Department of Chemistry Gc University Lahore

EVALUATION OF PHYTOMEDICINAL ATTRIBUTES AND METABOLITES’ IDENTIFICATION IN LEAVES OF HYOPHORBE INDICA AND HYOPHORBE LAGENICAULIS

James William SESSION 2015-2018 Registration No. 2015-GCUL-PhD-CHEM-62

DEPARTMENT OF CHEMISTRY GC UNIVERSITY LAHORE

EVALUATION OF PHYTOMEDICINAL ATTRIBUTES AND METABOLITES’ IDENTIFICATION IN LEAVES OF HYOPHORBE INDICA AND HYOPHORBE LAGENICAULIS

Submitted to GC University Lahore in partial fulfillment of the requirements

for the award of the degree of

DOCTOR OF PHILOSOPHY IN CHEMISTRY

BY James William SESSION 2015-2018 Registration No. 2015-GCUL-PhD-CHEM-62

DEPARTMENT OF CHEMISTRY GC UNIVERSITY LAHORE

DECLARATION

PLAGIARISM UNDERTAKING

RESEARCH COMPLETION CERTIFICATE

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CERTIFICATE OF APPROVAL

DEDICATION

My Parents (Late), beloved wife, my sons Shah Rukh

James, Hammad James and my daughter Aman James for their prayers and sacrifices to support me and to boost my

morale for the achievement of this endeavor.

ACKNOWLEDGEMENT

I am very thankful to Almighty God for providing me this opportunity to complete my

Doctorate in the field of chemistry at the beautiful, historical, richest and best endowed campus of Government College University, Lahore, Pakistan.

This experience was very virtuous, and I enjoyed healthy learning during my research work. The working not only improved my laboratory skills but also enhanced and updated the knowledge in the field. It was not as simple as it seems today but there were untiring efforts behind that made the things smooth and positive. Inspite of my efforts it was God’s grace that made me able to complete this endeavor.

I would like to offer the heartiest gratitude to my PhD supervisor Prof. Dr. Peter John, who supported and guided me beyond the imagination. His affection and supervision were full of comfort and ease to run the work at a smooth and steady pace. I would also like to pay special thanks to the worthy Prof. Dr. Ahmad Adnan, Chairperson and

Professor/Dean, Faculty of Chemistry and Life Science, Government College

University, Lahore, Pakistan. His continuous support and guidance always provided me an urge to learn and to accomplish the achievements heartedly and devotedly. I offer my special thanks to Dr. Muhammad Waseem Mumtaz, Assistant Professor of

Chemistry at the University of Gujrat, Pakistan, for his affection, technical support and professional guidance.

I am also very thankful to Prof. Dr. Hamid Mukhtar, Director, the Institute of

Industrial Biotechnology University, Lahore, for his technical assistance and special motivation.

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TABLE OF CONTENTS

DECLARATION ...... i PLAGIARISM UNDERTAKING ...... ii RESEARCH COMPLETION CERTIFICATE ...... iii CERTIFICATE OF APPROVAL ...... iv DEDICATION ...... v ACKNOWLEDGEMENT ...... vi LIST OF FIGURES ...... xi LIST OF TABLES ...... xv LIST OF ABBREVIATIONS ...... xvi AFFIRMATION ...... xix ABSTRACT ...... xx

INTRODUCTION...... 1

1.5.1 Synthetic Drugs for Diabetes Mellitus Type-2 ...... 8 1.5.2 The Dietary Enzyme Inhibitors ...... 9 1.5.3 Plant-Based Treatment of Diabetes Mellitus Type-2 ...... 10

LITERATURE REVIEW ...... 18

EXPERIMENTAL WORK ...... 32

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3.7.1 The DPPH Assay ...... 36 3.7.2 Total Antioxidant Power Assay ...... 37 3.7.3 Iron Chelating Activity ...... 37

3.8.1 The α-Glucosidase Inhibition Activity ...... 38 3.8.2 The α-amylase Inhibitory Assay ...... 38 3.8.3 Acetylcholine Esterase Inhibition Assay ...... 39

3.9.1 The 1HNMR Prediction of Metabolites ...... 39 3.9.2 UHPLC-QTOF-MS/MS Analysis ...... 40

3.11.1 Oral Glucose Tolerance Test ...... 41 3.11.2 Anti-Hyperglycemic Assessment ...... 41 3.11.3 Hypolipidemic Activity ...... 43

RESULTS AND DISCUSSION ...... 45

4.4.1 DPPH Activity ...... 51 4.4.2 Total Antioxidant Power Assay (TAP)...... 54 4.4.3 The β-Carotene Bleaching Assay ...... 56 4.4.4 Iron Chelating Activity ...... 58

4.5.1 The α-Glucosidase Inhibition Assay ...... 60 4.5.2 The α-Amylase Inhibition ...... 63 4.5.3 The Acetylcholine Esterase Inhibition ...... 65

4.6.1 The 1HNMR Based Identification of Metabolite Class ...... 67 4.6.2 Metabolite Identification by UHPLC-QTOF-MS/MS ...... 72

4.9.1 The Blood Glucose Level ...... 98 4.9.2 Blood Haemoglobin Level ...... 105 4.9.3 Hypolipidemic Assessment in Diabetic Mice ...... 107

CONCLUSION ...... 115

LIST OF FIGURES

Fig. 1-1 The role of ROS and antioxidant defense in diabetes mellitus type 2...... 7 Fig. 1-2 Hyophorbe lagenicaulis ...... 16 Fig. 1-3 Hyophorbe indica ...... 16 Fig. 3-1 Schematic diagram of methodology ...... 44

Fig. 4-1 IC50 values of H. indica extracts for DPPH scavenging ...... 52

Fig. 4-2 IC50 values of H. lagenicaulis extracts for DPPH scavenging ...... 53 Fig. 4-3 Antioxidant power of H. indica leaf extracts ...... 54 Fig. 4-4 Antioxidant power of H. lagenicaulis leaf extracts...... 55 Fig. 4-5 The % inhibition of β-carotene by leaf extracts of H. indica ...... 57 Fig. 4-6 The % inhibition of β-carotene by leaf extracts of H. lagenicaulis ...... 58 Fig. 4-7 Iron chelating activity of H. indica leaf extracts ...... 59 Fig. 4-8 Iron chelating activity of H. lagenicaulis leaf extracts ...... 60 Fig. 4-9 The α-glucosidase of H. indica leaf extracts ...... 62 Fig. 4-10 The α-glucosidase of H. lagenicaulis leaf extracts ...... 62 Fig. 4-11 The α-amylase inhibtion of H. indica leaf extracts ...... 64 Fig. 4-12 The α-amylase inhibtion of H. lagenicaulis leaf extracts ...... 64 Fig. 4-13 The acetylcholine esterase inhibition by H. indica leaf extracts ...... 66 Fig. 4-14 The acetylcholine esterase inhibition by H. lagenicaulis leaf extracts ...... 66 Fig. 4-15 Main 1HNMR spectrum of 60% ethanolic leaf extract of H. lagenicaulis 68 Fig. 4-16 Expanded 1HNMR spectrum (6-9 ppm) of 60% ethanolic leaf extract of H...... 68 Fig. 4-17 Expanded 1HNMR spectrum (3-6 ppm) of 60% ethanolic leaf extract of H...... 69 Fig. 4-18 Expanded 1HNMR spectrum (1-2.6 ppm) of 60% ethanolic leaf extract of ...... 69 Fig. 4-19 Main 1HNMR spectrum of 60% ethanolic leaf extract of H. indica ...... 70 Fig. 4-20 Expanded 1HNMR spectrum (6-8 ppm) of 60% ethanolic leaf extract of H. indica...... 70 Fig. 4-21 Expanded 1HNMR spectrum (3-6 ppm) of 60% ethanolic leaf extract of H. indica...... 71 Fig. 4-22 Expanded 1HNMR spectrum (1-3 ppm) of 60% ethanolic leaf extract of H. indica...... 71

Fig. 4-23 Chromatogram of 60% ethanolic extract of H. indica ...... 73 Fig. 4-24 Mass spectrum of citric acid...... 75 Fig. 4-25 Fragmentation of citric acid ...... 75 Fig. 4-26 Mass spectrum of procyanidin B3 ...... 76 Fig. 4-27 Mass spectrum of procyanidin B2 ...... 76 Fig. 4-28 Mass spectrum of procyanidin B1 ...... 77 Fig. 4-29 Fragmentation pattern of procyanidin ...... 78 Fig. 4-30 Mass spectrum of epicatechin ...... 79 Fig. 4-31 Mass spectrum of catechin ...... 79 Fig. 4-32 Fragmentation pattern of epicatechin ...... 80 Fig. 4-33 Mass spectrum apigenin-C-hexocide-C-hexocide ...... 80 Fig. 4-34 Fragmentation pattern of apigenin-C-hexocide-C-hexocide ...... 81 Fig. 4-35 Mass spectrum of kaempferol ...... 82 Fig. 4-36 Mass fragmentation pattern of kaempferol ...... 82 Fig. 4-37 Mass spectrum of quinic acid derivative ...... 83 Fig. 4-38 Mass spectrum of gallic acid...... 83 Fig. 4-39 Fragmentation pattern of gallic acid...... 84 Fig. 4-40 Chromatogram of 60% ethanolic extract of H. lagenicaulis ...... 84 Fig. 4-41 Mass spectrum of citric acid...... 87 Fig. 4-42 Fragmentation pattern of citric acid ...... 87 Fig. 4-43 Mass spectrum of trimethoxyflavone derivative ...... 88 Fig. 4-44 Mass spectrum of kaempferol...... 88 Fig. 4-45 Fragmentation pattern of kaempferol ...... 89 Fig. 4-46 Mass spectrum of rutin ...... 89 Fig. 4-47 Fragmentation pattern of rutin...... 90 Fig. 4-48 Mass spectrum of hesperetin-5-O-glucoside ...... 90 Fig. 4-49 Fragmentation pattern of hesperetin-5-O-glucoside ...... 91 Fig. 4-50 Mass spectrum of kaempferol coumaroyl glucoside ...... 91 Fig. 4-51 Fragmentation pattern of kaempferol coumaroyl glucoside ...... 92 Fig. 4-52 Mass spectrum of luteolin 3-glucoside...... 92 Fig. 4-53 Fragmentation pattern of luteolin 3-glucoside ...... 93 Fig. 4-54 Mass spectrum of isorhamnetin-3-O-rutinoside ...... 93 Fig. 4-55 Fragmentation pattern of isorhamnetin-3-O-rutinoside ...... 94

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Fig. 4-56 Cytotoxicity evaluation of 60% ethanolic plant extracts of H. indica and H...... 96 Fig. 4-57 The effect of temperature on DPPH radical scavenging % ...... 98 Fig. 4-58 The impact of H.indica leaf extracts on blood glucose level of mice (NG = normal mice group, DG = diabetic mice group, MG = metformin group, HG = half dose group, FG = full dose group)...... 99 Fig. 4-59 The impact of H.lagenicaulis leaf extracts on blood glucose level of mice. (NG = normal mice group, DG = diabetic mice group, MG = metformin group, HG = half dose group, FG = full dose group)...... 100 Fig. 4-60 The impact of H.indica leaf extracts on blood haemoglobin level of mice (NG = normal mice group, DG = diabetic mice group, MG = metformin group, HG = half dose group, FG = full dose group)...... 105 Fig. 4-61 The impact of H.lagenicaulis leaf extracts on blood haemoglobin level of mice (NG = normal mice group, DG = diabetic mice group, MG = metformin group, HG = half dose group, FG = full dose group)...... 106 Fig. 4-62 The impact of H.indica leaf extracts on total cholestrol (TC), high density lipoproteins (HDL) and low density lipoproteins (LDL) of mice (NG = normal mice group, DG = diabetic mice group, MG = metformin group, HG = half dose group, FG = full dose group)...... 107 Fig. 4-63 The impact of H.lagenicaulis leaf extracts on total cholestrol (TC), high density lipoproteins (HDL) and low density lipoproteins (LDL) of mice (NG = normal mice group, DG = diabetic mice group, MG = metformin group, HG = half dose group, FG = full dose group)...... 108 Fig. 4-64 Changes in body weight (g) of mice treated with H. indica (NG = normal mice group, DG = diabetic mice group, MG = metformin group, HG = half dose group, FG = full dose group)...... 110 Fig. 4-65 Changes in body weight (g) of mice treated with H. lagenicaulis (NG = normal mice group, DG = diabetic mice group, MG = metformin group, HG = half dose group, FG = full dose group)...... 111 Fig. 4-66 Water uptake (mL) by mice treated with H. indica(NG = normal mice group, DG = diabetic mice group, MG = metformin group, HG = half dose group, FG = full dose group)...... 112

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Fig. 4-67 Water uptake (mL) by mice treated with H. lagenicaulis(NG = normal mice group, DG = diabetic mice group, MG = metformin group, HG = half dose group, FG = full dose group)...... 113

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LIST OF TABLES

Table 1.1 Taxonomic classification of the Hyophorbe lagenicaulis and Hyophorbe indica...... 15 Table 3.1 Nutritive proportion of nutrients in kcal/g ...... 34 Table 3.2 Detail of solvent system used for extraction ...... 35 Table 3.3 Experimental conditions during in-vivo study ...... 42 Table 4.1 The results of the proximate analysis for H. indica and H. lagenicaulis ..... 45 Table 4.2 Mineral composition of H. indica and H. lagenicaulis ...... 46 Table 4.3 Extract yield of H. indica and H. lagenicaulis ...... 47 Table 4.4 Total phenolic content of H. indica and H. lagenicaulis...... 49 Table 4.5 Total flavonoid content of H. indica and H. lagenicaulis...... 51 Table 4.6 Chromatographic and mass spectrometric data of compounds in H. indica, including retention time (Rt) and major fragment peaks ...... 74 Table 4.7 The retention times (Rt) and mass spectrometric data of major compounds in H. lagenicaulis ...... 85 Table 4.8 The identified compounds and their classes ...... 95

LIST OF ABBREVIATIONS

AMPK Adenosine monophosphate kinase

ATP Adenosine triphosphate

ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)

AChE Acetylcholine esterase

ASE Ascorbic acid equivalent

BHA Butylated hydroxyanisole

BGL Blood glucose level

DM Diabetes mellitus

DMT1 Diabetes mellitus type 1

DMT2 Diabetes mellitus type 2

DNA Deoxyribonucleic acid

DPPH 2,2-diphenyl picrylhydrazil

DE Dried extract

DNS Dinitrosalicylic acid

EDTA Ethylenediaminetetraacetic acid

FRAP Ferric reducing antioxidant power

FG Full dose group

GLUT Glucose transport protein

GPX Glutathione peroxidase

GC-MS Gas chromatography mass spectrometry

GAE Gallic acid equivalent

HDL High density lipoprotein

HG Half dose group

1HNMR Proton nuclear magnetic resonance

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Hb Haemoglobin

KH2PO4 Potassium dihydrogen phosphate

LDL Low density lipoprotein

DG Diabetic group

NG Normal mice group

DG Diabetic untreated group

PE Plant extract

MG Metformin group m/z Mass to charge ratio

HG Half dose group

FG Full dose group

ORAC Oxygen radical absorbance activity

PNGP Para nitrophenyl glucopyranoside

PNPG 4-Nitrophenyl β-D-glucopyranoside

QTOF Quadrupole time of flight

ROS Reactive oxygen species

RE Rutin equivalent

SGOT Serum glutamic oxaloacetic transaminase

SOD Superoxide dismutase

SGPT Serum glutamic pyruvic transaminase

TBHQ Tertiary butylhydroquinone

TPC Total phenolic contents

TFC Total flavonoid contents

TMS Tetramethylsilane

TSP Trisodium phosphate

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TAP Total antioxidant power

TC Total cholesterol

UHPLC Ultra-high performance liquid chromatography

VLDL Very low-density lipoprotein

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AFFIRMATION

The work reported in this thesis has not been published elsewhere, except for the following:

1. William, J., John, P., Mumtaz, M. W., Ch, A. R., Adnan, A., Mukhtar, H., Sharif,

S., Raza, S. A. (2018). Antioxidant activity, Hypoglycemic potential and

metabolite profiling of Hyophorbe indica leaf extract. Pakistan Journal of

Pharmaceutical Sciences, 31(6 (Supplementary), 2737-2742.

2. William, J., John, P., Mumtaz, M.W., Ch, A.R., Adnan, A., Mukhtar, H., Sharif,

S., Raza, S.A. and Akhtar, M.T., (2019). Antioxidant activity, α-glucosidase

inhibition and phytochemical profiling of Hyophorbe lagenicaulis leaf

extracts. PeerJ, 7, p.e7022.

3. Antioxidant and Antidiabetic Potential of Hyophorbe indica and Hyophorbe

lagenicaulis from Pakistan at National symposium on emerging trends in the

extraction of plant bio-actives for Nutra-pharmaceutical developments held on

November 1-2, 2017 at University of Sargodha, Pakistan (HEC approved and

supported)

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ABSTRACT

Herbal medicines need to be explored and developed with the occurrence of drug resistance and high rate of metabolic dysfunctions. The treatment by naturally occurring substances has always been of advantages and this fact has been accepted globally. The current research was performed to explore the Hyophorbe indica and

Hyophorbe lagenicaulis of family Arecaceae for their possible medicinal significance and metabolites’ identification. As per available literature, limited scientific evidence is available on these plants in spite of their traditional use as a health tonic. The hydroethanolic extract of aerial parts of both plants were prepared by using pure ethanol, water and their mixtures of various combinations. For extract preparation, the leaves were quenched with liquid nitrogen and subjected to freeze-drying. The freeze- dried leaf powder was extracted by submerging in pure water, 20% ethanol, 40% ethanol, 60% ethanol, 80% ethanol and pure ethanol for 48 hours and shook for 2 hours and sonicated for 30 minutes. The plant extracts were filtered and the extra solvent was evaporated under vacuum. Extracts were stored at low temperature in refrigerator for further use. The prepared extracts were evaluated for total phenolic contents, flavonoid contents, 2,2-diphenyl-1-picrylhydrazyl scavenging, total antioxidant power assay, α-glucosidase and α-amylase inhibitions. Proton magnetic resonance (1HNMR) and ultra-high-performance liquid chromatography equipped with the mass spectrometer (UHPLC-QTOF-MS/MS) were used for phytochemical identification in most potent extract. The extract yields indicated that 60% ethanolic extract produced relatively higher amounts of extracts. The findings revealed that

60% ethanolic extracts of H. indica and H. lagenicaulis exhibited the highest total phenolic contents of 208.77±2.11 and 178.56 ± 1.47 mg gallic acid equivalent per

ABSTRACT gram dried extract, respectively. The maximum total flavonoid contents of

173.90±2.30 and 133.96 ± 1.19 mg rutin equivalent per gram dried extract were also obtained for 60% ethanolic extracts of H. indica and H. lagenicaulis, respectively.

The 2,2-diphenyl picrylhydrazil activity in terms of IC50 indicated that 60% ethanolic extracts of H. indica and H. lagenicaulis exhibited highest antioxidant activity of

35.35 ± 0.189 µg/mL and 43.11 ± 0.96 μg/mL, respectively. The total antioxidant power of 330.26 ± 3.13 ascorbic acid equivalent per gram plant extract (ASE/g PE) was obtained for 60% ethanolic extract of H. indica, which was the highest among all extracts. The total antioxidant power of 239.33 ± 3.78 ASE/g PE was obtained for

60% ethanolic extract of H. lagenicaulis. The β-carotene linoleic acid assay also reflected that 60% ethanolic extracts were the most active fraction to stop the β- carotene color bleaching. The highest α-glucosidase activity of 36.52 ± 0.08 μg/mL in terms of half minimum inhibitory concentration (IC50) was observed for 60% H. indica leaf extract while it was 41.25 ± 1.25 μg/mL for H. lagenicaulis. The 60% ethanolic extract of H. indica and H. lagenicaulis possessed an IC50 value of 58.2 ±

1.25µg/mL and 60.58±3.24 µg/mL respectively, to inhibit α-amylase activity. In case of acetylcholine esterase assay, the 60% extracts were most effective but not as effective as the extracts were for α-glucosidase and α-amylase. Metabolite profiling indicated the presence of citric acid, procyanidin B1, procyanidin B2, procyanidin B3, apigenin-c-hexocide-c-hexocide, gallic acid, kaempferol and derivatives of kaempferol and quinic acid were identified in 60% ethanolic extract of H. indica.

