuric acid reagents

55

3.4.5 Anesaldehyde-sulphuric acid

55

3.4.6 Iodine vapor

56

3.4.7 Saponin froth test

56

3.4.8 Tannin and phenolic compound

56

3.5

Determination of the total phenolic contents

57

3.6

Determination of the total flavonoid contents

57

viii

3.7

Angiotensin Converting Enzyme (ACE) Inhibition Activity

57

3.7.1 Preparation of Angiotensin Converting Enzyme (ACE) enzyme extract

3.8

58

3.7.2 Angiotensin Converting Enzyme (ACE) assay

59

3.7.3 Preparation of Hippuric acid (HA) standard curve

60

Animal Study

60

3.8.1 Sub-Acute Toxicity test of water extracts from leaves and rhizomes of Tacca integrifolia on Spontaneously Hypertensive Rats(SHR)

60

3.8.2 Spontaneously Hypertensive Rats (SHR) treatment of hypertension using water extracts from leaves and rhizomes of Tacca integrifolia compared with Captopril as positive reference standard 3.9

61

Antioxidant activity

62

3.9.1 DPPH Radical Scavenging Activity

62

3.9.1.1

DPPH radical scavenging activity of standard ascorbic acid

3.9.1.2

63

DPPH radical scavenging activity of extract from leaves and rhizomes of Tacca integrifolia

64

3.9.2 Ferric Reducing Power Assay (FRAP)

66

3.9.3 Metal Chelating Assay

66

3.10

Brine Shrimp Lethality Assay (BSLA)

68

3.11

Statistical analysis

69

ix

CHAPTER 4 RESULTS

70

4.1

Extraction of plant chemical compounds

70

4.2

Isolation of chemical compounds

71

4.2.1

Thin Layer Chromatography (TLC)

71

4.2.2

Column Chromatography (CC)

90

4.2.3

High Performance Liquid Chromatography (HPLC)

4.3

Determination of chemical compounds by LCMS/MS

95 106

4.3.1 Liquid Chromatography Mass Spectrometry/Mass Spectrometry (LCMS/MS) for leaves extract of Tacca integrifolia

107

4.3.1 Liquid Chromatography Mass Spectrometry/ Mass Spectrometry (LCMS/MS) for rhizomes extracts of Tacca integrifolia 4.4

121

Phytochemical detection of chemical compounds

128

4.4.1 Saponin froth test

128

4.4.2 Tannin and phenolic compounds

129

4.5

Determination of the total phenolic contents

129

4.6

Determination of the total flavonoid content

132

4.7

Angiotensin Converting Enzyme (ACE) Bioassay

136

4.7.1 Determination of Angiotensin Converting Enzyme (ACE) inhibition by Captopril as positive reference standard

136

4.7.2 Determination of Angiotensin Converting Enzyme (ACE) inhibition of leaves extracts of Tacca integrifolia

139

4.7.3 Determination of Angiotensin Converting Enzyme (ACE) inhibition of rhizome extracts of Tacca integrifolia

142

4.7.4 Determination of ACE inhibition of compounds isolated from extracts of Tacca integrifolia using TLC

147

x

4.8

4.7.5 Standard curve of Hippuric acid

156

Animal Study

157

4.8.1

Sub-acute Toxicity Test of water extracts from leaves and rhizome of Tacca integrifolia on Spontaneously Hypertensive Rats (SHR)

157

4.8.2 Anti-hypertension treatment of SHR with water extract from leaves and rhizome of Tacca integrifolia 4.9

163

Antioxidants

170

4.9.1 DPPH radical scavenging activity

170

i)

Ascorbic acid as positive reference standard

ii)

DPPH radical scavenging activity of leaves extract of Tacca integrifolia

iii)

4.9.2 Ferric reducing power assay

177

177

Reducing power of leaves extract of Tacca integrifolia

iii)

175

Butylated Hydroxyanisole (BHA) as positive reference standard

ii)

172

DPPH radical scavenging activity of rhizome extract of Tacca integrifolia

i)

170

177

Reducing power of rhizome extract of Tacca integrifolia

4.9.3 Metal Chelating Power Assay i)

Ethylenediaminetetraacetic acid (EDTA) as standard

ii)

Metal Chelating activity of leaves extract of Tacca integrifolia

180 182 182

183

xi

iii)

Metal Chelating activity of rhizome extract of Tacca integrifolia

4.10

Brine Shrimp Lethality Assay (BSLA)

186 189

4.10.1 Brine Shrimp Lethality Assay (BSLA) of extract from leaves of Tacca integrifolia

190

4.10.2 Brine Shrimp Lethality Assay (BSLA) of extract from rhizomes of Tacca integrifolia

191

CHAPTER 5 DISCUSSION

193

CONCLUSION

209

REFERENCES

211

APPENDIXES

222

xii

LIST OF FIGURES

Figure 2.00

Mechanism mediating hypertension

Figure 2.01

Renin Angiotensin-Aldosterone System (RAAS) role regulation of blood pressure

Figure 2.02

10

Mechanisms of Angiotensin Converting Enzyme (ACE) in hypertension and angioederma

Figure 2.03

9

13

Role of ACE inhibitors and angiotensin type 1 (AT1) receptor blockers

16

Figure 2.04

Chemical structures of ACE inhibitors

17

Figure 2.05

Interaction of oxygen free radicals and antioxidants

20

Figure 2.06

Common examples of Reactive Oxygen Species (ROS)

25

Figure 2.07

Effect of reactive oxygen species (ROS) on various organs leading to hypertension.

26

Figure 2.08

Antioxidants defense systems againts free radicals attack

28

Figure 2.09

Ascorbic acid and its oxidation products

30

Figure 2.10

Tacca integrifolia

31

Figure 2.11

Flower Anatomy of Tacca sp.

32

Figure 2.12

Tacca chantrieri and Tacca integrifolia

33

Figure 3.00

Leaves of Tacca integrifolia

37

Figure 3.01

Rhizomes of Tacca integrifolia

38

Figure 3.02

Soxhlet apparatus

40

Figure 3.03

Vacuum rotary evaporator

41

Figure 3.04

Thin Layer Chromatography (TLC) system

44

Figure 3.05

Diagram of TLC plate

45

Figure 3.06

The column chromatography

46

xiii

Figure 3.07

High Performance Liquid Chromatography (HPLC)

48

Figure 3.08

Solvent Filtration Kit

50

Figure 3.09

Mobile phase A and B in sonicator

51

Figure 3.10

Hydrolysis of the substrate Hippuryl- Histidyl- Leucine by angiotensin converting enzyme (ACE)

58

Figure 3.11

The chemical structure of the free stable radical DPPH

63

Figure 4.00

High Performance Liquid Chromatography (HPLC) of standard gallic acid

Figure 4.01

High Performance Liquid Chromatography (HPLC) of standard tannic acid

Figure 4.02

97

High Performance Liquid Chromatography (HPLC) of standard quercetin

Figure 4.03

96

98

High Performance Liquid Chromatography (HPLC) chromatogram of chloroform leaves extract from Tacca integrifolia

Figure 4.04

99

High Performance Liquid Chromatography (HPLC) chromatograms of chloroform rhizome extract of Tacca integrifolia

Figure 4.05

100

High Performance Liquid Chromatography (HPLC) chromatograms of methanol leaves extract of Tacca integrifolia

Figure 4.06

101

High Performance Liquid Chromatography (HPLC) chromatograms of methanol rhizomes extract of Tacca integrifolia

102

xiv

Figure 4.07

High Performance Liquid Chromatography (HPLC) chromatograms of water leaves extract of Tacca integrifolia

Figure 4.08

103

High Performance Liquid Chromatography (HPLC) chromatograms of water rhizomes extract of Tacca integrifolia

104

Figure 4.09

HPLC Chromatogram of standard of Hippuric acid

105

Figure 4.10

LCMS/MS chromatogram of hexane leaves extract from Tacca integrifolia

Figure 4.11

LCMS/MS chromatogram of Proanthocyanidin Trimer from hexane leaves extract of Tacca integrifolia