Similarly, 60% ethanolic extract of H. lagenicaulis also possessed functional molecules like kaempferol, hesperetin-5-O-glucoside and kaempferol-coumaroyl- glucoside, isorhamnetin-3-O-rutinoside. Other identified compounds were mainly luteolin-3-glucoside, citric acid and derivative of trimethoxy flavone. The excellent

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ABSTRACT in-vitro α-glucosidase and α-amylase activities by 60% ethanolic extracts and phytochemicals identified in these extracts urged to move for in-vivo antidiabetic activities in alloxan monohydrate induced diabetic mice. The mice were injected with alloxan dose of 150 mg/kg of body weight. The diabetic mice were treated with metformin 250 mg/kg of body weight. The extract doses of 250 mg/kg of body weight and 450 mg/kg of body weight were applied. The extract dose of 450 mg/kg of body weight of 60% ethanolic extract of H. indica was very effective to bring the elevated blood glucose level of diabetic mice within the normal range after 28 days. Moreover, the same dose was also found effective to improve the blood hemoglobin and lipid profile of diabetic mice when compared with normal mice. The antidiabetic potential of leaf extracts was most probably related to the antioxidant properties of the compounds present. These compounds were probably helpful to reduce the level of stress and to regenerate the normal functioning of the body by streamlining the metabolic processes. The findings recommend H. indica and H. lagenicaulis as the new and potent sources of antioxidant and antidiabetic agents which may be utilized to enhance the functionalities of food for the management and prevention of diabetes mellitus, which has been prevailing much and is the cause of many other malfunction of human body.

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INTRODUCTION

Nature has blessed life with a systematic balance between various body functions for smooth working. The strong linkage between nature and health provides the foundations of life. The human needs care for the diversified pools of nature to sustain life. The human response in the form of aerobic respiration to produce required energy generates some species that may have negative impacts on the system. These species are known as reactive oxygen species (ROS). The metabolism maintains the energy homeostasis of the body through numerous biochemical processes. These metabolic processes are essential to sustain a normal and healthy body function. Hence, ROS production is a complementary part of metabolism.

Reactive Oxygen Species

The production of ROS is an essential feature of metabolism, but there are some additional factors being responsible for increasing the ROS exposure. The external factors mainly air pollutants also contribute in this regard [1]. The ROS are produced and regulated at various sites in mitochondria by the transfer of electrons and also by metal-catalyzed reactions. The ROS play actively in apoptosis, activation of signaling cascades and gene expression. The sources of ROS may be endogenous or exogenous.

The production of ROS is accelerated in phagocytic cells due to excessive consumption of oxygen [2]. The reduced form of coenzyme Q via semiquinone intermediate which transfers the electrons to molecular oxygen to form superoxide.

This ROS production is non-enzymatic in nature and become faster at a high metabolic rate [3]. In peroxisomes, hydrogen peroxide (H2O2) is produced due to the

INTRODUCTION transfer of the electrons from metabolites to oxygen. Similarly, the cytochrome P-450 and B-5 enzymes in endoplasmic reticulum produce ROS, and this process is enzymatic in nature.

The environmental factors are also an impactful external source of ROS. The exposure of the body to environmental factors like air pollutants (smoke, smog, ozone and free radicals), heavy metals, water toxins, alcohols, pesticides, ultraviolet radiations and certain drugs result in the high concentration of ROS in the system [4].

These reactive species are known to play important physiological functions related to cell responses to multiple stimuli. Their production directly reflects the metabolic function at cellular levels. They play a vital role in development by regulating the basic functions of the cell. The atomic structure of oxygen is responsible for the production of ROS by gaining an electron.

The production of such harmful chemically reactive species is balanced by the antioxidant defense mechanism of the body. The excessive production of these species is very harmful to the living system. The indigenous and exogenous antioxidants balance the overproduction of ROS to maintain the homeostasis.

The singlet oxygen, hydroxyl radical, hydrogen peroxide and superoxide anion are major reactive species which are produced as a result of metabolic processes and are hazardous to the living cell. The ROS significantly impact the structure-based functions of biomolecules at the cellular and organism level. These alterations in biomolecules are responsible for a variety of diseases, including diabetes, cancer and neurodegenerative diseases [5, 6].

INTRODUCTION

Antioxidant Defense

The ROS are counterbalanced by antioxidants present in living systems.

Antioxidants are substances which capture the ROS to maintain the redox equilibrium. The antioxidants are of two types, synthetic and natural. The enzymes in the body act as natural antioxidants to pose a check on ROS production. The enzyme superoxide dismutase is well-known antioxidant, plays a vital role to prevent oxidative stress. Similarly, glutathione peroxidases and reductases are also natural antioxidants in the body. Some necessary vitamins like vitamin A and E are also important molecules with the antioxidative effect, which works endogenously [7].

•− Superoxide dismutase (SOD) is an important enzyme to scavenge O2 and has three isoforms;

• Cytoplasmic Cu/Zn SOD

• Mitochondrial Mn/Zn SOD

• Extra Cellular Cu/Zn SOD

Glutathione peroxidases are important antioxidant enzymes having selenium which catalyze the conversion of lipid peroxides and H2O2 to water and alcohols.

These enzymes terminate the ROS pathway and maintain the homeostasis [8].

Glutathione reductase also inhibits the oxidative burden to maintain the reducing regime of the cell. These enzymes are natural antioxidant defense line of the body.

Besides these molecules, some synthetic compounds are also there with well- established antioxidant properties.

The synthetic antioxidants have impressive potential to capture the ROS.

These organic compounds are industrially very important and are being used in the food industry. The butylated hydroxyanisole, butylated hydroxytoluene, tertiary-butyl

INTRODUCTION hydroquinone (TBHQ) are industrially important synthetic antioxidants of polyphenolic nature. These antioxidants are widely in use for commercial purposes, including the food industry. Vegetable oils are frequently stabilized with synthetic antioxidants. However, the toxicity of these compounds is a serious issue. The toxicity associated with these antioxidants is of great concern among consumers.

Therefore, the search for novel antioxidants is continued by exploring medicinal plants, which are a rich source of antioxidants with low or no toxicity.

The natural antioxidants are getting importance due to their effective role to counter ROS and oxidative stress with least toxicity level. The plant phenolics are very effective antioxidants with no or negligible toxicity. The consumption of diet rich in antioxidants may provide a safe and effective choice to reduce the level of

ROS and to minimize the oxidative stress for disease prevention and management.

Oxidative Stress

A balance between ROS and antioxidant defense is usually maintained, however, oxidative stress is developed when there is an imbalance between ROS and antioxidants. The ROS are continuously produced as a result of metabolic activity, and overproduction creates a significant difference in ROS and antioxidants. The difference in concentration of ROS and antioxidant defense system is not a good sign and may be associated with serious forthcoming problems. This situation is the start of oxidative stress which later on may be intensified. The oxidative stress is the precursor of many health disorders. The state of oxidative stress is harmful as it causes damage to the tissues, cellular structures, DNA, and other biomolecules to disturb the metabolic pathways. The damages to living systems by ROS and oxidative

INTRODUCTION stress results in the pathogenesis of chronic diseases like diabetes,

Alzheimer’s disease, cancer, ageing and arthritis [9].

There are many factors responsible for the overproduction of ROS and the development of a state of oxidative stress. Biochemical processes and environmental factors like pollutants, smoking and exposure to toxic substances are the major contributing features towards establishing high ROS leading to the state of oxidative stress [10].

Reactive Oxygen Species, Oxidative Stress and Metabolic Disorders

Metabolic disorders including insulin resistance, diabetes mellitus and cardiovascular disorders are collectively termed as metabolic syndrome. The ROS overproduction leads to damage proteins and nucleic acids which alters the cellular functions, energy metabolism, cell signaling and inflammation. Various studies have suggested that oxidative stress and inflammation are the leading cause of chronic ailments, including diabetes mellitus, obesity and cardiac disorders. Among metabolic disorders, diabetes mellitus is one of the leading ailments, which is affecting people all over the globe. Diabetes mellitus is known as a metabolic disorder which is characteristically recognized by the high level of blood glucose [11]. The diabetes is mainly of two types, the type 1 and type 2 (DMT1 and DMT2). The DMT2 is expanding at a fast rate all over the world and growing exponentially. A study has expected 592 million diabetic people globally by 2035 considering the current pace of expansion [12]. Pakistan is also suffering from this metabolic menace and bearing the expanding socioeconomic burden in this regard. The existing scenario indicates the presence of 11.77% diabetic patients in Pakistan. The prevalence of DMT2 is 14.81%

INTRODUCTION in urban areas of Pakistan, and 10.34% in a rural area. This data also indicated that the prevalence was more in male as compared to female [13].

The modern lifestyle, high caloric food intake, lack of exercise and obesity are the major causes of DMT2 initiation and prolongation. All these factors contribute in the production of ROS, which ultimately develop oxidative stress. The oxidative stress is responsible for diabetes due to changes in enzymatic functions, lipids, DNA, proteins and antioxidant enzymes of the body. The insulin is a hormone which regulates the glucose concentration in the blood. Glucose uptake by the majority of cells is a function of insulin action. Insulin is secreted by the pancreas to maintain the blood glucose level. An increase or decrease in blood glucose level generates hyperglycemia or hypoglycemia, respectively. Proper functioning of the pancreas to secrete insulin is necessary for energy and glucose homeostasis. Insulin production from the pancreas is a responsive phenomenon based upon glucose level in the blood.

When the response of cells to insulin is decreased due to any reason, uptake of glucose by cells is suffered. This state is known as insulin insensitivity which further develops the symptoms of DMT2 [14]. Insulin insensitivity puts pressure on the pancreas to increase insulin production, which may result in the loss of beta cells of the pancreas. The pancreas destruction leads to a reduction in insulin production.

Insulin binds with the cells to open gates for glucose entrance. The GLUT-4 plays an important role in glucose uptake by cells as it binds with glucose to facilitate its entrance. Certain proteins like kinase B activates GLUT-4 production. Any disturbance in insulin activity and GLUT-4 production stops the glucose entry into the cells [15]. This overproduction of ROS may activate the protein kinase signaling to reduce the response of cell receptor sites towards insulin, leading to insulin resistance, a major factor in DMT2 initiation and progression [16]. Insulin not only regulates

INTRODUCTION blood glucose level but also fat building. The oxidative stress is very obvious to develop and propagate DMT2. Similarly, oxidative stress is very aggressive in prolongation of diabetes and related side complications.

Fig. 1-1 The role of ROS and antioxidant defense in diabetes mellitus type 2. The glycation of biomolecules is a well-known chemical process which steadily disturbs the normal body functions. The high blood glucose levels result in the glycation of many proteins and antioxidant enzymes, which reduces the antiradical potential of system and increases the chances of oxidative damage. The reduction in antioxidant enzymes activity by the glycation process may be due to structural variation [17].

The exposure to exogenous sources of ROS may also be another reason to produce oxidative stress. The glycated end products are also responsible for many side complications. Under the diabetic situation, the non-enzymatic glycation of hemoglobin takes place, which reduces the hemoglobin functional efficiency. The glycated hemoglobin causes a microvascular blockage, especially in retinal and nephridial blood capillaries. The glycation of proteins also restricts their working

INTRODUCTION capability leading to abnormalities in normal physiological functions.

Similarly, modifications in high-density lipoproteins and low-density lipoproteins are also observed under diabetic conditions. The side complications during DMT2 due to oxidative damages to biomolecules mainly include retinopathy, kidney dysfunction, micro and cardio-vascular disorders, causing socio-economic burden. It not only builds economic pressure on the patients but also reduces their working capabilities.

Treatment and Management of Diabetes Mellitus Type-2

Different modes of treatment are in use all over the world to treat and manage

DMT2. These treatments mainly include allopathic medicinal system and herbal or plant-based medicinal system.

1.5.1 Synthetic Drugs for Diabetes Mellitus Type-2

There is a lot of focus on the treatment and management of DMT2 throughout the globe. Many synthetic drugs are available in the market to treat hyperglycemia effectively. The sulfonylureas, thiazolidinedione and metformin, are the main pharmaceutics to bring the high blood glucose level to a certain acceptable limit.

These drugs are used to control hyperglycemic conditions, but their mechanism of action is different from each other. Three compounds of thiazolidine class, namely pioglitazone, rosiglitazone and troglitazone, are used in clinical practice to treat diabetes. The troglitazone may cause toxicity in the liver, and regular liver test functions are advised to patients using it [18]. The sulfonylureas increase the intercellular Ca ++ ion concentration in β-cells of the pancreas to increase insulin secretion. However, the hypoglycemia and weight gain are the major side effects of sulfonylureas [19].

INTRODUCTION Metformin is widely used to control blood glucose level.

Metformin acts on the liver and reduces glucose production from the liver by activating adenosine monophosphate kinase (AMPK). The stomach disorders, weight loss, weakness, heartburn and gas are the most common side complications with it

[20].

No doubt, these drugs are very effective to control blood glucose level, but their side effects are of great concern for consumers. The side effects of these drugs mainly include weight loss, high low-density lipoprotein (LDL), fatigue and digestive disorders. Moreover, these drugs are not effective to reverse the damages like β-cell dysfunction, hyperlipidemia and insulin resistance. This scenario urges to discover alternate treatment of DMT2 in a safe, secure and cheaper way.

1.5.2 The Dietary Enzyme Inhibitors

The α-glucosidase inhibitors are considered as an efficient route to control postprandial glucose elevation. The complex carbohydrates are digested in the stomach and are converted into simpler ones. The simplified carbohydrates are absorbed through the intestine to become part of the bloodstream. The α-glucosidases are the enzymes which hydrolyze the carbohydrates in the intestine converting them into glucose. The glucose is absorbed to enter into the bloodstream. The inhibition of

α-glucosidase results in the delay of carbohydrate hydrolysis and consequently decline in postprandial glucose level. Similarly, α-amylase works on starch and glycogen to simplify them for absorption. The inhibition of α-amylase also slows down the digestion of starch, and its conversion into glucose becomes low. As a result, the blood glucose level remains under control [21]. The compounds which occupy some functional, active sites of protein may reduce the activity of protein

INTRODUCTION molecules. This inhibition is usually reflected in terms of binding energies.

The hydrophobic or hydrophilic both types of molecular interactions exist between phytochemicals and enzymes. These interactions at active site residues are studied to predict the enzyme inhibitory potential of compounds.

1.5.3 Plant-Based Treatment of Diabetes Mellitus Type-2

The plants are believed as a rich source of compounds responsible for the momentous decrease in dietary enzyme activity. Plant-based treatments are cost- effective and more acceptable among people due to less toxicity and availability.

Phytochemicals are metabolites produced in plants as a function of various metabolic activities. They possess many biological activities like antioxidant, antidiabetic by inhibiting α-glucosidase and α-amylase action. These compounds are also known to inhibit acetylcholine esterase enzyme activity. Phenolics are defined as a group of similar type of compounds which mainly include phenolic acids along with flavonoids and flavanols. The functional groups of these compounds interact with the dietary enzymes to restrict their activity. They are one of the important metabolites and have a vital therapeutic role in treating diabetes. The gallic acid, ellagic acid, ferulic acid, ascorbic acid, quercetin, rutin, corrilagin, isorhamnetin, ferujol and many more are well known for their phytomedicinal properties [22]. These are an essential component of many medicinal plants ranging from grasses to trees. Fruits and vegetables are also reported to have these functional metabolites. The plants having these compounds are very effective remedy to control hyperglycemia. Unlike synthetic drugs, plant extracts, and herbal formulations provide additional benefits like the repair of β-cells of the pancreas, improved insulin secretion, glucose absorption and hypolipidemia [23]. The plants are well accepted among consumers to

INTRODUCTION treat diabetes due to their safe nature, availability and low cost, especially in Asian and African countries. The plant-based medication of DMT2 recovers the physiological health of patients by improving the secondary aspects of the disease.

The phytochemicals from plants are not only enzyme inhibitors but are also very effective antioxidants. The antioxidant potential of plants is associated with many medicinal properties. The antioxidant compounds present in plants scavenge free radicals and ROS to eliminate the chances of oxidative stress, which in turn reduces the risks to develop DMT2. The in-vitro evaluation of antioxidant activity may be performed by adopting some widely accepted assays like diphenyl- picrylhydrazyl (DPPH) method, total antioxidant power assay (TAP), ferric reducing power assay (FRAP) and β-carotene linoleic assay etc.

The initial screening of plant extracts may be used as an indicator for potential medicinal outcomes. Usually, the high antioxidant potential of plant extract reflects the medicinal prospective. Sometimes the antioxidant activity of the specific extract is used to proceed further to assess its particular activity and metabolite identification.

The extraction process may impart an effective role in the phytomedicinal behavior of the plants. High extract yields may contain high amounts of phenolic compounds and flavonoid contents. Greater concentration of phytochemicals in plant extracts may enhance their medicinal activities.

Various solvents are employed for the extraction of phytochemicals. The solvent extraction is widely used to extract bio-actives from plants and is the most studied aspect of extraction methodology. The solvent polarity is one of the major factors which influences the extraction of metabolites from plant sources. The solvent regime includes aqueous, polar and nonpolar solvents for the purpose of extraction.

The ethanol is proved to be very effective for maximum extraction of phytochemicals

INTRODUCTION of medicinal importance [24]. The hydro-ethanol as solvent also imparts suitable polarity for enhancing the extraction of phenolic and flavonoid compounds

[25]. However, not a single solvent alone can resolve the purpose of high extract yields. That is why the impacts of different solvent compositions are assessed on extract yields and phytochemical contents.

The issues of low extract yields can be solved by screening the preliminary role of extraction solvent in different compositions [17]. Some additional approaches also impart contribution to enhance extraction efficiencies, which are reflected in the form of medicinal potential. Modern techniques like ultrasonication and freeze-drying are widely used to enhance the extract yields from plants. Ultrasonication involves the use of ultrasound radiations to rupture the cell walls of plant cells to release bounded metabolites quite easily. Ultrasound radiations provide energy to blast the plant matrix for maximum release of phytochemicals. However, longer exposure may damage the molecules and activity may be lost due to temperature. Similarly, freeze-drying helps to protect the maximum quantity of metabolites for further extraction [26].

Identification of secondary metabolites is an essential feature of studies, which involves plant extracts and isolates.

Metabolomics

The identification of biologically effective metabolites of plants is of keen interest to locate the reasons behind the medicinal properties of plant extracts. The phytochemical identification remains an area of great importance to develop novel approaches for disease cure and management in a natural way. Recently, metabolomics has emerged as an innovative practice to study the whole metabolome.

INTRODUCTION The liquid or gas chromatography attached with mass spectrometry and nuclear magnetic resonance spectroscopy (NMR) are adopted to study the metabolites under the umbrella of metabolomics. The mass spectrum is helping tool to identify the metabolites by considering their fragmentation patterns. The quadrupole time of flight, coupled with a mass spectrometer (QTOF-MS/MS) has revolutionary aspects in the field of metabolite identification with the complete authenticity [27].