Figure 4.12

111

LCMS/MS chromatogram of 1,3,5-triOQA from chloroform leaves extract of Tacca integrifolia

Figure 4.18

110

LCMS/MS chromatogram of Proanthocyanidin Trimer from chloroform leaves extract of Tacca integrifolia

Figure 4.17

110

LCMS/MS chromatogram of p hydroxybenzoic acid from chloroform leaves extract of Tacca integrifolia

Figure 4.16

109

LCMS/MS chromatogram of chloroform leaves extract from Tacca integrifolia

Figure 4.15

109

LCMS/MS chromatogram of Proanthocyanidin trimer from petroleum ether leaves extract of Tacca integrifolia

Figure 4.14

108

LCMS/MS chromatogram of petroleum ether leaves extract from Tacca integrifolia

Figure 4.13

108

111

LCMS/MS chromatogram of 2(3,4-Dihydroxyphenyl)-7hydroxy-5-benzene propanol from chloroform leaves extract of Tacca integrifolia

Figure 4.19

112

LCMS/MS chromatogram of methanol leaves extract from Tacca integrifolia

113

xv

Figure 4.20

LCMS/MS chromatogram of Quinic acid from methanol leaves extract of Tacca integrifolia

Figure 4.21

LCMS/MS chromatogram of 3-Caffeoquinic acid from methanol leaves extract of Tacca integrifolia

Figure 4.22

119

LCMS/MS chromatogram of Proanthocyanidin from water leaves extract of Tacca integrifolia

Figure 4.32

118

LCMS/MS chromatogram of Phenolic Acid Conjugate from water leaves extract of Tacca integrifolia

Figure 4.31

118

LCMS/MS chromatogram of salicylic acid from water leaves extract of Tacca integrifolia

Figure 4.30

117

LCMS/MS chromatogram of Protocatechuic acid from water leaves extract of Tacca integrifolia

Figure 4.29

117

LCMS/MS chromatogram of Quinic acid from water leaves extract of Tacca integrifolia

Figure 4.28

116

LCMS/MS chromatogram of water leaves extract from Tacca integrifolia

Figure 4.27

115

LCMS/MS chromatogram of Proanthocyanidin from methanol leaves extract of Tacca integrifolia

Figure 4.26

115

LCMS/MS chromatogram of Isoflavone glycosides from methanol leaves extract of Tacca integrifolia

Figure 4.25

114

LCMS/MS chromatogram of Dicaffeolquinic acid conjugate from methanol leaves extract of Tacca integrifolia

Figure 4.24

114

LCMS/MS chromatogram of phydroxybenzoic acid from methanol leaves extract of Tacca integrifolia

Figure 4.23

113

119

LCMS/MS chromatogram of Proanthocyanidin trimer from water leaves extract of Tacca integrifolia

120

xvi

Figure 4.33

LCMS/MS chromatogram of hexane rhizome extract from Tacca integrifolia

Figure 4.34

LCMS/MS chromatogram of Proanthocyanidin Trimer from hexane rhizome extract of Tacca integrifolia

Figure 4.35

127

LCMS/MS chromatogram of Proanthocyanidin from water rhizome extract of Tacca integrifolia

Figure 4.45

126

LCMS/MS chromatogram of Dicaffeoquinic acid conjugate from water rhizome extract of Tacca integrifolia

Figure 4.44

126

LCMS/MS chromatogram of water rhizome extract from Tacca integrifolia

Figure 4.43

125

LCMS/MS chromatogram of Gypenoside from methanol rhizome extract of Tacca integrifolia

Figure 4.42

125

LCMS/MS chromatogram of methanol rhizome extract from Tacca integrifolia

Figure 4.41

124

LCMS/MS chromatogram of Gypenoside from chloroform rhizome extract of Tacca integrifolia

Figure 4.40

124

LCMS/MS chromatogram of Triterpenoids Saponins from chloroform rhizome extract of Tacca integrifolia