The high-performance liquid chromatography equipped with QTOF-MS/MS is a highly efficient technique with great sensitivity and reliability in plant analysis. The structure elucidation by this technique provides the most reliable information about a specific compound in a mixture. The UHPLC-QTOF-MS/MS is known to give authentic information about the structure of compounds in the complex mixtures like plant extracts. The benefits of UHPLC-QTOF-MS/MS are highest resolution, greatest achievable sensitivity, fastest analysis speed and improved efficiency.

Attraction and Efficacy of Phytotherapy

The plants are well known for their medicinal properties and have gained importance among people due to many reasons. Various studies have explored nature for the treatment of diseases. Therefore, the origin of many drugs goes back to plant products [28]. Recent developments in functional foods and nutraceuticals present in plants as an excellent tool to prevent or cure disease pathogenesis. The phytomedicinal system is being developed on scientific grounds keeping in view the knowledge of ethnopharmacology. Decoctions and teas of many plants are frequently used as a tonic to cure and manage many ailments.

The plant fractions and isolates were reported showing tremendous medicinal characteristics. Many in-vitro and in-vivo studies on plants have ascertained their

INTRODUCTION potential to cure chronic ailments with no or negligible toxicity. A number of plants have been explored for this purpose.

The cost-effectiveness, availability and trust in plant-based medicines have served the people to a great extent and still under expansion as per modern time and scale. Pakistan is the country in Southeast Asia and enriched with a variety of plants.

These plants range from medicinal and food domain to ornamental groups. This variety of plants provides a great deal to explore them for possible or hidden medicinal potential.

Family Arecaceae

The Arecaceae is a botanical family comprising of mainly perennial flowering plants. The plants of this family are climbers, shrubs, and stem-less plants, which are commonly known as palms. Family Arecaceae is also known as palm family and is composed of 181 genera and about 2600 species. Some palm species were reported to have tremendous medicinal applications including aphrodisiac, anti-diarrhea, urinary and vaginal infections, to stop bleeding from cuts and antidote for poison [29]. Palm oil phenolics present in palm fruit juice were reported to have potent anti- hyperglycemic and anti-lipidemic properties with improvements in insulin secretions and lipid profile [30]. Most of the palms of this family are used as traditional medicines in many African, American and Asian countries [31, 32].

The genus Hyophorbe is composed of five species, namely Hyophorbe lagenicaulis, Hyophorbe indica, Hyophorbe amaricaulis, Hyophorbe vaughanii, and

Hyophorbe verschaffeltii [33].

The H. lagenicaulis and H. indica are being grown in Pakistan as ornamental plants, and at the same time, these plants are also in use to treat diabetes in the

INTRODUCTION traditional medicinal system of Pakistan. However, very limited information was available on the medicinal properties and phytoconstituents of

Hyophorbe lagenicaulis and Hyophorbe indica. The findings may be of great use for pharmacological development and progress in the natural way of disease treatment.

The taxonomic classification of both plants is given in Table 1.1

Table 1.1 Taxonomic classification of the Hyophorbe lagenicaulis and Hyophorbe indica

Classification Hyophorbe lagenicaulis Hyophorbe indica

Kingdom Plantae Plantae

Phylum Spermatophyta Spermatophyta

Subphylum Angiospermae Angiospermae

Class Monocotyledonae Monocotyledonae

Order Arecales Arecales

Family Arecaceae Arecaceae

Genus Hyophorbe Hyophorbe

Species Hyophorbe lagenicaulis Hyophorbe indica

INTRODUCTION

Fig. 1-2 Hyophorbe lagenicaulis

Fig. 1-3 Hyophorbe indica

INTRODUCTION

Objectives

The present research work was designed to cover the following domains:

• The optimized fractionation of Hyophorbe lagenicaulis and Hyophorbe indica

• To quantify total phenolic and flavonoids in extracts

• To determine the antioxidant activities

• To evaluate the α-glucosidase and α-amylase inhibitions by extracts

• To confirm the antidiabetic potential of Hyophorbe lagenicaulis and

Hyophorbe indica in albino mice model

• To study the effect of plant extract on lipid profile

• To identify the metabolites of functional importance

• To predict the feasibility of plants as a potential candidate of medicinal

significance

The research results may be helpful to explore these plants as a suitable candidate with a potential role in functional foods with antidiabetic attributes. These findings may open new ways to reduce the socioeconomic burden associated with diabetes in the Pakistan.

LITERATURE REVIEW

Starkov compared the ROS generation capacity of mitochondria and discussed

ROS based signaling at the intracellular level. The ROS overproduction led to oxidative stress, which was responsible for many health disorders. This study also introduced the concept of different ROS species under the headings of generated scavenge and emitted ROS. It was suggested that the most probable strategy to reduce the hazards of ROS was the improvement in the ROS defense system of the body. The non-enzymatic antioxidants may be employed for the purpose [34].

Wright et al. reported oxidative stress as the major cause of many critical health implications like insulin insensitivity, β cells dysfunction and DMT2. The aspects of side complication due to prolong diabetic condition regarding vascular spoilage were also discussed. The overloaded glucose level in the blood was mainly due to excessive dietary intake and sedentary life format, which led to the production of ROS. The reaction of glucose with proteins resulted in the production of glycated end products. It was found proper management of hyperglycemia was suggested to control the vascular dysfunctions at micro and macro levels [35].

Nasri et al. reviewed the role of herbal medicines to control the hyperglycemia as an important therapeutic route for diabetes management. Plants have gained attention to treat diabetes due to their no or negligible toxicity. The antioxidant properties of plants have made them an effective tool to cure chronic ailments by avoiding the side effects of synthetic drugs [23].

Sani et al. evaluated Allium sativum L (Garlic) bulbs, Allium ascalonicum L

(Persian shallot) bulb and Salvia officinalis L (Sage) leaves for possible antidiabetic effect. The impact of methanolic extracts of these plants was studied on SOD,

LITERATURE REVIEW

Glutathione peroxidase (GPX) and catalytic activity in plasma. The triglycerides, total cholesterol high density lipoprotein (HDL), LDL and very low-density lipoprotein

(VLDL) were also measured in plasma of diabetic rats. The findings were compared with standard drug metformin under the same experimental conditions. It was observed that metformin exhibited mild antioxidant and hypolipidemic impact. The phytochemical analysis indicated the presence of alkaloids, glycosides and saponins in Allium sativum L and Allium ascalonicum L extracts. The total phenolic contents were also determined, and their findings revealed that the antioxidant and hypolipidemic properties of these plants made them workable choice to treat diabetes

[36].

Elgindi et al. carried out the isolation and characterization of phyto- metabolites from leaves of Hyophorbe verschaffeltii along with the determination of antioxidant activity. The air-dried leaves were macerated in 70% methanol. The aqueous fraction was used to separate and isolate the compounds by a merger of chromatographies. The isolated organic compounds were characterized by 1HNMR and C13-NMR. The antioxidant activity of 70% methanolic extract was evaluated in

CCl4 induced hepatic injury-based method. The serum enzyme levels (alanine aminotransferase and aspartate aminotransferase) were also measured. Superoxide dismutase and malondialdehyde in the liver were also investigated to assess the level of oxidative stress. The identified compounds included quercetin, luteolin, cannignin, and brisbagenin. The extract dose of 200 mg/kg reflected significant improvements in alanine aminotransferase and aspartate aminotransferase and a notable decline in superoxide dismutase in the rats. Their study revealed that Hyophorbe verschaffeltii extract upon consumption reduced the level of oxidative stress by improving the antioxidant defense of the body [37].

LITERATURE REVIEW

Firdous discussed the developing scenario regarding diabetes and its side complications. The increase in population, rapid urbanization, obesity development and sedentary lifestyle were considered as major factors behind diabetes expansion.

The author mentioned the role of plants as a pool for polyphenols, tannins, terpenoids, alkaloids and flavonoids. These phytochemicals served as an approach to develop new antidiabetic compounds. Many compounds were isolated from plants, and these were proved as effective antidiabetic agents [38].

Yessoufou et al. evaluated the impact of Picralima nitida seeds, Nauclea latifolia roots and stem and Oxytenanthera abyssinica leaves on glucose level of pregnant rats. These plants were used as folk medicine in Africa to treat diabetes during pregnancy. All extracts possessed impressive antioxidant activities might be due to phenolic contents. The most potent antioxidant plant was Picralima nitida, and its ethanolic extract also exhibited a significant decrease in hyperglycemia in rats.

Similarly, butanolic extract of Nauclea latifolia and ethanolic extract of Oxytenanthera abyssinica also effectively controlled the blood glucose level of diabetic pregnant rats. The immunosuppressive activity on T-cell proliferation was also shown by all extracts; however, the Picralima nitida butanol fraction was the most potent probably due to linoleic acid or alkaloids. The biological activities proved the pharmacological role of these plants [39].

Tiwari et al. investigated the impact of oxidative damage in diabetic oriented health problems. The role of composite extract having a combination of antidiabetic plants and fruits at small dose was assessed on glucose level and advanced oxidation products in diabetic rats. Composite extract dose of 100 mg/kg body weight rehabilitated the biochemical indicators due to the antioxidant potential. Their

LITERATURE REVIEW findings optimized the extract dose of composite extract to control diabetes significantly [40].

Ahmad et al. assessed the antidiabetic potential of methanolic extract of Vinca rosea. The 14 days trial of diabetic rats was carried out with an extract dose of 500 and 300 mg/kg. The high extract dose exhibited a substantial reduction in blood glucose compared with a low dose of 300 mg/kg. Improvements in body weight and lipid profile were also observed. The histopathological studies indicated the restoration of β-cells which was the possible reason behind the antidiabetic activity of

Vinca rosea [41].

Agnaniet et al. compared the antidiabetic potentials of Nauclea diderrichii (De

Wild.) Merr. (ND) and Sarcocephalus pobeguinii Hua ex Pellegr. (SP), which were used as folk medicines in Gabon to treat various ailments. However, their uses or treatment of diabetes was more prominent. Their findings showed that the leaf extract of ND was more effective than bark extract and acarbose to inhibit α-glucosidase. The high-performance liquid chromatography (HPLC) fractions of both ND and SP were also evaluated for the same under a similar set of conditions, and results showed the high potential to inhibit α-glucosidase. This inhibition ranged from 80% to 90% at 0.1 mg/mL concentration. The most potent fraction also possessed the highest amounts of phenolic compounds. A direct relationship was observed between α-glucosidase inhibition and phenolic compounds. Their study confirmed the effective ethnopharmacological role of both plants to treat diabetes [42].

Prihantini et al. studied the antioxidant and α-glucosidase inhibitory potential of crude methanolic extracts of few subtropical plants. Various antioxidant assays were used to find antioxidant potential including DPPH, hydrogen peroxide and β- carotene assay. Total phenolic contents were also calculated. The methanolic extract

LITERATURE REVIEW of Elaeocarpus sylvestris, Distylium racemosum, Acer mono Maxim and Liquidambar styraciflua possessed notable phenolic contents, antioxidant potential and α- glucosidase inhibition. These plants were proved as a potential source of antioxidants and antidiabetic agents [43].

Lee et al. investigated the stems and leaves of five medicinal plants for antioxidant and α-glucosidase inhibition. The leaves of Neptunia oleracea exhibited the lowest IC50 value of 35.45 and stem possessed IC50 value of 29.72 µg/mL for

DPPH activity. The findings of α-glucosidase inhibition were also very impressive for

Neptunia oleracea leaves and stem with IC50 values of 19.09 and 19.74 µg/mL, respectively. The total phenolic contents (TPC) of Neptunia oleracea leaves, and stem were found to be 40.88 and 21.21 mg GAE/g extract. They observed that

Strobilanthes crispus harvested from two different sites showed variations in phenolic distribution, antioxidant and α-glucosidase inhibitory properties. The results revealed that phenolic contents were the major contributing factor towards antioxidant activity and enzymatic inhibition. Their study suggested that Neptunia oleracea could be used to treat diabetes [44].

Rehman et al. evaluated the extract of Cassia nemophila pod for antioxidant and antidiabetic potential. The antioxidant activity of the ethanolic extract was determined by DPPH assay while the antidiabetic potential was quantified by glucose uptake by yeast assay and glucose adsorption assay. Maximum DPPH scavenging of

43.3% was observed at 80 µg/mL. The yeast cells appeared with improved glucose absorption in the presence of extract. Similarly, glucose adsorption capacity increased with an increase in glucose concentration. Their study proved Cassia nemophila pod as a potent source of antioxidant and antidiabetic compounds [45].

LITERATURE REVIEW

Bothon et al. evaluated the 50% hydro-alcoholic extracts of Polygonum senegalensis and Pseudocedrela kotschyi for the presence of phytochemicals and α- glucosidase inhibition. The DPPH assay, FRAP, oxygen radical absorbance capacity

(ORAC) and cell-based dichloro-dihydro-fluorescein diacetate assay were used. The antibacterial activity was performed against Bacillus subtilis, Clostridium difficile, Enterococcus faecalis, Staphylococcus aureus by measuring minimum inhibitory concentration. The IC50 values of antioxidant activities and α-glucosidase inhibition remained notable and antibacterial activities were low. They found that both plants could be a good option for diabetes treatment [46].

Murugan et al. investigated total phenolic contents, nutritional composition, antioxidant activity, α-glucosidase and α-amylase inhibitory properties of leaves and fruits of Breynia retusa. The vitamin C, vitamin E, phenolic contents and flavonoids were also determined. The fruit contained higher starch content, total proteins, minerals and amino acids than leaves. The leaf extract showed high antioxidant activity as indicated by DPPH, FRAP and lipid peroxidation assays. The methanolic extracts of leaves effectively controlled the activity of α-amylase and α-glucosidase enzymes. Their findings provided a promising way to develop functional foods with antidiabetic properties [47].

Chatterji and Fogel studied an herbal formulation (SR2004) in Israel for its impacts on blood glucose level and HbA1c of diabetic patients. The SR2004 was composed of Artemisia dracunculus, Cinnamomum zeylanicum, Morus alba, Urtica dioica, and Taraxacum officinale. The DMT2 patients (119) were administered

SR2004 in addition to routine medicinal therapy for 12 weeks. The side effects of

SR2004 were also monitored. The 87% of patients came under follow up on completion of the 12-week study period. The reduction in HbA1c from 9.0% to 7.1%

LITERATURE REVIEW was observed, which was 22% and statistically significant (ρ<0.0001). The average blood glucose levels were reduced from 211 mg/dL to 133 mg/dL, which was 37% being significant (ρ<0.0001). The average total cholesterol was reduced by 13%, but this reduction was non-significant (ρ<0.0001). The serum triglycerides were reduced by 40% being significant (ρ<0.0001). 12% of patients did not make any response to

SR2004 formulation. A reduction of 30% on insulin dependence was also observed in

5 patients. In a fraction of patients (02), the reversal of retinopathic complication was observed. No adverse side effects of herbal formulation were observed during 12- week clinical investigation except slight abdominal disorders in 16 patients. The

SR2004 was proved as a promising herbal solution to treat DMT2 and improvement in microvascular disorders suggested for looking into the mechanism of action of this novel phytotherapeutic approach [48].

Konsue et al. evaluated Mimosa pudica aqueous and hydroethanolic leaf extracts and its influence on various hematological parameters. The red blood cells,

Hb, platelets, hematocrit, corpuscular hemoglobin, white blood cells, lymphocytes and monocytes of healthy and streptozotocin-induced diabetic rats. Extract doses at

125, 250 and 500 mg/kg body weight (b.w) were administered, and the dose of 250 mg/kg b.w reduced fasting blood glucose concentration of diabetic rats which was statistically significant (ρ<0.05). The extracts did not exhibit any impact on red blood cells, corpuscular hemoglobin, hemoglobin, platelets, hematocrit, white blood cells, lymphocytes and monocytes. However, a reduction in white blood cells and corpuscular volume was noted. Their findings confirmed the traditional use of

Mimosa pudica to treat diabetes [49].

Mard et al. carried out an in-vivo trial for the antidiabetic and antilipidemic effects of Phoenix dactylifera leaf extract and its fractions in Wistar rat model. The

LITERATURE REVIEW alloxan-induced diabetic mice were grouped into 8 categories out of which one group served as control. The other 7 groups were treated with extract doses of 100, 200, 400 mg/kg body weight and with fractions at 50, 100 and 200 mg/kg body weight. One group was administered glibenclamide at 4 mg/kg bodyweight for 14 days. The blood glucose testing was made on 1st, 6th and 14th days. Overnight fasted rats were sacrificed to measure plasma insulin level, triglyceride and cholesterol. A significant rise in plasma insulin was noted while a remarkable decrease in water consumption, triglycerides and cholesterol were observed when compared with the control group

(ρ<0.01) [50].

Bolsinger et al. studied the role of phytochemicals in the prevention of metabolic disorders. They reported that phenolics from the juice of palm fruit may be helpful to treat diabetes. The study made use of Nile rats as type 2 diabetic model.

The experiment was composed of the 4-36-week period, and palm fruit juice was added into the diets at doses to ensure intake of 170-720 mg gallic acid equivalents/kg body weight per day. Random fasting glucose level and change in body weight were measured during the study period. The palm fruit juice concentration substantially controlled the hyperglycemia and showed improvement in lipid profile as compared with the control diabetic rat group. They found that the palm fruit juice may be used as a dietary component to increase insulin secretion [30].

Raza et al. determined the anti-hyperglycemic potential of Conocarpus erectus leaves. The hydroethanolic leaf extracts of Conocarpus erectus were explored for total phenolic and flavonoid contents, antioxidant and antidiabetic activities. The maximum phenolic and flavonoids were detected in 60% ethanolic fraction. The same fraction was administered orally to diabetic mice, and blood glucose was noted on a weekly basis. The extract sufficiently reduced the blood glucose of diabetic mice than

LITERATURE REVIEW the control animal group. Their research results confirmed the antidiabetic potential of

Conocarpus erectus leaves as a rich pool of biologically functional metabolites [25].

Saadullah et al. investigated the antidiabetic role of Conocarpus lancifolius by measuring the α-glucosidase inhibition and in-vivo determination of HDL, LDL, creatinine, urea, triglyceride, serum glutamic pyruvic transaminase (SGPT) and serum glutamic oxaloacetic transaminase (SGOT) in diabetic rabbits. The rabbits were made diabetic by injection of alloxan. The methanolic extract dose of 200 mg/kg body weight decreased the blood glucose level, total cholesterol, triglycerides and LDL significantly (ρ<0.05). The qualitative phytochemical analysis confirmed glycosides, saponins, terpenoids and tannins in the extract. The Presence of saponins might be a key factor responsible for antidiabetic potential [51].

Riyanto and Wariyah determined the hypoglycemic potential of Aloe vera

(instant) in diabetic Wistar rats. Standard diet was made enriched with fresh Aloe vera which was stored for 0-8 weeks at the interval of 2 weeks. The diet was given to rats under examination. The blood glucose level was monitored after every 7 days for a period of 4 weeks. They found that the Aloe vera significantly reduced blood glucose level from 214.00 mg/dL to 97.57 mg/dL [52].

Zaiton et al. investigated the probable antidiabetic effect of Ferula assafoetida extract against β-cells dysfunction in alloxanated diabetic rats. The plant extract exhibited a substantial decrease in blood glucose and a considerable increase in serum insulin level. The blood glucose level of treated diabetic rats was reduced from

10.28±0.85 mML to 6.75±0.31 m/mL upon administration of the extract. A notable increase in insulin level was also observed, which reflected the improvement in pancreas functioning. Their results suggested the antidiabetic role of this plant extract

[53].

LITERATURE REVIEW

Ahmed et al. studied the antioxidant and α-glucosidase inhibition potential of

Clinacanthus nutans extract, and related fractions along with phytochemical profiling.