Figure 4.39

123

LCMS/MS chromatogram of chloroform rhizome extract from Tacca integrifolia

Figure 4.38

123

LCMS/MS chromatogram of Proanthocyanidin trimer isomer from petroleum ether rhizome extract of Tacca integrifolia

Figure 4.37

122

LCMS/MS chromatogram of petroleum ether rhizome extract from Tacca integrifolia

Figure 4.36

122

127

LCMS/MS chromatogram of Proanthocyanidin trimer from water rhizome extract of Tacca integrifolia

128

xvii

Figure 4.46

Standard curve of Gallic acid

130

Figure 4.47

Standard curve of Quercetin

133

Figure 4.48

ACE inhibition of captopril in ACE assay

137

Figure 4.49

ACE inhibition of leaves extracts from Tacca integrifolia

141

Figure 4.50

ACE activity of leaves extract from Tacca integrifolia

142

Figure 4.51

ACE inhibition of rhizome extracts from Tacca Integrifolia

145

Figure 4.52

ACE activity of rhizome extract from Tacca integrifolia

146

Figure 4.53

Histogram of percentage of ACE inhibition of chemical compounds isolated from hexane leaves extract of Tacca integrifolia

Figure 4.54

148

Histogram of percentage of ACE inhibition of chemical compounds isolated from petroleum ether leaves extract of Tacca integrifolia

Figure 4.55

149

Histogram of percentage of ACE inhibition of chemical compounds isolated from chloroform leaves extract of Tacca integrifolia

Figure 4.56

150

Histogram of percentage of ACE inhibition of chemical compounds isolated from hexane rhizome extract of Tacca integrifolia

Figure 4.57

152

Histogram of percentage ACE inhibition of chemical compounds isolated from petroleum ether rhizome extract of Tacca integrifolia

Figure 4.58

153

Histogram of percentage of ACE inhibition of chemical compounds isolated from chloroform rhizome extract

Figure 4.59

of Tacca integrifolia

154

Standard curve of Hippuric acid (HA)

156

xviii

Figure 4.60

Histogram of body weight of SHR sub-acute toxicity test of water leaves extract of Tacca integrifolia

Figure 4.61

158

Histogram of body weight of SHR sub-acute toxicity test from water rhizome extract of Tacca integrifolia

160

Figure 4.62

Histogram of mean body weight of SHR

165

Figure 4.63

Graph of mean blood pressure (mmHg) measurement of SHR

Figure 4.64

DPPH inhibition of leaves extracts from Tacca integrifolia

Figure 4.65

181

Metal chelating activity of leaves extract of Tacca integrifolia

Figure 4.68

179

Graph of Ferric Reducing Power Assay of rhizome extract of Tacca integrifolia

Figure 4.67

174

Graph of Ferric Reducing Power Assay of leaves extract of Tacca integrifolia

Figure 4.66

167

185

Metal chelating activity of rhizome extract of Tacca integrifolia

188

xix

LIST OF TABLES

Table 2.00

Several of antioxidant vitamins, polyphenols and flavonoids

22

Table 2.01

Chemical compounds of Tacca species

35

Table 3.00

DPPH radical scavenging activity of standard ascorbic acid

64

Table 3.01

DPPH radical scavenging activity of extracts of Tacca integrifolia

65

Table 3.02

Metal Chelating Assay of EDTA

67

Table 3.03

Metal Chelating Assay of extract of Tacca integrifolia

68

Table 4.00

Colour observation of leaves and rhizome extracts of Tacca integrifolia

Table 4.01

Solvent system used in Thin Layer Chromatography (TLC) of extracts from leaves and rhizome of Tacca integrifolia

Table 4.02

80

Thin Layer Chromatography of hexane extract from rhizome of Tacca integrifolia

Table 4.07

77

Thin Layer Chromatography of methanol extract from leaves of Tacca integrifolia

Table 4.06

75

Thin Layer Chromatography of chloroform extract from leaves of Tacca integrifolia

Table 4.05

73

Thin Layer Chromatography of petroleum ether extract from leaves of Tacca integrifolia

Table 4.04

71

Thin Layer Chromatography of hexane extract from leaves of Tacca integrifolia

Table 4.03

70

82

Thin Layer Chromatography of petroleum ether extract from rhizome of Tacca integrifolia

84

xx

Table 4.08

Thin Layer Chromatography of chloroform extract from rhizome of Tacca integrifolia

Table 4.09

Thin Layer Chromatography of methanol extract from rhizome of Tacca integrifolia

Table 4.10

93

Compounds identified from TLC of extracts from Tacca integrifolia

Table 4.13

91

Thin Layer Chromatography of water extract from rhizome of Tacca integrifolia

Table 4.12

88

Thin Layer Chromatography of water extract from leaves of Tacca integrifolia

Table 4.11

86

94

Compound detected in Liquid Chromatography Mass Spectrometry/Mass Spectrometry (LCMS/MS) of leaves extract from Tacca integrifolia

Table 4.14

107

Compound detected in Liquid Chromatography Mass Spectrometry/Mass Spectrometry (LCMS/MS) of rhizome extract from Tacca integrifolia

121

Table 4.15

Saponin froth test

129

Table 4.16

Colour changes in tannin and phenolic compound test

129

Table 4.17

Absorbance of Gallic acid

130

Table 4.18

Total phenolic content from leaves extract of Tacca integrifolia

Table 4.19

131

Total phenolic content from rhizome extract of Tacca integrifolia

131

Table 4.20

Absorbance of Quercetin

132

Table 4.21

Total flavonoid content from leaves extract of Tacca integrifolia

134

xxi

Table 4.22

Total flavonoid content from rhizome extract of Tacca integrifolia

Table 4.23

135

Total Phenol and Total Flavonoid content from leaves and rhizome extracts of Tacca integrifolia

135

Table 4.24

ACE inhibition and activity of standard of Captopril

136

Table 4.25

ACE inhibition and ACE activity of hexane leaves extract of Tacca integrifolia

Table 4.26

ACE inhibition and ACE activity of petroleum ether leaves extracts of Tacca integrifolia

Table 4.27

143

ACE inhibition and ACE activity of methanol rhizome extract of Tacca integrifolia

Table 4.34

143

ACE inhibition and ACE activity of chloroform rhizome extract of Tacca integrifolia

Table 4.33

143

ACE inhibition and ACE activity of petroleum ether rhizome extracts of Tacca integrifolia

Table 4.32

141

ACE inhibition and ACE activity of hexane rhizome extract of Tacca integrifolia

Table 4.31

140

ACE inhibition and ACE activity of water leaves extract of Tacca integrifolia

Table 4.30

139

ACE inhibition and ACE activity of methanol leaves extract of Tacca integrifolia

Table 4.29

139

ACE inhibition and ACE activity of chloroform leaves extract of Tacca integrifolia

Table 4.28

138

144

ACE inhibition and ACE activity of water rhizome extract of Tacca integrifolia

144

xxii

Table 4.35

ACE inhibitions and activity of the chemical compounds isolated from hexane leaves extract of Tacca integrifolia

Table 4.36

148

ACE inhibitions and activity of the chemical compounds isolated from petroleum ether leaves extract of Tacca integrifolia

Table 4.37

ACE inhibitions and activity of the chemical compounds isolated from chloroform leaves extract of Tacca integrifolia

Table 4.38

151

ACE inhibitions and activity of the chemical compounds isolated from hexane rhizome extract of Tacca integrifolia

Table 4.40

150

ACE inhibitions and activity of the chemical compounds isolated from methanol leaves extract of Tacca integrifolia

Table 4.39

149

151

ACE inhibitions and activity of the chemical compounds isolated from petroleum ether rhizome extract of Tacca integrifolia

Table 4.41

153

ACE inhibitions and activity of the chemical compounds isolated from chloroform rhizome extract of Tacca integrifolia