Hexane, butanol, aqueous and ethyl acetate fractions were extracted from the methanolic extract. The DPPH and FRAP assays were adopted to judge the antioxidant potential. The α-glucosidase activity was used to determine the antidiabetic activity of extract fractions. The phytochemical analysis was performed by GC-QTOF-MS. The extract yield of 12.63 ± 0.98% by weight was obtained through methanolic extraction. The aqueous residual fraction produced 52.25 ± 1.01% extract yield. The highest DPPH radical scavenging of 79.98 ± 0.31% was given by ethyl acetate fraction followed by 63.07 ± 0.11% scavenging by an aqueous fraction.

The FRAP assay findings presented methanolic extract as the most powerful to reduce the radicals with a value of 141.89 ± 0.87 μg AAE/g. The ethyl acetate showed the second-highest FRAP value of 133.6 ± 0.2987 μg AAE/g. The α-glucosidase inhibitory potential of 72.16 ± 1.0% and 70.76 ± 0.49% by butanol and ethyl acetate fraction, respectively. It was observed that total phenolic and flavonoid contents significantly influenced the antioxidant activity and α-glucosidase inhibition (ρ<0.05).

This study recommended the ethyl acetate and butanol fraction of Clinacanthus nutans as a rich source of functional medicinal agents to counter oxidation and hyperglycemia. The phytochemical analysis indicated the presence of organic acids

(α-hydroxyl acids, dicarboxylic acids), terpenes, inositols, fatty acids, coumarins, glycosides, phytosterols, polysiloxanes, phenolics and polyols [41].

Hafeez et al. investigated the antioxidant, antidiabetic and antimicrobial activities of Ajuga bracteosa. Various extracts (ethyl acetate, n-hexane, chloroform, methanol, acetone and aqueous) of Ajuga bracteosa roots were evaluated for antibacterial activity through agar well diffusion assay. Antioxidant activity was

LITERATURE REVIEW measured by iron chelating activity, DPPH assay and 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid) (ABTS) method. The DPPH assay showed the antioxidant activity ranging from 61.92% to 88.84%, and ABTS assay values ranged from 0.11% to 38.82%. The chloroform and n-hexane extracts exhibited the highest α- glucosidase activity, and their IC50 values were 29.92 µg/mL and 131.7 µg/mL, respectively. The n-hexane extract possessed the highest inhibition of E. Coli, E. amnigenus and S. aureus. The phytochemical analysis indicated the significant amounts of phenols, flavonoids, tannins, saponins, glycosides and terpenoids. They concluded that Ajuga bracteosa roots were a good source to develop the antioxidant, antimicrobial and antidiabetic agents [54].

Sassi et al. evaluated the antioxidant activity of flowers, leaves, stem and root extracts of Chrysanthoglossum trifurcatum. The α-glucosidase inhibitory activity and phytochemical analysis were also made. The essential oil collected from aerial parts was also investigated. Extraction was made by using methanol, petroleum ether and ethyl acetate. The extracts were subjected to phytochemical analysis to check the presence of phenols, flavonoids, flavonols and tannins. The essential oil was also analyzed by gas chromatography mass spectrometry (GC-MS) to identify and quantify the metabolites. The DPPH and ABTS assays were used to determine the antioxidant activity of extracts and oil. The methanolic extract exhibited highest contents of phytochemicals and essential oil, the analysis indicated the presence of limonene (29.21%), γ -terpinene (12.96%), 4-terpenyl acetate (12.18%) and α -pinene

(5.76%). The petroleum ether leaves, root extracts and essential oil showed promising

α-glucosidase inhibitions in terms of IC50 values. Their findings suggested that the functional activities and phytochemicals present in Chrysanthoglossum trifurcatum were of dietary importance to treat diabetes type 2 [55].

LITERATURE REVIEW

Russo et al. proved that cholinesterase is an enzyme, which hydrolyses choline esters and is known to play an important role in neural disorders. The anticholinesterase, antidiabetic and antioxidant properties of fourteen Smallanthus sonchifolius (yacon) varieties were determined and compared. The phytochemical analysis of leaves was also performed. Antioxidant activity was determined by adopting common antioxidant assays like DPPH, FRAP and lipid peroxidation. Anti- cholinesterase was measured by noting the inhibition of acetylcholinesterase and butyrylcholinesterase. The inhibition of α-amylase and α-glucosidase was measured to assess the antidiabetic activity of extracts. Metabolite profiling was performed by high performance liquid chromatography with diode array detection (HPLC-DAD). The

4,5-di-O-caffeoylquinic acid (CQA) and 3,5-di-O-CQA were responsible for the α- amylase and α-glucosidase inhibition while flavonoids seemed to be responsible for acetylcholinesterase and butyryl cholinesterase inhibition [56].

Al-Zuaidy et al. studied the antidiabetic and antioxidant aspects of ethanolic extracts of Melicope lunu‐ankenda. The α-glucosidase enzyme inhibition was assessed as an index of the antidiabetic property while DPPH assay was utilized for antiradical properties. The 1HNMR and UHPLC equipped with tandem mass spectrometer were used to identify the metabolites in extracts. Their results showed that 60% ethanolic fraction exhibited maximum enzyme inhibition with an IC50 value of 37 μg/mL and DPPH scavenging (IC50 of 48 μg/mL). The findings of the in-vivo trial indicated that extract dose of 400 mg/kg b.w significantly reduced the blood sugar (62.75%). The metabolite profiling confirmed the presence of isorhamnetin, skimmianine, scopoletin, and melicarpinone in the extract. The study provided a useful lead to introduce antidiabetic functionalities in food and phytotherapy for diabetes management [57].

LITERATURE REVIEW

Al-Zuaidy et al. also investigated the Melicope lunu‐ankenda extract to see the impact on insulin level, insulin sensitivity, serum glucose level and insulin resistance in obese diabetic rats. The study revealed that rats having 400 mg/kg b.w extract were associated with a significant reduction in triglyceride, cholesterol and LDL, where a notable increase in HDL was observed. The variations in liver enzymes and renal injury biomarkers were also measured. Metabolomics studies clearly indicated the role of Melicope lunu‐ankenda extract to mitigate the discrepancies in glucose, lipid and protein metabolism [58].

Quispe et al. evaluated α-glucosidase, and aldose reductase inhibition assays for 30 herbal extracts in-vitro. The Hypericum laricifolium Juss was characterized as the most potent herb among all as it exhibited 97.2 ± 2.0%, and 56.9 ± 5.6% inhibitions of α-glucosidase and aldose reductase, respectively. The 81.9 ± 2.5% and

58.8 ± 4.6% inhibitions were observed for DPPH and ABTS assays respectively. This was the only herbal extract which exhibited more than 50% inhibition for all mentioned assays. Phytochemical profiling by HPLC indicated the presence of protocatechuic acid, chlorogenic acid, caffeic acid and flavonoids like quercetin, and kaempferol in the herbal extract, however quercetin showed the highest inhibition of enzymes and free radicals. This study suggested the greater potential of Hypericum laricifolium Juss to treat diabetes and its related side complications [59].

Jabir et al. reported the identification of phytochemicals from Heracleum candolleanum seeds. The antidiabetic activity was determined by measuring the inhibition of α-amylase. The seed extract contained phytochemicals mainly phenols and anthraquinones. These metabolites were responsible for anti-α-amylase properties of seed extract [60].

LITERATURE REVIEW

Musa et al. unleashed the inhibitory potential of grape seed, white tea and green tea extracts against α-glucosidase and α-amylase rather than α-amylase [61]. spectroscopically. The acarbose was used as standard drug. Their results revealed that grape seed extract was associated with the highest inhibition of α-glucosidase and α- amylase. This study concluded that catechin 3-gallates were potent inhibitor of α- glucosidase

Mumtaz et al. studied the role of antioxidant potential of the plants to mitigate the state of oxidative stress for the prevention of hyperglycemia. This approach was considered as an effective tool to manage diabetes. The antioxidant activity (DPPH assay) and antidiabetic activity (α-glucosidase and α-amylase inhibition) of Ficus benjamina leaf extracts were performed. The most prominent antioxidant activity with an IC50 value of 63.71± 2.66 µg/mL was shown by 80% ethanol extract. Similarly, α- glucosidase and α-amylase inhibitory properties of 80% ethanolic extract were also high with IC50 values of 116.01 ± 3.83 µg/mL and 152.66 ± 7.32 µg/mL, respectively.

The 1HNMR based metabolite profiling identified 31 metabolites in the leaf extract.

Their study suggested that Ficus benjamina could be used to develop novel functional foods and nutraceuticals with antidiabetic attributes [62].

EXPERIMENTAL WORK

This section includes chemicals/reagents, the brief detail of methodology for

the material collection, extraction, antioxidant activities, enzyme inhibitory assays,

phytochemical analysis and in-vivo trials.

Chemicals and Reagents

The chemicals and reagents were procured mainly from Sigma and Merck.

These chemicals were of analytical grade. The different chemicals and reagents

included, methanol, ethanol, sodium carbonate (Na2CO3), gallic acid (C7H6O5),

sodium hydroxide (NaOH), liquid nitrogen, hydrochloric acid (HCl), ascorbic acid

(C6H8O6), trichloromethane (CHCl3), Folin Ciocalteu reagent, sodium nitrite

(NaNO2), aluminium chloride (AlCl3), sodium chloride (NaCl), rutin (C27H30O16),

phosphoric acid (H3PO4), 2,2-diphenyl-1-picrylhydrazyl (DPPH), p-nitrophenol

glucopyranoside, disodium phosphate (Na2HPO4), monosodium phosphate

(NaH2PO4), α-glucosidase, tris (hydroxymethyl) aminemethane (Tris), acarbose

(C25H43NO18), sulphuric acid (H2SO4), ammonium molybdate ((NH4)2MoO4),

butylated hydroxyanisole (C11H16O2), β-carotene, linoleic acid, tween

40(polyoxyethylene sorbitan monopalmitate).

Collection and Identification

The plant specimens were collected and identified through specimen voucher

GC. Herb. Bot. 3599 and GC .Herb. Bot. 3600 for Hyophorbe lagenicaulis and

Hyophorbe indica, respectively. Fresh leafy parts of H. lagenicaulis and H. indica were harvested, washed and subjected to proximate analysis.

EXPERIMENTAL WORK

Proximate Analysis

The moisture content (%) of fresh leaves was calculated by heating the aerial parts of H. indica and H. lagenicaulis at 105°C for 24 hours. The moisture content was computed by measuring the difference of weights before and after oven drying.

Muffle furnace with temperature programming was used to find ash content. 100 g of leafy material was heated with temperature programming (100 °C for 3 hours, 300 °C for 3 hours, 600 °C for 9 hours and 950 °C for 3 hours). The weight of remaining ash was calculated, and values were expressed in %w/w [63].

A reported method with slight modification was followed for protein content estimation [64]. The 100 g of leafy material was digested in sulfuric acid at 370°C. A chunk of potassium sulphate was also added. After complete digestion, the reaction mixture was cooled to ambient temperature and diluted to 100 mL of distilled water.

+ The NaOH was added to convert NH4 ions into NH3 gas which was collected in a flask having 100 mL of 5% boric acid. The collected NH3 was titrated with HCl (0.1

N), and the endpoint was noted by using pH electrode (Sensorex). The following mathematical relationship was used for Nitrogen (%) calculation,

(mL of acid for sample − mL of acid used for blank) × (0.1N) × (1.4007) %N = Sample in grams

The %N obtained was multiplied by a factor 6.25 to calculate protein content (%).

The crude fat (%) was calculated by hexane extraction from 5 g leaves in soxhlet apparatus for 6 hours. The carbohydrates were calculated by routine difference method.

Carbohydrates (%) = 100% − (Ash Content + Moisture Content + Crude Fat + Protein Content)

EXPERIMENTAL WORK

The nutrition status in kcal/g of plant material was computed by considering the following energy values.

Table 3.1 Nutritive proportion of nutrients in kcal/g

Sr. No Nutrients Energy value kcal/g

1 Proteins 4

2 Carbohydrates 4

3 Lipids 9

The formula to calculate the nutritive value (Nv) is given as under,

kcal Nv ( ) = (9 × Fat Content) + (4 × Protein Content) + (4 × Carbohydrate Content) g

Mineral Composition

The mineral composition of leafy parts of H. indica and H. lagenicaulis was determined by a reported method [65]. 1.0 g of each sample was digested in 10 mL of concentrated HNO3 and HClO4 (1:1). The mixture was heated at 150°C for 120 minutes and up to 200°C to enhance the digestion efficiency. The digestion was considered as completed by the appearance of transparent color. The reaction mixture was cooled, and diluted by distilled water and filtered by Whatman filter paper no. 42, and again diluted to make volume 100 mL.

The trace metals (Zn, Fe, Pb, Ni, Co and Mn) were determined by Atomic Absorption

Spectrophotometer (Perkin Elmer Analyst 800). The calcium (Ca) and magnesium

(Mg) were determined by Ethylenediaminetetraacetic acid

EXPERIMENTAL WORK

(EDTA) method while sodium and potassium were determined by emission spectrometry (Flame Photometer Corning 410).

Green Extract Preparation

The fine leafy powder was lyophilized on Christ Alpha 1-4 LD Germany for

48 hours. The freeze-dried material was extracted with different compositions of ethanol and water. The detail is given below in Table 3.2.

Table 3.2 Detail of solvent system used for extraction

Solvent Compositions Water Ethanol

1 100% 0%

2 80% 20%

3 60% 40%

4 40% 60%

5 20% 80%

6 0% 100%

The solvents were added to the freeze-dried extracts and ultrasonicated. Extracts were transferred to the shaker for 2 hours shaking. The filtration was carried out to remove insoluble material and small amount of solvent present in extracts was removed on the rotary evaporator. The amounts of freeze-dried extracts produced were measured to calculate the extract yields (%).

EXPERIMENTAL WORK

Total Phenolic and Flavonoid Contents

An already established method was utilized for total phenolic contents with minor changes [66]. 0.001 g of plant extract was suspended in methanol. 0.25 µL of this suspension was mixed with Folin Ciocalteu reagent followed by 2 mL of 10%

Na2CO3. After 120 minutes of storage at room temperature, absorbance was taken at

765 nm. The standard curve of gallic acid was drawn. The findings in gallic acid equivalent (GAE) mg/g dried extract have been reported.

For determination of total flavonoid contents (TFC), an already reported scheme was followed after some minor changes [67]. Initially, plant extracts (0.1 mg) in 2 mL of methanol were diluted with distilled water, followed by the addition of 0.5 mL of 5% solution of NaNO2. 10% AlCl3 was added to the mixture and allowed to stand for 10 minutes time period. 1M solution of NaOH was added and shook to note absorbance at 510 nm on a spectrophotometer. The values of TFC in rutin equivalent mg/g dried extract (RE mg/DE) have been presented.

In-vitro Antioxidant Activities

Methods like 2,2-diphenyl-1-picrylhydrazyl (DPPH), total antioxidant power and β-carotene linoleic acid assays were used for this purpose.

3.7.1 The DPPH Assay

The DPPH assay is a widely used method to find antioxidant activity.

An established method with slight changes was adopted for this purpose [68]. The 50-

200 ppm of methanolic dilutions of plant extract (1mg/mL) were mixed with 10 mL of DPPH reagent solution of 0.001M strength. The reaction mixture was allowed to stay for 30 minutes at room temperature in the dark. After this specific time, samples

EXPERIMENTAL WORK were subjected to absorbance measurement at 520 nm. For comparison, butylated hydroxyanisole (BHA) was utilized as standard and IC50 values were calculated.

3.7.2 Total Antioxidant Power Assay

Total antioxidant power assay was used to determine the antioxidant power of plant extracts. This method is based upon the formation of Phosphomolybdenum complex, which absorbs at 695 nm. The 250 µg of the extract was dissolved in 1 mL of methanol and was mixed with 4.0 mL of reagent solution. The reagent solution was formed by mixing 0.60 M sulphuric acid, 4.0 mM ammonium molybdate and 28.0 mM sodium phosphate. The obtained samples were allowed to incubate at 95ᵒC for 90 minutes. The absorbance of samples was noted at 695 nm after cooling of the samples

[69]. The ascorbic acid standard was used to draw the standard curve for calculation and findings for each gram extract were calculated in equivalence to ascorbic acid

(ASE/g DE)

3.7.3 Iron Chelating Activity

Metal chelating activity was performed by adopting a previous protocol [70].

The FeSO4 (2 mM) was prepared, and 100 µL of this was mixed with 0.2 mL of 5.0 mM ferrozine followed by mixing with 2.0 mg/mL of plant extract. The mixture was allowed to stand for 10 minutes at room temperature. The spectrophotometer was tuned at 562 nm for absorbance. Greater absorbance values reflected low chelating activity. The EDTA was used as a positive control. The iron-chelating activity was measured by the following equation,

EXPERIMENTAL WORK

As Iron Chelation = (Act − ) × 100 Act

The Act represents the absorbance of control while As is the absorbance of the sample.

Enzyme Inhibition Assays

3.8.1 The α-Glucosidase Inhibition Activity

The 200 ppm of methanolic extract was mixed with 70 µL of 50 mM phosphate buffer. 1 unit/mL of α-glucosidase was added to the plant extract.

Incubation of samples at 37°C was carried out for a period of 10 minutes and 5.0 mM of 4-Nitrophenyl β-D-glucopyranoside (PNPG) was added to the reaction mixture.

The mixture was left for 30 minutes to complete the reaction. The absorbance at 405 nm was measured. The standard drug acarbose was used as an enzyme inhibitor. The results were reported in terms of IC50 values in µg/mL [71].

A − A Inhibition % = b s × 100 Ab

Where Ab is the absorbance of blank and As is the absorbance of the sample. The

standard compound Acarbose was used as a reference. The results were computed in

IC50 (µg/mL).

3.8.2 The α-amylase Inhibitory Assay

The previously reported method was employed to measure the inhibition of α-amylase activity by plant extracts [72]. The 1-10 mg of each extract was suspended in methanol, and 250 μL of these dilutions were added to porcine α-amylase, 0.5 mg in

5.0 mL of phosphate buffer (0.02 M) having pH 6.9. The obtained mixtures were allowed to stay for 10 minutes at 25°C temperature. After 10 minutes incubation,

EXPERIMENTAL WORK dinitrosalicylic acid (DNS) was added, followed by further incubation of 5 minutes.

Each resultant mixture was further diluted by distilled water (5.0 mL). The absorbance was noted at a wavelength of 540 nm. Acarbose was used as standard and a blank was also run under the same conditions.

The percentage inhibitions were calculated by using the equation given below.

A − A Inhibition % = b s × 100 Ab

where As is the absorbance of the sample and Ab represents the absorbance of the

blank. Final values were computed in IC50 values with units µg/mL.

3.8.3 Acetylcholine Esterase Inhibition Assay

Briefly, acetylthiocholine iodide (25 µL) was added to 3 mM of DTNB IN

Tris-HCl buffer. 50 µL of each extract was added to reaction mixture. The reaction was started by adding 25 µL of acetylcholine esterase enzyme and absorbance was taken at 405 nm [56].

Metabolite Profiling

The metabolite prediction was made by 1HNMR, and identification was made

by UBHPLC-QTOF-MS/MS analysis.

3.9.1 The 1HNMR Prediction of Metabolites

Most effective leaf extracts were suspended in CH3OH-d4 and KH2PO4 buffer of pH 6.0 in D2O containing trisodium phosphate (TSP) (0.1 %). The sample was vortex for 1 minute and ultrasonicated for 15 minutes at 35 °C. The solution was projected to centrifugation at 13000 rpm for l0 minutes. The 600 μL of supernatant

EXPERIMENTAL WORK was subjected to 1HNMR analysis [73, 74]. INOVA 500 MHz spectrometer (Varian

Inc., CA), at 499.887 MHz frequency was used for spectrum generation at 26 °C with tetramethylsilane (TMS) as the internal standard. The spectrum was bucketed with

Mest Renova 11.0.