Table 4.42

154

ACE inhibitions and activity of the chemical compounds isolated from methanol rhizome extract of Tacca integrifolia

155

Table 4.43

Absorbance of Hippuric acid (HA)

156

Table 4.44

Body weight measurement of SHR on Sub-acute toxicity test of water leaves extract of Tacca integrifolia

Table 4.45

157

Body weight measurement of SHR on Sub-acute toxicity test of water rhizome extract of Tacca integrifolia

159

Table 4.46

Liver function test of SHR undergoing Sub-acute toxicity test

161

Table 4.47

Renal function test of SHR undergoing Sub-acute toxicity test

162

xxiii

Table 4.48

Mean Body Weight of SHR

164

Table 4.49

Mean Systolic Blood pressure of SHR

166

Table 4.50

Liver Function test of blood serum collected from SHR undergoing anti-hypertension treatment

Table 4.51

168

Renal Function test of blood serum collected from SHR undergoing anti-hypertension treatment

169

Table 4.52

DPPH radical scavenging activity of ascorbic acid

171

Table 4.53

DPPH radical scavenging activity of leaves hexane extract from Tacca integrifolia

Table 4.54

DPPH radical scavenging activity of leaves petroleum ether extract from Tacca integrifolia

Table 4.55

175

DPPH radical scavenging activity of rhizome chloroform extract from Tacca integrifolia

Table 4.61

175

DPPH radical scavenging activity of rhizome petroleum ether extract from Tacca integrifolia

Table 4.60

174

DPPH radical scavenging activity of rhizome hexane extract from Tacca integrifolia

Table 4.59

173

DPPH radical scavenging activity of leaves water extract from Tacca integrifolia

Table 4.58

173

DPPH radical scavenging activity of leaves methanol extract from Tacca integrifolia

Table 4.57

173

DPPH radical scavenging activity of leaves chloroform extract from Tacca integrifolia

Table 4.56

172

176

DPPH radical scavenging activity of rhizome methanol extract from Tacca integrifolia

176

xxiv

Table 4.62

DPPH radical scavenging activity of rhizome water extract from Tacca integrifolia

176

Table 4.63

Reducing Power of Butylated Hydroxynisole (BHA)

177

Table 4.64

Reducing power of hexane leaves extract from Tacca integrifolia 178

Table 4.65

Reducing power of petroleum ether leaves extract from Tacca integrifolia

Table 4.66

Reducing power of chloroform leaves extract from Tacca integrifolia

Table 4.67

180

Reducing power of methanol rhizome extract from Tacca integrifolia

Table 4.73

180

Reducing power of chloroform rhizome extract from Tacca integrifolia

Table 4.72

180

Reducing power of petroleum ether rhizome extract from Tacca integrifolia

Table 4.71

179

Reducing power of hexane rhizome extract from Tacca integrifolia

Table 4.70

178

Reducing power of water leaves extract from Tacca integrifolia

Table 4.69

178

Reducing power of methanol leaves extract from Tacca integrifolia

Table 4.68

178

181

Reducing power of water rhizome extract from Tacca integrifolia

181

Table 4.74

Metal Chelating activities of EDTA

183

Table 4.75

Metal Chelating activities of hexane leaves extracts from Tacca integrifolia

183

xxv

Table 4.76

Metal Chelating activities of petroleum ether leaves extracts from Tacca integrifolia

Table 4.77

Metal Chelating activities of chloroform leaves extracts from Tacca integrifolia

Table 4.78

189

Probit analysis table of hexane extracts from leaves of Tacca integrifolia

Table 4.88

189

Number of dead shrimp in BSLA of rhizomes extract from Tacca integrifolia

Table 4.87

187

Number of dead shrimp in BSLA of leaves extract from Tacca integrifolia

Table 4.86

187

Metal Chelating activities of water rhizome extracts from Tacca integrifolia

Table 4.85

187

Metal Chelating activities of methanol rhizome extracts from Tacca integrifolia

Table 4.84

186

Metal Chelating activities of chloroform rhizome extracts from Tacca integrifolia