3.9.2 UHPLC-QTOF-MS/MS Analysis

The plant extract in favourable solvent was filtered and projected to UHPLC-

QTOF-MS/MS (AB Sciex 5600-1, equipped with Eksigent UHPLC system). The instrument scanning range was selected from 50-1200 m/z for MS/MS. The Thermo

Hypersil Gold column with dimensions of 100 mm × 2.1 mm × 3 µm was used. Water and acetonitrile having 0.1% formic acid and 5 mM ammonium formate was used for gradient elution. The programming was started from 10% acetonitrile and reached to

90% acetonitrile. The solvent flow of 0.25 mL/min was used and the ion spray voltage was -4500 V in negative mode.

Cytotoxicity Evaluation

The plant extract was subjected to cytotoxicity assay based upon the hemolytic process as per reported scheme [75]. The 1000 µg/mL extract was added to the solution of NaCl (0.85%) and 2% human erythrocyte suspension. The mixture was allowed to stand for 30 minutes. The centrifugation was carried out, and the supernatant was subjected to measure absorbance at 540 nm. Positive control (0.1%

Triton) and negative control (phosphate buffer saline) were used. The measurements were made in triplicate, and hemolytic percentages were calculated by the following formula.

EXPERIMENTAL WORK

Abs of sample − Abs of negative control Hemolytic % = × 100 Abs of positive control

The Anti-Hyperglycemic and Hypolipidemic Activities

To determine the anti-hyperglycemic and hypolipidemic activities, diabetic mice model was used. Initially, 60 healthy mice (8 weeks old) were selected and kept at 28°C ± 2.0°C temperature for ten days. The average value of relative humidity was

68.75% ± 4.5% during the study period. The permission to carry the animal trial was granted by the ethical committee of GC University Lahore. After ten days period of acclimatization, mice were weighed on a digital balance.

3.11.1 Oral Glucose Tolerance Test

The 18 hours fasted normal mice were administered 2.0 g of glucose per kg of body weight after administering vehicle, drug and plant extract as per designed doses.

The blood glucose levels were noted by glucometer through ruptured tail at 0, 30, 60 and 120 minutes of the treatment [76].

3.11.2 Anti-Hyperglycemic Assessment

The 24 hours fasted mice were injected with alloxan monohydrate at a dose of

150 mg/kg body weight. The blood glucose level (mg/dL) was measured by glucometer to ensure the induction of diabetes. Mice with blood glucose level >200 mg/dL were categorized as diabetic [77]. 60 mice were divided into two groups, each having 30 mice for H. indica and H. lagenicaulis set of experiment. The distribution of mice, temperature and humidity conditions of the animal house are given in Table

3.3.

EXPERIMENTAL WORK

30 mice of each group were further splitted into 5 groups of 6 each. The details of sub-groups are given as under.

NG = Normal group (Mice having no injection of alloxan)

DG = Diabetic group (Mice having no treatment)

MG = Metformin group (Treated with metformin dose at 250 mg/kg b.w)

HG = Half dose group (Treated with plant extract dose of 250 mg/kg b.w)

FG = Full dose group (Treated with plant extract dose of 450 mg/kg b.w)

The metformin dose of 250 mg/kg b.w was selected on the basis of previously

reported works of similar nature. Numerous studies involving in-vivo antidiabetic

studies reported the use of metformin at 250 mg/kg b.w. Similarly, plant extract at

250 mg/kg b.w and 450 mg/kg b.w were used. These doses were also set on the

basis of previously reported studies using the same ranges of doses. [25, 58, 78, 79]

Table 3.3 Experimental conditions during in-vivo study

Name of Plant No. of Average body Average Relative Light mice weight (g) temperature humidity % and dark ᵒC cycle (h)

H. indica 30 28.53 ± 1.80 28°C ± 2.0 68.75% ± 4.5 12

H. lagenicaulis 30 28.66±1.75 28°C ± 2.0 68.75% ± 4.5 12

The blood glucose concentrations were measured by rupturing lateral vein in mice tail after every seven days for a period of 28 days. The water consumption and changes in body weight (g) were also recorded.

EXPERIMENTAL WORK

3.11.3 Hypolipidemic Activity

After the completion of 28 days trial, the blood from the lateral vein of mice was subjected for determination of various hematological parameters which included blood hemoglobin (Hb), total cholesterol (Tc), high-density lipoproteins (HDL) and low-density lipoproteins (LDL).

Statistical Analysis

The Statistic 10.0 software was used to find out the level of significance among values. ANOVA was used to evaluate the impact of treatments on results outcome. The standard deviation was applied for multiple values.

EXPERIMENTAL WORK

Optimized Fractions TPC & TFC by Hydroethanolic In-vitro Antioxidant Solvents and Antidiabetic activity

Metabolite Profiling

Fig. 3-1 Schematic diagram of methodology

RESULTS AND DISCUSSION

This chapter includes the findings and scientific outcomes of present study.

The results are given in the form of tables, figures and diagrams. The statistical analysis, where required, was also performed to see the level of significance.

Proximate Analysis

The protein content, fat content, carbohydrate content, moisture content and ash content were determined. The nutritive value was calculated by estimating protein content, fat content and total carbohydrates in H. indica and H. lagenicauliss, as shown in Table 4.1.

Table 4.1 The results of the proximate analysis for H. indica and H. lagenicaulis

Name of Moisture Ash Protein Fat Carbohydrate% Nutritive Plant content% content% content% content% value (kcal/g)

H. indica 17.26 6.12 14.10% 1.95% 55.10 294.35

H. 16.50 5.95 11.33% 1.88% 59.25 299.24

lagenicaulis

The handsome amounts of protein and carbohydrate were present in both plants. The proteins and carbohydrates, both are an essential component to support the living system of plants and animals. The nutritive value was calculated on the basis of proximate analysis. The results indicated that both the plants exhibited almost similar nutritive values. The proteins and carbohydrates were observed as a major contributing factor in the nutritive value of both plants. The proximate analysis provided interesting information about the major contents of plant material.

RESULTS AND DISCUSSION

The results of mineral composition are given in Table 4.2. Some essential metals were determined through various techniques, and results were represented in mg/kg.

Table 4.2 Mineral composition of H. indica and H. lagenicaulis

Name of Pb Ni Co Mn Fe Zn Ca Mg Na K Plant

(mg/kg)

H. indica 0.067 0.018 BDL 0.015 09.10 2.5 95.4 14.96 71.40 10.24

H. 0.055 0.020 BDL 0.011 7.22 2.11 122.2 17.17 85.20 6.25 lagenicaulis

The mineral composition analysis revealed that aerial parts of both plants possessed sufficient amounts of essential metals like Zn, Ca, Mg, K and Mn. The minerals were reported in mg/kg or ppm. The Zn is an essential nutrient for the proper functioning of animal and plant systems. The Ca, Mg, K and Mn were also present in suitable amounts which may add additional nutritive and medicinal aspects. The Zn plays an important role in non-enzymatic membrane stabilization to control diabetes.

The Zn deficiency was reported to have a close link with insulin insensitivity [80]. The

Co was not detected in both plants. The Pb was detected at a very low concentration which might not be harmful. The variations in element concentrations depend upon the soil properties and water quality used for irrigation.

Extract Yields

The extraction from plants is an important aspect while considering the phytotherapeutic approaches. The extraction provides the phytochemicals generally responsible for the medicinal properties of plants. The high extract yields often contain higher contents of secondary metabolites. The role of pretreatment and extraction

RESULTS AND DISCUSSION methodology is very crucial to enhance extract yield from plant materials. The strength of extraction methodology contributes a lot in the extract yield and phytochemical based bioassays [81]. The comparison of extract yield produced by various hydroethanolic solvent compositions from freeze-dried and ultrasonicated leafy materials is given in Table 4.3.

Table 4.3 Extract yield of H. indica and H. lagenicaulis

Solvent type Extract yield (%) of H. indica Extract yield (%) of H. lagenicaulis

20% Ethanol 15.53 ± 0.07 d 14.31 ± 0.2 de

40% Ethanol 19.07 ± 0.16 b 16.33 ± 0.32 c

60% Ethanol 22.63 ± 0.19 a 20.46 ± 0.25 a

80% Ethanol 18.46 ± 0.07 c 18.05 ± 0.13 b

100% Ethanol 15.55 ± 0.06 d 15.10 ± 0.15 d

The results indicated that changing solvent composition affected the extract yields considerably. The 60% ethanol provided maximum extract yield of 22.63 ±

0.19% followed by 40% ethanol for which the extract yield was 19.07 ± 0.16%. The lowest extract yields were observed when 20% and 100% ethanol were used for extraction. In case of extraction from H. indica, extract yield from 60% ethanol was

18.66% higher than the 40% ethanol, 22.58% higher than 80% ethanol and 45.71% from 20% ethanol. The statistical analysis revealed that extract yields from 60% ethanol were significantly higher than all other solvent types used for extraction (ρ

<0.05). The combination of water and ethanol worked well for the extraction purpose.

Both components were responsible for the polarity of the solvent system. The role of

RESULTS AND DISCUSSION polarity associated with solvent components is considered a decisive factor behind the extraction efficiency. The solvent polarity due to its composition was the most probable reason behind the differences in extract yield [82]. The water added softness in tissues of plant material and alcohol dissolved the likely compounds, mainly polar.

The hydroxyl groups of polyphenols were probably responsible for the transfer of compounds from plant matrix to solvent.

Various solvents are being used to extract the phytochemicals. However, alcohol-based solvent systems are frequently used due to many benefits of alcohol like polarity, safety, less toxicity etc. The properties of ethanol may be improved by adding some other components like water. Some previous studies also supported the role of hydroethanol as a competent solvent for enhancing extraction [58]. Another study reported the impact of 60% ethanol on extract yield from Vernonia amygdalina. The maximum extract yield of 22.86 ± 0.35 from Vernonia amygdalina was obtained using

60% ethanol [83]. The freeze-drying and ultrasonication both were also notable contributing factors in extraction optimization as indicated by a recent study which obtained high extract yield of 21.5 ± 0.03% from leaves of Conocarpus erectus [25].

The ultrasonication has been proved as an excellent tool for optimized extraction as it ruptures the cell wall to remove metabolites. The ultrasonication has solved the issues of low extract yields associated with conventional extraction methodologies from plant materials [84].

Total Phenolic and Flavonoid Contents (TPC)

The polyphenols are a major contributing factor towards the medicinal properties of plants. The high extract yields may contain relatively higher amounts of phenolics. The values of total phenolic content are summarized in Table 4.4.

RESULTS AND DISCUSSION

Table 4.4 Total phenolic content of H. indica and H. lagenicaulis.

Solvent Type H. Indica H. lagenicaulis

(TPC in mg GAE/g DE)

20% Ethanol 105.55 ± 1.05 e 78.09 ± 1.36 d

40% Ethanol 154.50 ± 2.15 b 109.63 ± 1.67 c

60% Ethanol 208.77 ± 2.11 a 178.56 ± 1.47 a

80% Ethanol 140.44 ± 2.09 c 144.67 ± 2.31 b

100% Ethanol 119.24 ± 1.54d 109.62 ± 0.44c

The impact of solvent was obvious from the results. Different solvents produced different amounts of phenolics. In the case of H. indica, the highest TPC

(208.77 ± 2.11 mg GAE/g DE) were obtained when extracted with 60% ethanol. The second largest phenolic contents were observed when 40% of ethanol was used. The lowest TPC (105.55 ± 1.05 mg GAE/g DE) were generated by 20% ethanol.

Similar findings regarding TPC were observed for H. lagenicaulis. The 60% extract of H. lagenicaulis contained the highest phenolic content (178.56 ± 1.47 mg

GAE/g DE) while 80% ethanol yielded 144.67 ± 2.31 mg GAE/g DE. The lowest TPC

(78.09 ± 1.36 mg GAE/g DE) was obtained when extracted with 20% ethanol. It was observed that 40% ethanol extracted the TPC from H. indica more effectively as compare to H. lagenicaulis for which 80% ethanol was more efficient. The statistical analysis revealed that TPC for both H. indica and H. lagenicaulis generated by 60% ethanol were substantially higher than other fractions (ρ <0.05). Phenolic content are considered as an important functional component of plants and majority medicinal,

RESULTS AND DISCUSSION and antioxidant characteristics of plants are due to these compounds. A previous study has reported the antioxidative potential and phenolic content of fifteen plants used in medicine in Bangladesh. The TPC of these plants were between 0.04 to 6.01 mg

GAE/gDE [22].

The values of TFC are given in Table 4.5. The estimation of flavonoids in H. indica leaf extracts revealed that 60% ethanolic extract has shown highest flavonoid content (173.90 ± 2.30 mg Rutin/g DE) followed by 40% ethanolic extract (118.11 ±

1.01 mg Rutin/g DE), 80% ethanolic extract (117.60 ± 1.78 mg Rutin/g DE), 100% ethanolic extract (102.88 ± 1.22 mg Rutin/g DE) and 20% ethanolic extract (95.75 ±

1.25 mg Rutin/g DE), respectively.

The TFC for H. lagenicaulis ranged from 68.94 ± 1.6 mg Rutin/g DE to 133.96

±1.19 mg Rutin/g DE. The highest TPC value of 133.96 ±1.19 mg Rutin/g DE was obtained by 60% ethanol, followed by 80% ethanol (115.51 ±0.90 mg Rutin/g DE).

The TFC yield by 60% ethanol was significantly higher when compared with the remaining extracts (ρ < 0.05).

Flavonoids are a well-known class of polyphenolic origin. They are also known to possess high medicinal functionalities and biological impacts. The plants are a rich source of natural flavonoids. Their concentration may vary in the same species from different areas. Further different parts of a plant may have different status of flavonoid concentration. Quercetin, rutin, kaempferol etc. are well known and deeply studied flavonoids.

The flavonoids and their derivatives are associated with strong antioxidant and anti-diabetic attributes. By virtue of inherited biological significance, flavonoids are important ingredients of functional foods and nutrapharmaceuticals. The studies have

RESULTS AND DISCUSSION shown that natural flavonoids are very effective to increase insulin sensitivity and to control hyperlipidemia [85].

Table 4.5 Total flavonoid content of H. indica and H. lagenicaulis.

Solvent Type TFC in mg GAE/g DE

H. indica H. lagenicaulis

20% Ethanol 95.75 ± 1.25 d 68.94 ± 1.61 e

40% Ethanol 118.11 ± 1.01 b 92.02 ± 1.72 d

60% Ethanol 173.90 ± 2.30a 133.96 ± 1.19a

80% Ethanol 117.60± 1.78 b 115.51± 0.90 b

100% Ethanol 102.88 ± 1.22c 100.90 ± 1.59c

Antioxidant Activities

4.4.1 DPPH Activity

DPPH assay is a widely accepted method to determine antioxidant activity.

The DPPH is a radical which shows remarkable stability and absorbs at 517 nm. The stabilization of DPPH by proton results in a reduction of absorbance, which indicates the antioxidant potential of particular extract or compound. Polyphenols have the ability to grant stability in DPPH radical by donating proton or sharing electron. That is why the plants having functional molecules usually show antioxidant activity.

The results of DPPH scavenging assay for H. indica extracts in terms of IC50 value are given in Fig. 4.1.

RESULTS AND DISCUSSION

The highest DPPH scavenging of 35.35 ± 0.189 μg/mL was given by 60% ethanolic fraction followed by 80% fraction (IC50 = 43.78 ± 0.076 μg/mL). The lowest antioxidant potential was exhibited by 20% ethanolic extract.

The DPPH activity (IC50 value) for H. lagenicaulis extracts is represented in

Fig. 4.2. The lowest IC50 value (43.11 ± 0.96 μg/mL) was witnessed for 60% ethanolic extract, which indicated the highest DPPH scavenging. The second highest antioxidant potential was possessed by 80% ethanolic extract (50.41 ± 0.77 μg/mL). The 20% ethanolic extract was the least efficient to scavenge DPPH radical. The statistical findings highlighted that antioxidant potential of 60% ethanolic extract was significant among all other extracts (ρ < 0.05).

80 f 70 e 60 d 50

g/mL) c μ 40 b 50 a 30

0 20% 40% 60% 80% 100% BHA

ethanolic ethanolic ethanolic ethanolic ethanolic DPPHscavenging (IC Extracts and BHA

Fig. 4-1 IC50 values of H. indica extracts for DPPH scavenging

RESULTS AND DISCUSSION

80 f e 70

60 d c 50 b 40 a

g/mL) μ

20 50

(IC DPPH scavenging DPPHscavenging 0 20% 40% 60% 80% 100% BHA ethanolic ethanolic ethanolic ethanolic ethanolic Extracts and BHA

Fig. 4-2 IC50 values of H. lagenicaulis extracts for DPPH scavenging

The statistical analysis revealed that IC50 value 60% extracts for DPPH activity

(both for H. indica and H. lagenicaulis) were significant with ρ<0.05 when compared with values of remaining extracts. The DPPH activity presents the potential of a substance or extract to snub the free radicals and is the leading assay to assess the antioxidant potential. The DPPH radical may get stabilized by electron or proton transfer mechanism when in contact with polyphenols. The extent of radical stabilization is reflected as the antioxidant potential of the plant extract. The DPPH activity provides a preliminary assessment to move further for other biological and medicinal properties. The phenolics and flavonoids play a leading role in antioxidant activity. The free radicals and ROS are leading cause of oxidative damages to biomolecules, including lipids, proteins and DNA in the living system. The antioxidants bring these detrimental species in control, and DPPH assay is the basic tool to get primary information about the antioxidant and medicinal potential of plants.

RESULTS AND DISCUSSION

However, many other assays are also available, which may give useful information about the antioxidant properties of plants in a more quantitative way.

4.4.2 Total Antioxidant Power Assay (TAP).

This assay is based upon the reduction of Mo(VI) to Mo(V) by antioxidants.

The green color complex is formed as a result of reduction which absorbs at 695 nm.

Greater absorbance value reflects greater antioxidant capacity [86]. Fig. 4.3 indicated the results of TAP assay for H. indica extracts.

The 60% ethanolic extract showed the highest antioxidant capacity

(330.26±3.13 ASE/g DE). The second highest TAP value was found for 80% extract, which showed 260.52±2.13 ASE/g DE. The least TAP value was calculated for 20% ethanolic extract (188.7±3.54 ASE/g DE). The 60% ethanolic extract comparatively showed higher capacity than all other extracts as indicated by ANOVA (ρ<0.05). The

TAP values for H. lagenicaulis were found highest in 60% ethanolic extract (Fig. 4.4)

400

350 a

300 b 250 c d e 200

150

100

Antioxidant power ASE/g power Antioxidant DE 0 20% ethanol 40% ethanol 60% ethanol 80% ethanol 100% ethanol

Extracts

Fig. 4-3 Antioxidant power of H. indica leaf extracts

RESULTS AND DISCUSSION

300

250 a

200 b c c 150

d 100

0 20% ethanolic 40% ethanolic 60% ethanolic 80% ethanolic 100%

Antioxidant power ASE/g DE DE ASE/g power Antioxidant ethanolic

Extracts

Fig. 4-4 Antioxidant power of H. lagenicaulis leaf extracts

The 60% ethanolic extract of H. lagenicaulis showed TAP value of 239.33 ±

3.78 ASE/g DE. The second maximum activity was shown by 80% ethanolic extract for which the value was 189.33±2.51 ASE/g DE. The 20% ethanolic extract showed least antioxidant power. The statistical comparison indicated that 60% fraction was significantly powerful antioxidant fraction among all other extracts (ρ<0.05).