Table 4.83

186

Metal Chelating activities of petroleum ether rhizome extracts from Tacca integrifolia

Table 4.82

185

Metal Chelating activities of hexane rhizome extracts from Tacca integrifolia

Table 4.81

184

Metal Chelating activities of water leaves extracts from Tacca integrifolia

Table 4.80

184

Metal Chelating activities of methanol leaves extracts from Tacca integrifolia

Table 4.79

184

190

Probit analysis table of petroleum ether extracts from leaves of Tacca integrifolia

190

xxvi

Table 4.89

Probit analysis table of chloroform extracts from leaves of Tacca integrifolia

Table 4.90

Probit analysis table of methanol extracts from leaves of Tacca integrifolia

Table 4.91

192

Probit analysis table of methanol extracts from rhizomes of Tacca integrifolia

Table 4.96

192

Probit analysis table of chloroform extracts from rhizomes of Tacca integrifolia

Table 4.95

191

Probit analysis table of petroleum ether extracts from rhizomes of Tacca integrifolia

Table 4.94

191

Probit analysis table of hexane extracts from rhizomes of Tacca integrifolia

Table 4.93

191

Probit analysis table of water extracts from leaves of Tacca integrifolia

Table 4.92

190

192

Probit analysis table of water extracts from rhizomes of Tacca integrifolia

192

xxvii

LIST OF SYMBOLS AND ABBREVIATIONS

α-TOH

Alpha-Tocopherol

β-CAR

Beta-carotene

ACE

Angiotensin Converting Enzyme

ACEI

Angiotensin Converting Enzyme Inhibitor

AlCl2

Aluminum chloride

AP-I

activated protein-I

AscO-

Ascorbate

AT1

Angiotensin II Type 1

AT2

Angiotensin II Type 2

BHA

Butylated Hydroxyanisole

BHT

Butylated Hydroxytoluene

BiONO3

Bismuth nitrate

Bp

Blood pressure

BSLA

Brine Shrimp Lethality Assay

CAT

catalase

CAP

Captopril

CVD

Cardiovascular Disease

DPPH

1,1-diphenyl-2-picrylhydrazyl

EDTA

Ethylenediaminetetraacetic acid

ENA

Enalapril

FeCl2

Ferric Chloride

GPX

gluthathione peroxidase

H2 O2

Hydrogen peroxide

HA

Hippuric acid

xxviii

HAc

Glacial acetic acid

HCl

Hydrochloric acid

HHL

Hippuryl-L-Histidyl-L-Leucine

HL

histidyl-L-leucine

HPLC

High Performance Liquid Chromatography

IC50

Half maximal Inhibitory concentration

K+

Potassium

KI

Potassium Iodide

LC50

Median Lethal concentration

LCMS/MS

Liquid Chromatography Mass Spectrometry /Mass Spectrometry

LDL

Low Density Lipoprotein

MAPKs

mitogen-activated protein kinase

Na+

Sodium

NaCl

sodium chloride

NADPH

Nicotinamide adenine dinucleotide Phosphate-oxidase

NANO3

Sodium nitrate

NAOH

Sodium hydroxide

NFκB

nuclear transcription factor kappa-β

NSAIDs

nonsteroidal anti-inflammatory drugs

O2

Oxygen

1

Single oxygen

O2

O2˙-

superoxide anion radicals

OD

Optical Density

OH˙

Hydroxyl radicals

OS

Oxidative Stress

xxix

RAAS

Renin Angiotensin Aldosterone System

Rf

Retention factor

ROS

Reactive Oxygen Species

SHR

Spontaneously Hypertensive Rats

SOD

Superoxide dismutase

TCA

Trichloroacetic acid

TFA

Trifluoroacetic acid

TLC

Thin Layer Chromatography

xxx

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ABSTRACT Tacca integrifolia Ker-Gawl is belongs to the family of

ABSTRACT Tacca integrifolia Ker-Gawl is belongs to the family of Taccaceae and locally known as “Belimbing Tanah”. It has been used traditionally for...

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