The TAP values for H. indica were better than H. lagenicaulis extracts. The antioxidant potential may vary even for the same plant due to many factors. The climate, soil fertility, water quality, gene expression, stress and plant age may be decisive factors to control the antioxidant capacities of plants. The physiological and molecular responses of plants to environmental conditions may regulate the gene expressions, which further lead to change in phytochemicals and antioxidant potential of plants [87, 88]. An investigation revealed the total antioxidant capacity of 90 mg

ASE/Gde for Anchomanes difformis (family Araceae) based upon phspho-

RESULTS AND DISCUSSION molybdenum complex formation method which was less than the values of the current study [89].

The total antioxidant capacity values of methanolic, hexanolic and aqueous extracts of Adiantum caudatum leaves were reported. The methanolic extract exhibited the highest TAP values, however less than the value of 60% ethanolic extract of H. indica. The study also indicated that aqueous extract exhibited the least antioxidant capacity, which supported the findings of current work [90]. The aqueous phase has a high polarity which may be a misfit with the polarity of metabolites and therefore, the water as a solvent is not as successful as alcohol. The antioxidant power of plant extract is obviously due to the presence of polyphenols.

The phenolic and flavonoids contribute to the antioxidant properties of plant extracts. The plant phenolics are well studied natural metabolites and are responsible for antioxidant activities of plants. Substantial pieces of evidence are available on the antioxidant role of phenolics. The phenolics scavenge ROS and other oxidants to reduce or eliminate the chances of oxidative stress-related diseases. The structural features of these metabolites contribute to their action against oxidants. The phenolics are considered as an important dietary components for maintaining the ROS homeostasis [91].

4.4.3 The β-Carotene Bleaching Assay

The results of this assay for H. indica and H. lagenicaulis leaf extracts are given in Fig. 4.5 and Fig. 4.6.

The β-carotene bleaching potential is a very useful indicator of antioxidant activity, especially when the lipids and fats stability and shelf life is under consideration. The reactive oxygen species attack the unsaturation sites of carotene to

RESULTS AND DISCUSSION produce peroxides. The plants having antioxidants inhibit the oxidation of carotene.

The carotene bleaching appears in the fainting of yellow color. The maintenance of yellow shade in the presence of plant extracts or antioxidants reflects the antioxidant potential of substances added in the reaction mixture.

The results indicated that 60% ethanolic extracts of both plants showed the highest % inhibition for β-carotene bleaching. The 60% ethanolic extract of H. indica inhibited the β-carotene bleaching by 78.50%. Similarly, the 60% ethanolic extract of

H. lagenicaulis inhibited the β-carotene bleaching by 72.38%. The statistical analysis indicated that both values were significantly higher, as shown by ρ<0.05.

90 a 80

70 b

60 c de 50 e 40

Inhibition % Inhibition 30

0 20% ethanolic 40% ethanolic 60% ethanolic 80% ethanolic 100% ethanolic Extracts

Fig. 4-5 The % inhibition of β-carotene by leaf extracts of H. indica

RESULTS AND DISCUSSION

80 a 70 b 60 c

% 50 d e 40

30 Inhibition 20

0 20% ethanolic 40% ethanolic 60% ethanolic 80% ethanolic 100% ethanolic Extracts

Fig. 4-6 The % inhibition of β-carotene by leaf extracts of H. lagenicaulis

4.4.4 Iron Chelating Activity

Iron chelating activity results are presented in Fig. 4.7 and Fig. 4.8 for H. indica and H. lagenicaulis, respectively.

RESULTS AND DISCUSSION

H. indica 90 a b 80 c 70 60 d 50 e 40 30

chelating activity (%) activity chelating 20 -

10 Iron 0 20% ethanolic 40% ethanolic 60% ethanolic 80% ethanolic 100% ethanolic Extracts

Fig. 4-7 Iron chelating activity of H. indica leaf extracts

The results indicated that 60% ethanolic extracts of both plants exhibited substantial iron-chelating potential. Iron metal (Fe) may produce free radicals by gain or loss of electrons which contribute to oxidative stress. The chelating agents in plants are effective to reduce the Fe based production of free radicals. Plants are well known to pose a chelating effect due to the presence of certain phytochemicals [92]. The role of chelating therapy is very essential because transition metals are known to cause various anomalies, especially in metabolic syndrome. The iron-chelating activity of both plant extracts may be of therapeutic use to reduce Fe load-based disorders and related health issues.

RESULTS AND DISCUSSION

80 H. lagenicaulis

70 a

60 b b

50 c

40 d

chelating activity (%) activity chelating -

20 Iron

0 20% ethanolic 40% ethanolic 60% ethanolic 80% ethanolic 100% ethanolic

Fig. 4-8 Iron chelating activity of H. lagenicaulis leaf extracts

Enzyme Inhibition Assays

4.5.1 The α-Glucosidase Inhibition Assay

The results of α-glucosidase inhibition for H. indica and H. lagenicaulis are given in Fig. 4.9 and Fig. 4.10.

The results of α-glucosidase inhibition by hydroethanolic extracts of H. indica revealed that 60% ethanolic extract showed maximum enzyme inhibition (IC50 = 36.52

± 0.08 μg/mL). The lowest inhibition of α-glucosidase was observed for 20% ethanolic extract, as indicated by its IC50 value (66.55 ± 0.08 μg/mL). The results of enzyme inhibition were compared statistically and noted that 60% of ethanolic extract was a significantly potent inhibitor of the enzyme among all extracts (ρ<0.05). The comparison of 80% and 40% ethanolic extracts indicated that these were non- significant regarding enzyme inhibition (ρ>0.05).

RESULTS AND DISCUSSION

It was noteworthy that no extract could reach acarbose for which the IC50 was

26.13 ± 0.01 μg/mL. Similar findings were observed for H. lagenicaulis extracts, where also 60% extract exhibited the highest α-glucosidase inhibition (IC50 = 41.25 ±

1.25 μg/mL). However, in contrast to H. indica, 80% and 40% ethanolic extracts were statistically significant as indicated by letters as superscript. Again, no extract could match the enzyme inhibition shown by acarbose.

The α-glucosidases are the enzymes which are essential for the release of glucose from complex carbohydrates. The reversible inhibition of α-glucosidase in a competitive manner delays the digestion of carbohydrates and reduces the glucose uptake by the intestine. This mechanism of action reduces the postprandial glucose level [93]. The use of α-glucosidase inhibitors in diets is must due to carbohydrate-rich diets in Pakistan. It reflected that the dietary intake of enzyme’s inhibitors is very essential to keep blood glucose level in control after-meal [94]. The phenolics of plants are involved in hypoglycemia, most probably due to inhibition of α- glucosidases [95].

RESULTS AND DISCUSSION

70 e d 60

50 c c 40 b

30 a

(µg/mL) 50

IC 10

0 20% ethanol 40% ethanol 60% ethanol 80% ethanol 100% Acarbose ethanol Extracts

Fig. 4-9 The α-glucosidase of H. indica leaf extracts

80 f 70 e 60 d

50 c b

g/mL) μ 30 a

value ( value 50

IC 10

0 20%Ethanolic 40%Ethanolic 60%Ethanolic 80%Ethanolic 100% Acarbose Ethanolic Extracts

Fig. 4-10 The α-glucosidase of H. lagenicaulis leaf extracts

RESULTS AND DISCUSSION

The active site residues which are basically amino acid residue of α- glucosidase are very important entities. The structure-based efficiency of the enzyme is a key point. The occupation of these functional sites by any molecule and their mixture, i.e. plant extract alters the activity of the enzyme. This modification in the structure of enzyme results in activity loss.

4.5.2 The α-Amylase Inhibition

The α-amylase inhibition results for H. indica and H. lagenicaulis are given in

Fig. 4.11 and Fig. 4.12 respectively. The findings explored that the 60% ethanolic extract was the most potent fraction to inhibit the in-vitro activity of the α-amylase enzyme. The 60% ethanolic extract of H. indica possessed the IC50 value of 58.2±1.25

µg/mL and 60% ethanolic extract of H. lagenicaulis possessed the IC50 value of

60.58±3.24 µg/mL. The 60% ethanolic extracts of both plants were proved as significantly different among all extracts shown by statistical analysis (ρ<0.05). The

40% and 60% extracts of H. indica were statistically non-significant (ρ>0.05) but that of H. lagenicaulis were statistically significant (ρ<0.05). However, no extract could reach the inhibitory activity of standard drug acarbose. The possible cause of α- amylase inhibition was the availability of phytochemicals (phenolics and flavonoids) in the plant extracts [21].

RESULTS AND DISCUSSION

120

e 100 cd d c

g/mL) 80 c μ b

60 value ( value

40 a

50 IC 20

0 Aqueous 20% ethanolic 40% ethanolic 60%ethanolic 80% ethanolic 100%ethanolic Acarbose

Extracts

Fig. 4-11 The α-amylase inhibtion of H. indica leaf extracts

120 f 100 e d c 80 b 60 a

40 value (µg/mL) value

20 50

IC 0 20% ethanolic 40% ethanolic 60%ethanolic 80% ethanolic 100%ethanolic Acarbose

Extracts

Fig. 4-12 The α-amylase inhibtion of H. lagenicaulis leaf extracts

RESULTS AND DISCUSSION

The α-amylase inhibitors are also known as starch blockers because they act on the starch present in food. These inhibitors stop or slow down the intestinal digestion of starch and restrict its splitting into simpler sugars for absorption. This phenomenon reduces the blood glucose level after a meal [96]. The in-vitro inhibition of α-amylase activity is considered as an important tool to investigate the antidiabetic potential of plant extracts. A study reported that ethanolic extract of Phyllanthus amarus was more effective than hexane fraction to inhibit the in-vitro activity of α-amylase [97].

Another study linked the α-amylase inhibitory potential of the plants to phenolics, flavonoids, and antioxidant capacity and a mild to weak linkage was observed [98].

The polyphenols from plants are not only responsible for eliminating the oxidative stress but also has the ability to bind proteins which may be another factor to restrict the α-amylase activity [99]. The values of TPC, TFC, DPPH assay, other antioxidant activities, α-glucosidase and α-amylase inhibitory properties emphasized to explore most active fraction (60% ethanolic) for metabolites/phytochemicals being the main factor.

4.5.3 The Acetylcholine Esterase Inhibition

The results of acetylcholine esterase inhibition are given in Fig. 4.13 and Fig.

4.14. The results were mutually compared and statistically analyzed.

RESULTS AND DISCUSSION

300

250 d c b a a 200

150

(µg/mL) 50 50

IC 100

0 20% ethanolic 40% ethanolic 60% ethanolic 80% ethanolic 100% ethanolic Extracts

Fig. 4-13 The acetylcholine esterase inhibition by H. indica leaf extracts

300 e d c 250 b a

200

150

(µg/mL)

50 IC 100

0 20% ethanolic 40% ethanolic 60% ethanolic 80% ethanolic 100% ethanolic Extracts

Fig. 4-14 The acetylcholine esterase inhibition by H. lagenicaulis leaf extracts

RESULTS AND DISCUSSION

The 60% ethanolic extract of H. indica exhibited maximum acetylcholine esterase activity with an IC50 value of 201 µg/mL but not significantly different from

80% ethanolic extract. However, the 60% ethanolic extract of H. lagenicaulis showed an IC50 value of 220 µg/mL. The basic mechanism of acetylcholine esterase assay is the dissociation of acetylthiocholine iodide to produce thio-nitrobenzoate ion, which forms the yellow color complex with DTNB. This yellow complex absorbs at 470 nm.

The plant extracts which reduce the formation of this complex may have the potential to restrict the activity of acetylcholine esterase. The role of acetylcholine esterase is very obvious in Alzheimer’s disease by interacting with amyloid-β to increase its deposits. Hence acetylcholine esterase inhibitors may reduce the intensity of neural disorders [100]. The results of the current study revealed that both plants exhibited reasonable acetylcholine esterase inhibition. This enzyme inhibition indicated the presence of some phytochemicals with ability to block the activity of this neural enzyme.

Metabolite Profiling

4.6.1 The 1HNMR Based Identification of Metabolite Class

The 1HNMR spectra confirmed the presence of various metabolites as indicated by specific peaks in respective regions. The chemical shift values (δH) in various regions predicted the presence of metabolites like amino acids, sugars, fatty acids and polyphenols. The main 1HNMR spectrum and expanded regions are represented from Fig. 4.15 to 4.22 for both plant extracts.

RESULTS AND DISCUSSION

Aromatic

Carbohydrates

Organic acids

Fig. 4-15 Main 1HNMR spectrum of 60% ethanolic leaf extract of H. lagenicaulis

Aromatic Region

Fig. 4-16 Expanded 1HNMR spectrum (6-9 ppm) of 60% ethanolic leaf extract of H. lagenicaulis

RESULTS AND DISCUSSION

Carbohydrate Region

Fig. 4-17 Expanded 1HNMR spectrum (3-6 ppm) of 60% ethanolic leaf extract of H.

Organic Acid Region (Fatty acids and Amino acids)

Lagenicaulis

Fig. 4-18 Expanded 1HNMR spectrum (1-2.6 ppm) of 60% ethanolic leaf extract of

H. lagenicaulis

RESULTS AND DISCUSSION

Carbohydrates

Aromatic Organic acids

Fig. 4-19 Main 1HNMR spectrum of 60% ethanolic leaf extract of H. indica

Aromatic Region

Fig. 4-20 Expanded 1HNMR spectrum (6-8 ppm) of 60% ethanolic leaf extract of H.

indica

RESULTS AND DISCUSSION

Carbohydrate Region

Fig. 4-21 Expanded 1HNMR spectrum (3-6 ppm) of 60% ethanolic leaf extract of H.

indica

Organic acid Region including Fatty Acids and Amino Acids

Fig. 4-22 Expanded 1HNMR spectrum (1-3 ppm) of 60% ethanolic leaf extract of H.

indica

RESULTS AND DISCUSSION

The region ranging from 1-2.5 ppm, was recognized as the organic acid region, which included amino acids and fatty acids along with some peaks associated with carbohydrates. The chemical shift value ranging from 3.5 ppm to 4 ppm was a typical carbohydrate range. However, some typical peaks like duplets may appear from 5 ppm to 6 ppm representing the sugars like glucose and fructose. The region from 6-9 ppm is known as an aromatic region and exhibit peaks of polyphenols (phenolic acids and flavonoids). The significant peaks were observed for 60% ethanolic extracts of H. lagenicaulis and H. indica in all important regions of 1HNMR spectra. This technique has emerged as an innovative approach to profile primary and secondary metabolites, both on a qualitative and quantitative scale. The 1HNMR technique is now considered as an important tool in the recent field of plant metabolomics. However, the quantitative assessment of metabolites in complex plant extract is a very difficult task and need more care and deep insight into the structural environment of metabolites

[27]. Peaks in aromatic regions of 1HNMR spectra of 60% ethanolic extract of both plants indicated the existence of polyphenols, might be responsible for biological properties of these plant extracts. This technique successfully screened out the possible metabolites, but their identification and confirmation were carried out by UHPLC-

QTOF-MS/MS technique.

4.6.2 Metabolite Identification by UHPLC-QTOF-MS/MS

The secondary metabolites were characterized by adopting UHPLC-QTOF-

MS/MS and the identified compounds in H. indica are given in Table 4.6 along with characteristic features. The chromatogram is represented in Fig. 4.23.

The peak at Rt 1.603 minutes, was of citric acid (m/z 191[M-H]-) whereas the

- main fragment peak at m/z 111 [M-CO2-H2O] was noted. The procyanidin B1,

RESULTS AND DISCUSSION procyanidin B2 and procyanidin B3 were characterized by parent ion peak of m/z 577

[M-H]- in the mass spectrum. The signal at m/z 425 was observed due to removal of an aromatic moiety of m/z 152. Epicatechin was detected at Rt 5.749 minutes with parent ion peak of m/z 289. Similarly, the catechin was detected at Rt 7.511 minutes with parent ion peak of m/z 289 while the characteristic peak of m/z 245 was also observed.

A peak at m/z 123 was noted due to removal of the fragment of m/z166.

Fig. 4-23 Chromatogram of 60% ethanolic extract of H. indica

Apigenin-c-hexocide-c-hexocide was noted at Rt 8.380 with parent ion peak at m/z 593 whereas some characteristic fragment ions were observed at 383 amu and 353 amu, respectively. Kaempferol appeared at Rt 9.114 minutes with m/z 285, which was a characteristic one. The fragment ion peaks at m/z 151 due to removal of the fragment of 134 amu. The peak at m/z 353 is characteristic of caffeoyl quinic acid and m/z for quinic acid. The fragments indicated the compound most probably as feruloyl-O-

RESULTS AND DISCUSSION sinapoyl-O-caffeoylquinic acid. Gallic acid was confirmed by the presence of its

- - characteristic peaks at m/z 169 [M-H] , m/z 125 [M-CO2-H] and m/z 79 [M-CO2-

– H2O-H] (Fig. 4.24-4.39).

Table 4.6 Chromatographic and mass spectrometric data of compounds in H. indica, including retention time (Rt) and major fragment peaks

Sr. Compound Rt Molecular ion Major Molecular No (min) peak (m/z) fragments (m/z) formula

1 Citric acid 1.482 191 173, 129, 111 C6H8O7

2 Procyanidin B3 5.438 577 451, 425, 407 C23H30O17

3 Epicatechin 5.749 289 245, 263, 123 C15H14O6

4 Procyanidin B2 6.557 577 451, 425, 407 C23H30O17

5 Catechin 6.695 933 577, 425, 355, C38H46O27 derivative 337, 209, 191, 147, 85

6 Procyanidin B1 7.222 577 451, 425, 407 C23H30O17

7 Catechin 7.511 289 271, 245, 203, C15H14O6 123

8 Apigenin-c- 8.380 593 503, 473, 395, C20H34O20 hexocide-c- 383, 353, 325 hexocide

9 Kaempferol 9.114 285 217, 175, 151, C15H10O6 133

10 Kaempferol 9.585 527 447, 364, 285, C17H20O19 derivative 241

11 Quinic acid 11.341 733 653, 353, 299, C32H30O20 derivative 285, 191

12 Gallic acid 21.401 169 125, 97, 79 C7H6O5

RESULTS AND DISCUSSION

Fig. 4-24 Mass spectrum of citric acid

Fig. 4-25 Fragmentation of citric acid

Citric acid appeared at Rt 1.482 minutes with characteristic parent ion peak at

- - m/z 191[M-H] and daughter ion peak at m/z 111[M-CO2-H2O] .

RESULTS AND DISCUSSION

Fig. 4-26 Mass spectrum of procyanidin B3

Fig. 4-27 Mass spectrum of procyanidin B2

RESULTS AND DISCUSSION

Fig. 4-28 Mass spectrum of procyanidin B1

RESULTS AND DISCUSSION

Fig. 4-29 Fragmentation pattern of procyanidin

RESULTS AND DISCUSSION

Fig. 4-30 Mass spectrum of epicatechin

Fig. 4-31 Mass spectrum of catechin

RESULTS AND DISCUSSION

Fig. 4-32 Fragmentation pattern of epicatechin

Fig. 4-33 Mass spectrum apigenin-C-hexocide-C-hexocide

RESULTS AND DISCUSSION

Fig. 4-34 Fragmentation pattern of apigenin-C-hexocide-C-hexocide

RESULTS AND DISCUSSION

Fig. 4-35 Mass spectrum of kaempferol

Fig. 4-36 Mass fragmentation pattern of kaempferol

RESULTS AND DISCUSSION

Fig. 4-37 Mass spectrum of quinic acid derivative

The peak at m/z 353 is characteristic of caffeoyl quinic acid and m/z for quinic acid. The fragments indicated the compound most probably as feruloyl-O-sinapoyl-O- caffeoylquinic acid [101].

Fig. 4-38 Mass spectrum of gallic acid.

RESULTS AND DISCUSSION

Fig. 4-39 Fragmentation pattern of gallic acid

The secondary metabolites identified in H. lagenicaulis with the help of

UHPLC-QTOF-MS/MS are given in Table 4.7 along with retention times, molecular ion peaks and fragment ions. The chromatogram is given in Fig. 4.40.

Fig. 4-40 Chromatogram of 60% ethanolic extract of H. lagenicaulis

RESULTS AND DISCUSSION

Table 4.7 The retention times (Rt) and mass spectrometric data of major compounds in

H. lagenicaulis

Sr. Compound Rt Molecular ion Major Molecular

No (min) peak (m/z) fragments (m/z) formula

1 Citric acid 1.603 191 111 C6H8O7

2 Trimethoxy 8.972 773 635, 609, 300 C40H38O16 flavone derivative

3 Kaempferol 9.110 285 151, 93 C15H10O6

4 Rutin 9.27 609 300, 271 C27H30O16

5 Hesperetin 5-O- 9.433 463 301,300, 271, C22H24O11 glucoside 97

6 Kaempferol- 9.689 593 285, 284, 255 C31H30O12 coumaroyl- glucoside

7 Luteolin 3- 9.724 447 285, 284, 255, C21H20O11 glucoside 227 8 Isorhamnetin-3-O- 9.995 623 543, 527, 427, C21H36O21 rutinoside 315, 314

The compound appeared at Rt of 1.603 minutes was identified as citric acid.

- The reason was the characteristic peaks at m/z 191[M-H] and at m/z 111[M-CO2-

- H2O] due to fragmentation. The compound at Rt 8.972 minutes was identified as trimethoxy flavone derivative. The signals at m/z 635, m/z 609 and m/z 300 were characteristic peaks for the said compound.

A peak at Rt 9.110 minutes with m/z 285 was the characteristic peak of kaempferol. The kaempferol upon removal of 134 amu, produced its fragments with m/z 151 and peak due to the removal of phenyl moiety appeared at m/z 93 amu. These

RESULTS AND DISCUSSION peaks in the mass spectrum confirmed the presence of kaempferol in 60% ethanolic extract.

Similarly, another important flavonoid, the rutin, was also identified in plant extract. The rutin appeared at Rt 9.27 minutes, and its main peak was detected at m/z

609. Rutin upon ionization generated fragments and the fragment of m/z 300 produced by elimination of mass residue of 309 amu. The daughter ion at m/z 300 further lost 29 amu to give a peak at m/z 271.

The presence of kaempferol-coumaroyl-glucoside was also confirmed by the mass spectrum data. The peak at m/z 593 was its characteristic parent peak whereas due to the fragmentation process, coumaroyl glucoside was removed to give a peak at m/z 285 and a peak was observed at Rt 9.724 minutes with m/z 447.

The leuteolin peak was produced at m/z 285 due to removal of glucose which identified the compound as leuteolin 3-glucoside. The peak at Rt 9.433 minutes (m/z

- - - 463 [M-H] , fragment ions at m/z 301 [M-glucose-H] , m/z 271 [m/z 301-CH2O-H]

- - m/z 255 [m/z 301-C2H2O-H] , m/z 149 [m/z 255-C6H2O2-H] ) confirmed the presence of hesperetin 5-O-glucoside. The compound appeared at Rt 9.995 minutes having a peak of m/z 623 was confirmed as isorhamnetin 3-O-rutinoside by studying its mass fragmentation pattern. Ionization process produced daughter ion peaks of m/z 315[M-

- - - 318-H amu] , m/z 300 [m/z 315-CH3-H] and m/z 284[m/z 315-CH3O-H] (Fig. 4.41-

4.55).

RESULTS AND DISCUSSION

Fig. 4-41 Mass spectrum of citric acid

Fig. 4-42 Fragmentation pattern of citric acid

RESULTS AND DISCUSSION

Fig. 4-43 Mass spectrum of trimethoxyflavone derivative

Fig. 4-44 Mass spectrum of kaempferol.

RESULTS AND DISCUSSION

Fig. 4-45 Fragmentation pattern of kaempferol

Fig. 4-46 Mass spectrum of rutin

RESULTS AND DISCUSSION

Fig. 4-47 Fragmentation pattern of rutin

Fig. 4-48 Mass spectrum of hesperetin-5-O-glucoside

RESULTS AND DISCUSSION

Fig. 4-49 Fragmentation pattern of hesperetin-5-O-glucoside

Fig. 4-50 Mass spectrum of kaempferol coumaroyl glucoside

RESULTS AND DISCUSSION

Fig. 4-51 Fragmentation pattern of kaempferol coumaroyl glucoside

Fig. 4-52 Mass spectrum of luteolin 3-glucoside

RESULTS AND DISCUSSION

Fig. 4-53 Fragmentation pattern of luteolin 3-glucoside

Fig. 4-54 Mass spectrum of isorhamnetin-3-O-rutinoside

RESULTS AND DISCUSSION

Fig. 4-55 Fragmentation pattern of isorhamnetin-3-O-rutinoside

The compounds identified in H. indica and H. lagenicaulis are well reported for their antioxidant and antidiabetic activities. These phytochemicals usually make a complex with the substrate (enzyme) to occupy the active site residues. Later, these interactions can modify the structure of the protein, which may reduce or completely vanish the enzyme activity. The inhibition of dietary enzymes by phytochemicals can be non-competitive, which is an additional feature. The major classes of identified compounds are given in Table 4.8.

RESULTS AND DISCUSSION

Table 4.8 The identified compounds and their classes

Sr. No Major Compounds Class of Compound

1 Citric acid Tricarboxylic acid

2 Procyanidin Condensed tannins

3 Epicatechin Flavonoid

4 Catechin Flavanol

5 Apigenin-c-hexocide-c-hexocide Flavanoid glycoside

6 Kaempferol Flavanol

7 Gallic acid Phenolic acid

8 Rutin Flavanoid

9 Hesperetin glucoside Flavanone

10 Luteolin glucoside Flavone

11 Isorhamnetin glucoside Flavonol

It is evident that these metabolites from leaf extracts have the potential to reduce the activity of α-glucosidase and α-amylase; however, their contribution in extract should be confirmed by animal trials to conclude the impact on scientific grounds.

RESULTS AND DISCUSSION

Cytotoxicity Evaluation

The toxicity of plant extracts must be evaluated before going for animal trials.

The toxicity of plants-based products is usually low or negligible, which provides them with an advantage over synthetic drugs or compounds. Cytotoxicity evaluation is an essential component of the study where extracts or specific substances are involved in pre-clinical and clinical trials. If the extract shows high toxicity, it may lose its importance in spite of its high medicinal potential. In this study, the hemolytic assay was conducted to compare the toxicity of extracts to that of triton-x100. The results for hemoglobin release are given in Fig. 4.56.

These results were given in hemolytic %, which meant the ability of the extract to destroy blood cells. These results revealed that both plant extracts exhibited negligible toxicity removing the risk factor for further pre-clinical trial.

100

Toxicity % Toxicity 30

0 H. l H.L PBSTriton x-100

Fig. 4-56 Cytotoxicity evaluation of 60% ethanolic plant extracts of H. indica and H.

RESULTS AND DISCUSSION

Lagenicaulis

Thermal Stability

Thermal stability of plant extracts is very important to get the maximum benefit from polyphenol-rich extracts. The temperature may deteriorate the functional molecules, which consequently reduces biological activities. The 60% ethanolic extracts of H. indica and H. lagenicaulis were subjected to thermal stability analysis in terms of DPPH activity. The extracts were stored at various temperature conditions, and antioxidant activities were determined. The results of the thermal stability test are given in Fig. 4.67. It was observed that the extracts exhibited sufficient thermal stability at elevated temperature. Although, there was a decrease in DPPH radical scavenging potential of extracts with increasing temperature. A gentle and smooth decline in DPPH scavenging activity was noted, but still, the radical scavenging percentages were acceptable. The H. indica leaf extract comparatively showed some resistance to temperature effect. The loss in DPPH scavenging activity was probably due to structural degradation of compounds. A study performed on six leafy vegetables to access their antioxidant potential at 100°C reported that the antioxidant activity was dose dependent, and polyphenol concentration was the major factor even at high temperature [102]. The thermal stability of plant extracts was acceptable and can be used in food processing with mild loss of activity at relatively high temperature.

RESULTS AND DISCUSSION

100 90 80 70 60 50 H. indica 40 H. lagenicaulis 30 20

10 DPPH radical scavenging % scavenging DPPHradical 0 0 20 40 60 80 100 Temperature °C

Fig. 4-57 The effect of temperature on DPPH radical scavenging %

Hypoglycemic and Hypolipidemic Potential of H. indica and H. lagenicaulis

4.9.1 The Blood Glucose Level

The hypoglycemic activity of plant extracts was evaluated by checking the blood glucose level (BGL) of diabetic mice in comparison with metformin. The impact of H. indica and H. lagenicaulis leaf extract doses on BGL of diabetic mice is represented in Fig. 4.57 and Fig 4.58, respectively. The values of BGL indicated that the extracts of both plants reduced the BGL to variable extents, and the effect was dose-dependent. The H. indica leaf extract at a high dose significantly brought the

BGL to safe value at completion of the four-week experimental period. The extract at half dose also reduced the BGL but less effectively than FG. When the impact of H. lagenicaulis on BGL of diabetic mice was assessed, it was observed that the extract even at the high dose was not effective as was the case with H. indica. The possible reason behind this phenomenon might be the variation in quality and quantity of phytochemicals present in leaves of both plants. The values of TPC and TFC of

RESULTS AND DISCUSSION identified metabolites clearly supported the notion of phytochemical distribution responsible for hypoglycemic potential. These polyphenols are known to improve the health indicators by adopting various pathways as per disease complication. On considering the impact of these phytochemicals to reduce the BGL, there may be some possible reasons. The reduction in blood glucose level may be due to the following main reasons,

• Blocking of active sites of α-glucosidase and α-amylase • Improved glucose uptake by cells • Delocalization of GLUT-4 • Reduction in oxidative stress • Regeneration of pancreatic β-cells

350

300

250 NG 200 DG 150 MG

100 HG (mg/dL) Blood Glocose Level Level BloodGlocose FG 50

0 0 1 2 3 4 Number of Weeks

Fig. 4-58 The impact of H.indica leaf extracts on blood glucose level of mice (NG =

normal mice group, DG = diabetic mice group, MG = metformin group, HG =

half dose group, FG = full dose group).

RESULTS AND DISCUSSION

350

300

250

200 NG

150 DG MG 100 HG

50 FG Blood Glucose Level mg/dL Level BloodGlucose

0 0 1 2 3 4

Number of Weeks

Fig. 4-59 The impact of H.lagenicaulis leaf extracts on blood glucose level of mice. (NG = normal mice group, DG = diabetic mice group, MG = metformin group, HG = half dose group, FG = full dose group).

The α-glucosidase and α-amylase inhibition is one of the most common observations. The plant polyphenols act as enzyme blockers by occupying specific active sites, which ultimately limit the enzyme activity. As a result, the digestion of carbohydrates is reduced, which appears in the reduction of blood glucose level.

The active site residues of dietary enzymes when occupied by polyphenols, the alteration in structure may occur which modify the functional ability of enzymes.

Consequently, the enzymatic activity is reduced, which is the most probable mechanism behind the carbohydrate hydrolyzing enzyme activity loss upon using the plant. When numerous compounds are present in an extract, the synergistic impact may take place to exert cumulative influence.

The translocation of GLUT-4 is also an important factor which results in glucose absorption by cells. The GLUT-4 translocation improves the entry of glucose

100

RESULTS AND DISCUSSION molecules through insulin into cells. This process is due to the rehabilitation of insulin sensitivity, which is usually lost in diabetic conditions. The plant extracts may enhance the GLUT-4 translocation both through insulin-dependent and insulin-independent pathways. As the GLUT-4 is the major transporter, which facilitates glucose intake by the muscle and adipose tissues. GLUT-4 impairment is observed with the development of insulin resistance. This impairment is the major hurdle in GLUT-4 translocation, which reduces the glucose intake by cells and tissues. The phytochemicals are known to re-equilibrate the GLUT-4 between the inner membrane and the plasma membrane.

The hydroxyl groups and aromatic portions of phytochemicals interact with the functional regions of ROS to exert antioxidant activity. The phenolic compounds in plant extracts are considered as major contributing entities towards antioxidant activities of plants.

The structural feature of polyphenols which are generally phenolic moieties or groups which interact with ROS and other free radicals to scavenge them to change their reactivity [103]. Most probably, the antioxidant potential of plant extract is the main operating factor, which reduces the ROS. The scavenging of ROS definitely reduces oxidative stress. The improvements in biochemical indicators or biomarkers as a result of the reduction of oxidative stress represent the improved health status which not only indicates the diabetes intensity reduction but also prevents the disease prolongation.

The improvement in pancreatic beta-cell functions and regeneration of beta cells is also another factor which cannot be ignored especially when plant-based extracts and compounds are involved. The compounds identified in the current study were reported to have antidiabetic potential along with the strong antioxidant activity.

The two important flavonoids, the kaempferol and rutin identified in the current study

101

RESULTS AND DISCUSSION are well reported to exert antidiabetic role by blocking the specific sites of α- glucosidase. The reduced activity of α-glucosidase results in the reduction of postprandial BGL after administering kaempferol and rutin [104].

A comparison of α-glucosidase inhibition for kaempferol and quercetin was conducted in a study which indicated that the IC50 value of kaempferol was lower than quercetin. It reflected that kaempferol as a strong inhibitor of α-glucosidase [105].

Another glycoside Rutin is very common in medicinal plants and herbs. Rutin is characterized by the presence of many hydroxyl groups which are involved in its medicinal activities. A study reported that rutin exhibited strong inhibition of α- glucosidase action and to manage metabolic syndrome like obesity and hyperglycemia

[106, 107]. Another study revealed that luteolin and its derivatives possessed α- glucosidase inhibition, surprisingly higher than acarbose [108]. Another flavonoid isorhamnetin-3-O-rutinoside identified in H. lagenicaulis was also studied as an α- glucosidase inhibitor in a previously reported investigation. The results of the study revealed that isorhamnetin-3-O-rutinoside showed substantially low IC50 value [109].

The search for novel α-glucosidase inhibitors is an ongoing strategy because the most effective inhibitor acarbose is associated with certain limitations or side complications. Many gastrointestinal disorders are associated with acarbose, which reduces its acceptability [110]. Another important aspect is the mode of action of acarbose against dietary enzymes. Acarbose was reported to inhibit α-glucosidase in a competitive way, whereas the plant extracts enriched in polyphenols followed non- competitive pathway. The pathway adopted by plant extract (non-competitive) was associated with many benefits, including structural facing a wide variety of sites rather than a single position. Moreover, the non-competitive pathway of action by

102

RESULTS AND DISCUSSION polyphenols being the active part of extract is independent of substrate concentration, while acarbose mode of action is concentration-dependent of α-glucosidase [111].

A study reported the antidiabetic role of Conocarpus erectus leaf extracts. The study involved the estimation of polyphenols, antioxidant and α-glucosidase inhibition, which proved the phenolics and flavonoids as a major contributing factor in medicinal potential of the plant [25]. A study also supported the role of polyphenols of plants to decrease glucose level in diabetes. The phytochemicals from plants not only reduced the BGL, but also led to the betterment of hematological indicators [112]. The principle mode of action of secondary metabolites or phytochemicals towards antioxidant and α-glucosidase inhibitory action of H. lagenicaulis was not yet confirmed. However, the most probable mode of action might be the synergic effect of metabolites usually responsible for medicinal potential of extracts of vegetal origin

[42]. Moreover, these functional molecules were also reported to alter the cellular functions in a positive way to enhance the system capacity, either to decrease the intensity of oxidative stress or to improve the repair of impaired physiological machinery under disease condition [109].

A significant hypoglycemic activity of 60% ethanol extract of H. indica owing to the presence of certain important compounds. The citric acid was identifying in the extract, and it is a week acid which is naturally present in many plants and fruits.

Procyanidins (B1, B2, and B3) were also present in the plant extract. Procyanidins are the tannins which are a subclass of flavonoids made up of epicatechin and catechin.

Similarly, apigenin-di-hexoxide was also identified in extract. The extract also contained kaempferol, a quinic acid derivative and gallic acid. The well-established antioxidant and antidiabetic properties of such molecules were due to functional group oriented interference with free radicals and active sites of the enzyme [103, 105]. The

103

RESULTS AND DISCUSSION decline in BGL of diabetic mice by 60% ethanol extract at high dose are quite comparable with the metformin-treated mice group. The physiological alterations are probably due to the interaction of bioactive molecules in plant extract with the α- glucosidase enzyme in the intestine of mice. The α-glucosidase inhibition by compounds is due to blocking of active sites of enzymes or modification in structure to reduce the activity [113]. A recent study compared the in-vitro hypoglycemic potential of Cinnamomum zeylanicum and Cumin cyminum. The results indicated the dose- dependent inhibition pattern against α-glucosidase enzyme by both species [114].

Similarly, the antidiabetic influence of Conocarpus erectus was evaluated in obese diabetic mice. Extract dose of 450 mg/kg b.w controlled the BGL significantly [25].

There may be many reasons behind the reduction in BGL of diabetic mice upon consumption of H. indica leaf extract including the elimination of oxidative stress, repair of the pancreas, enhancement of glucose uptake by cells, as reported by previous investigations [45, 115]. The elimination of oxidative stress removed the pressure of ROS on pancreatic cells, which might be an important factor behind the rejuvenation of pancreatic function and insulin secretion. The repair of beta cells of the pancreas is also linked with the same phenomenon. The antioxidant, anti-α- glucosidase and hypoglycemic characteristics of H. indica and H lagenicaulis leaf extracts may be attributed due to synergistic mode of action governed by the collective contribution of secondary metabolites present in plant extracts. The phenomenon at the cellular level mentioned here are operating simultaneously, but their intensity may vary, which should be investigated and correlated with screening out the interconnectivity.

104

RESULTS AND DISCUSSION

4.9.2 Blood Haemoglobin Level

The results of haemoglobin (Hb) levels of different mice groups to study the impact of H. indica and H lagenicaulis leaf extracts are represented in Fig. 4.59 and

Fig. 4.60, respectively.

The highest Hb levels were observed for NG mice, and lowest Hb levels were obtained for DG mice. This reflected that the high blood glucose level significantly affected the Hb of mice. The main reason behind the lowering of the Hb level of diabetic mice (DG) was probably the glycation of Hb [116].

10 a 9 8 b 7 c d d 6 5 4

3 haemoglobin (g/dL) haemoglobin 2

Blood 1 0 NG DG MG HG FG Treatment groups

Fig. 4-60 The impact of H.indica leaf extracts on blood haemoglobin level of mice

(NG = normal mice group, DG = diabetic mice group, MG = metformin group,

HG = half dose group, FG = full dose group).

105

RESULTS AND DISCUSSION

10 a 9 8 b 7 e d c 6 5 4

3 haemoglobin (g/dL) haemoglobin 2

1 Blood 0 NG DG MG HG FG Treatment groups

Fig. 4-61 The impact of H.lagenicaulis leaf extracts on blood haemoglobin level of

mice (NG = normal mice group, DG = diabetic mice group, MG = metformin

group, HG = half dose group, FG = full dose group).

It was obvious from results that the Hb of FG mice were significantly higher than Hb of all treated mice groups (DG, MG, and HG) for both plants (ρ<0.05). However, no extract dose could bring the Hb level of diabetic mice to the level of NG mice, but significant improvements were observed. The improvement in Hb levels might be due to the antidiabetic effect of plant extracts. The reduction in BGL and oxidative stress was also reported as a key factor to control the risks associated with diabetes like reduction in Hb [117]. The 60% ethanolic extracts of both plants effectively improved the Hb; however, the H. indica leaf extract was slightly better than H. lagenicaulis extract regarding improvement in Hb of diabetic mice.

106

RESULTS AND DISCUSSION

4.9.3 Hypolipidemic Assessment in Diabetic Mice

The Fig. 4.61 shows the comparison of TC, HDL and LDL of NMG, DUG,

MFG for H. indica extract-treated mice groups.

120

100

80 TC (mg/dL) 60 HDL(mg/dL)

40 LDL(mg/dL)

0 NG DG MG HG FG

Fig. 4-62 The impact of H.indica leaf extracts on total cholestrol (TC), high density

lipoproteins (HDL) and low density lipoproteins (LDL) of mice (NG = normal

mice group, DG = diabetic mice group, MG = metformin group, HG = half

dose group, FG = full dose group).

The average values of TC of NMG, DUG, MFG, LDG and HDG mice were

40.5 mg/dL,110.30 mg/dL,58.65 mg/dL,87.30 mg/dL and 65.70 mg/dL, respectively.

The TC of DUG animals was very high, indicating the adverse health status.

Metformin reduced the TC but could not be brought to the NMG level. Similarly, extract doses also influenced the TC of mice but could not bring the values upto the level of NMG mice. The reduction in TC was probably due to a reduction in HDL levels, as shown in Fig. 4.61. The HDL level of HDG mice was quite comparable to

107

RESULTS AND DISCUSSION

NMG and MFG mice. These findings revealed the significant impact of plant extracts on lipid profiles of diabetic mice.

The impact of H. lagenicaulis leaf extract on lipid profile is shown in Fig. 4.62

120

100

80 TC (mg/dL) 60 HDL(mg/dL)

40 LDL(mg/dL)

0 NG DG MG HG FG

Fig. 4-63 The impact of H.lagenicaulis leaf extracts on total cholestrol (TC), high

density lipoproteins (HDL) and low density lipoproteins (LDL) of mice (NG =

normal mice group, DG = diabetic mice group, MG = metformin group, HG =

half dose group, FG = full dose group).

The TC, HDL and LDL of animals of NG were found 43 mg/dL, 25.5 mg/dL and 15.5 mg/dL, respectively. However, these values for DG animals were very high and unhealthy. It was observed that metformin affected the values of TC, HDL and LDL by bringing them to acceptable range. The high dose of the plant extract (FG) significantly improved the lipid profile by lowering TC and HDL, while an immense increase in LDL was noticed. However, this reduction in TC was observed yet very high from TC of NG and MG animals but quite better than the TC of DG. The results

108

RESULTS AND DISCUSSION indicated that plant extract at high dose improved the lipid profile of diabetic animals.

The TC is considered as the combination of HDL and LDL. The H. indica leaf extract was more effective to control the elevated concentrations of TC and HDL in diabetic mice in comparison to H. lagenicaulis. The cholesterol is beneficial to general health, but its high level can cause serious health impacts like cardiac disorders. Diabetes usually results in high LDL, which is very dangerous because LDL represents saturated part of cholesterol. The LDL should be within limits to avoid complications under diabetic conditions. The disturbance in cholesterol balance and an increase in

LDL due to diabetes is called diabetic dyslipidemia. Dyslipidemia leads to cardiac disorders and atherosclerosis. The phytochemicals present in plants modify the basic mechanism to give healthy impacts. The polyphenols may alter the appetite signaling pathway and reduce appetite. On the other hand, these polyphenols also increase energy production by breaking the lipids. These phenomena may reduce the cholesterol level by minimizing the LDL concentration [118]. The decrease in BGL of diabetic mice is often linked with the improved lipid profile. There is a direct relationship between BGL and lipid metabolism. The normalization of blood glucose is also reflected as an improvement in TC, HDL and LDL values of diabetics. The different interconnected pathways working is modified by free radicals like free fatty acids to disturb the lipid profile. The phytochemical constituents of plant extracts encounter these free radicals to normalize the redox balance. The reversal of damaged pathology to the normal state was reported for plant-based treatments, where the deteriorating effects of synthetic drugs were observed [119]. A moderate reduction in

HDL value of diabetic mice upon administering Salvia officinalis leaf extract was also reported, which indicated that there was no lipolysis observed for the treatment [120].

109

RESULTS AND DISCUSSION

The flavonoids and phenolic compounds of plants are the key responsible entities to combat diabetic complications to bring the metabolic functions at normal [121].

Changes in Body Weight

The changes in body weight were measured and are shown in Fig. 4.63 and

4.64 for H. indica and H. lagenicaulis, respectively.

29.5 29 28.5 NG 28 DG 27.5 MG 27 Weight Weight grams in HD 26.5 FG 26 0 7 14 21 28 Days

Fig. 4-64 Changes in body weight (g) of mice treated with H. indica (NG = normal

mice group, DG = diabetic mice group, MG = metformin group, HG = half

dose group, FG = full dose group).

The results indicated that body weights of NG increased with the passage of time. The body weights of diabetic mice (DG) decreased from the normal value (NG) during the study period. The metformin and half dose of the extract (H. indica) showed some resistance against weight loss. However, the weights of FG mice relatively showed a stable trend than MG and HD animals.

The changes in body weight of mice treated with H. lagenicaulis were also monitored carefully. It was shown by results that the weight loss for DG was very

110

RESULTS AND DISCUSSION sharp after 14 days of having diabetes. The mice of HG follow the routine trend of increase in body weight. Contrary to H. indica, the mice treated with H. lagenicaulis did not show a significant check on weight loss. The resistance was shown by FG

29.5

28.5 NG 28 DG 27.5 MG

Weight Weight grams in 27 HD

26.5 FG

26 0 7 14 21 28 Days against weight loss and relatively better as compared to MG and HD group.

Fig. 4-65 Changes in body weight (g) of mice treated with H. lagenicaulis (NG =

normal mice group, DG = diabetic mice group, MG = metformin group, HG =

half dose group, FG = full dose group).

It was observed that H. indica restricted weight loss more effectively than H. lagenicaulis. The insulin resistance or deficiency stops the entry of glucose in tissues and cells, which is necessary to meet the energy requirement. To cope with the energy needs of the body, the burning of stored lipids and muscles to get the energy is considered as the major cause of weight loss. The nutritive value, as well as the pharmacological properties of both plant species, may be a possible cause to restrict the weight loss of diabetic mice. A study reported the role of Camellia sinensis leaves

111

RESULTS AND DISCUSSION in lowering of blood glucose level and inhibiting the bodyweight loss of albino mice.

The various hematological parameters and lipid profiles of diabetic mice were compared with the treatment group. The plant leaves successfully restored the normal body weight by altering the lipid profile [122].

Water Uptake by Mice

The results of water uptake by mice during the study period are given in Fig.

4.65 and Fig. 4.66 for H. indica and H. lagenicaulis leaf extract respectively.

8 7 6

5 NG 4 DG 3 MG 2 HD

Wateruptake mL/mouse in FG 1 0 0 7 14 21 28 Days

Fig. 4-66 Water uptake (mL) by mice treated with H. indica(NG = normal mice

group, DG = diabetic mice group, MG = metformin group, HG = half dose

group, FG = full dose group).

The results indicated that the water intake by diabetic mice groups was increased with the passage of time. The NG of H. indica treated experiment showed a sudden rise in water uptake during 14 to 21 days, which later on got a little smoother.

112

RESULTS AND DISCUSSION

The water consumption for FD and MD were comparable, whereas the NG mice showed a stable trend.

Fig. 4.66 represents the results of water uptake by mice treated with H. lagenicaulis. In this case, the water consumption by DG followed a sudden and continuously rising trend of water consumption by the end of 28 days period. The MG showed relatively lower water consumption than the FG group.

8 7 6

5 NG 4 DG 3 MG 2 HD

Wateruptake mL/mouse in FG 1 0 0 7 14 21 28 Days

Fig. 4-67 Water uptake (mL) by mice treated with H. lagenicaulis(NG = normal mice

group, DG = diabetic mice group, MG = metformin group, HG = half dose

group, FG = full dose group).

The DM is known to increase the thrust due to the high glucose level in the blood. The high sugar content of blood forces the kidney to overwork to get rid of extra sugar, which results in frequent urination. The high rate of urination results in water deficiency, which is met by drinking water. The relative lowering of water uptake by diabetic mice upon receiving extracts at different doses may be linked to a

113

RESULTS AND DISCUSSION reduction in BGL. It may be narrated that the lowering of BGL and water consumption both are interlinked.

114

CONCLUSIONS

CONCLUSION

The findings of current research work are impressive and added substantial information on the pharmacological role of H. indica and H. lagenicaulis. The extraction was optimized using various solvent compositions, and results indicated that 60% ethanol was the most effective solvent for extraction of phytochemicals from the leafy portion of H. indica and H. lagenicaulis. The polarity was probably the most important feature, which discriminated the extract yields and resultantly the polyphenol content. The lyophilization assisted ultrasonication was used and found quite effective to enhance extract yields and phytochemicals. The extracts were subjected to antioxidant activities (DPPH assay, total antioxidant power assay), enzyme inhibitory assays (α-glucosidase, α-amylase, acetylcholine esterase) and metabolite profiling. The 60% ethanolic extract was observed as the most potent. The relatively higher amounts of phytochemicals in 60% ethanolic extracts were responsible for the antioxidant activities, metal chelating potential, enzyme inhibitions and antidiabetic properties. The metabolite identification using 1HNMR and UHPLC-

QTOF-MS/MS added significant information in the limited phytochemical library of

H. indica and H. lagenicaulis. Kaempferol and its derivatives, leutolin-3-O-glucoside, gallic acid, hesparatin derivative, isorhamnetin, apigenin di-glucoside, rutin, procyanidin B3, procyanidin B2, procyanidin B1, epicatechin and catechin were among identified compounds and were most probably responsible for antidiabetic properties of H. indica and H. lagenicaulis. The in-vivo trials were conducted in alloxanated mice, and BGL were checked on a weekly basis. The lipid profile and blood hemoglobin was determined and compared at the end of experiment. The hypoglycemic and hypolipidemic impact of H. indica was more pronounced than H. lagenicaulis. The possible reason was the difference in quality and quantity of

115

CONCLUSIONS secondary metabolites. The successful in-vivo trials may be exploited for the development of antidiabetic formulations and functional diets. Both plant extracts reduced the blood glucose level of diabetic mice within the stipulated time period; however, the hypoglycemic impact of H. indica was relatively higher than H. lagenicaulis. Both plants can provide secondary metabolites with antioxidant and antidiabetic functionalities. The plant extracts having these metabolites may be opted to add antidiabetic properties in foods. Further studies may be carried out on isolation of these compounds. The present study has added valuable information on the phytochemical status of both plants. The moderate DPPH radical scavenging activity loss of potent extracts under different temperature storage was impressive. The negligible toxicity of H. indica and H. lagenicaulis was significant and a favorable feature, which enhanced the importance and suitability of both plants to develop food additive and herbal formulation with good hypoglycemic properties and moderate hypolipidemic attributes.

116

REFERENCES

[1] E. Birben, U.M. Sahiner, C. Sackesen, S. Erzurum, O. Kalayci, Oxidative stress and antioxidant defense, The World Allergy Organization Journal, 5 (2012) 9-19.

DOI: 10.1097/WOX.0b013e3182439613.

[2] J.T. Hancock, R. Desikan, S.J. Neill, Role of reactive oxygen species in cell signalling pathways, Biochemical Society Transactions, 29 (2001) 345-350. DOI:

10.1042/0300-5127:0290345.

[3] T. Finkel, N.J. Holbrook, Oxidants, oxidative stress and the biology of ageing,

Nature, 408 (2000) 239-247. DOI: 10.1038/35041687.

[4] A. Phaniendra, D.B. Jestadi, L. Periyasamy, Free radicals: properties, sources, targets, and their implication in various diseases, Indian Journal of Clinical

Biochemistry, 30 (2015) 11-26. DOI: 10.1007/s12291-014-0446-0.

[5] C.M.O. Volpe, P.H. Villar-Delfino, P.M.F. Dos Anjos, J.A. Nogueira-Machado,

Cellular death, reactive oxygen species (ROS) and diabetic complications, Cell death

& disease, 9 (2018) 119. DOI: 10.1038/s41419-017-0135-z.

[6] Z. Liu, Z. Ren, J. Zhang, C.C. Chuang, E. Kandaswamy, T. Zhou, L. Zuo, Role of

ROS and Nutritional Antioxidants in Human Diseases, Frontiers in Physiology, 9

(2018) 477. DOI: 10.3389/fphys.2018.00477.

[7] N.L. Madhikarmi, K.R. Murthy, Antioxidant enzymes and oxidative stress in the erythrocytes of iron deficiency anemic patients supplemented with vitamins, Iranian

Biomedical Journal, 18 (2014) 82-87.

[8] F. Tabet, R.M. Touyz, Chapter 30 - Reactive Oxygen Species, Oxidative Stress, and Vascular Biology in Hypertension, in: G.Y.H. Lip, J.E. Hall (Eds.)

Comprehensive Hypertension, Mosby, Philadelphia, 2007, pp. 337-347.

117

REFERENCES

[9] G. Pizzino, N. Irrera, M. Cucinotta, G. Pallio, F. Mannino, V. Arcoraci, F.

Squadrito, D. Altavilla, A. Bitto, Oxidative Stress: Harms and Benefits for Human

Health, Oxidative Medicine and Cellular Longevity, 2017 (2017). DOI:

10.1155/2017/8416763.

[10] G.S. Aseervatham, T. Sivasudha, R. Jeyadevi, D. Arul Ananth, Environmental factors and unhealthy lifestyle influence oxidative stress in humans--an overview,

Environmental Science and Pollution Research International, 20 (2013) 4356-4369.

DOI: 10.1007/s11356-013-1748-0.

[11] E. Abi-aad, R. Bechara, J. Grimblot, A. Aboukais, Preparation and characterization of ceria under an oxidizing atmosphere. Thermal analysis, XPS, and

EPR study, Chemistry of Materials, 5 (1993) 793-797. DOI: 10.1021/cm00030a013.

[12] L. Guariguata, D.R. Whiting, I. Hambleton, J. Beagley, U. Linnenkamp, J.E.

Shaw, Global estimates of diabetes prevalence for 2013 and projections for 2035,

Diabetes Research and Clinical Practice, 103 (2014) 137-149. DOI:

10.1016/j.diabres.2013.11.002.

[13] S.A. Meo, I. Zia, I.A. Bukhari, S.A. Arain, Type 2 diabetes mellitus in Pakistan:

Current prevalence and future forecast, Journal of the Pakistan Medical Association,

66 (2016) 1637-1642.

[14] S. Huang, M.P. Czech, The GLUT4 glucose transporter, Cell Metabolism, 5

(2007) 237-252. DOI: 10.1016/j.cmet.2007.03.006.

[15] J. Li, K.L. Houseknecht, A.E. Stenbit, E.B. Katz, M.J. Charron, Reduced glucose uptake precedes insulin signaling defects in adipocytes from heterozygous GLUT4 knockout mice, The FASEB Journal, 14 (2000) 1117-1125. DOI:

10.1096/fasebj.14.9.1117.

118

REFERENCES

[16] J.A. Kim, Y. Wei, J.R. Sowers, Role of mitochondrial dysfunction in insulin resistance, Circulation Research, 102 (2008) 401-414. DOI:

10.1161/circresaha.107.165472.

[17] M. Singh, A. Jha, A. Kumar, N. Hettiarachchy, A.K. Rai, D. Sharma, Influence of the solvents on the extraction of major phenolic compounds (punicalagin, ellagic acid and gallic acid) and their antioxidant activities in pomegranate aril, Journal of

Food Science and Technology, 51 (2014) 2070-2077. DOI: 10.1007/s13197-014-

1267-0.

[18] A.J. Scheen, Hepatotoxicity with thiazolidinediones: is it a class effect?, Drug safety, 24 (2001) 873-888. DOI: 10.2165/00002018-200124120-00002.

[19] E. Ritz, M. Adamczak, A. Wiecek, Chapter 2 - Carbohydrate Metabolism in

Kidney Disease and Kidney Failure, in: J.D. Kopple, S.G. Massry, K. Kalantar-Zadeh

(Eds.) Nutritional Management of Renal Disease, Academic Press2013, pp. 17-30.

[20] G. Rena, D.G. Hardie, E.R. Pearson, The mechanisms of action of metformin,

Diabetologia, 60 (2017) 1577-1585. DOI: 10.1007/s00125-017-4342-z.

[21] M.N. Wickramaratne, J.C. Punchihewa, D.B. Wickramaratne, In-vitro alpha amylase inhibitory activity of the leaf extracts of Adenanthera pavonina, BMC

Complementary and Alternative Medicine, 16 (2016) 466. DOI: 10.1186/s12906-016-

1452-y.

[22] A.B. Tukun, N. Shaheen, C.P. Banu, M. Mohiduzzaman, S. Islam, M. Begum,

Antioxidant capacity and total phenolic contents in hydrophilic extracts of selected

Bangladeshi medicinal plants, Asian Pacific Journal of Tropical Medicine, 7s1 (2014)

S568-573. DOI: 10.1016/s1995-7645(14)60291-1.

119

REFERENCES

[23] H. Nasri, H. Shirzad, A. Baradaran, M. Rafieian-Kopaei, Antioxidant plants and diabetes mellitus, Journal of Research in Medical Sciences, 20 (2015) 491-502. DOI:

10.4103/1735-1995.163977.

[24] Q.D. Do, A.E. Angkawijaya, P.L. Tran-Nguyen, L.H. Huynh, F.E. Soetaredjo, S.

Ismadji, Y.H. Ju, Effect of extraction solvent on total phenol content, total flavonoid content, and antioxidant activity of Limnophila aromatica, Journal of Food and Drug

Analysis, 22 (2014) 296-302. DOI: 10.1016/j.jfda.2013.11.001.

[25] S.A. Raza, A.R. Chaudhary, M.W. Mumtaz, A. Ghaffar, A. Adnan, A. Waheed,

Antihyperglycemic effect of Conocarpus erectus leaf extract in alloxan-induced diabetic mice, Pakistan Journal of Pharmaceutical Sciences, 31 (2018) 637-642.

[26] S. Oancea, D. Ghincevici, O. Ketney, The effect of ultrasonic pretreatment and sample preparation on the extraction yield of antioxidant compounds and activity of black currant fruits, 2014, 62 (2014) Acta Chimica Slovenica. DOI:

10.17344/acsi.2014.895.

[27] M.W. Mumtaz, A.A. Hamid, M.T. Akhtar, F. Anwar, U. Rashid, M.H. Al-

Zuaidy, An overview of recent developments in metabolomics and proteomics – phytotherapic research perspectives, Frontiers in Life Science, 10 (2017) 1-37. DOI:

10.1080/21553769.2017.1279573.

[28] A.N. Welz, A. Emberger-Klein, K. Menrad, Why people use herbal medicine: insights from a focus-group study in Germany, BMC Complementary and Alternative

Medicine, 18 (2018) 92. DOI: 10.1186/s12906-018-2160-6.

[29] C.B. Jin, Medicinal properties and common usages of some palm species in the

Kampung Peta community of Endau-Rompin National Park, Johor, Journal of

Tropical Medicinal Plants, 6 (2005) 79-83.

120

REFERENCES

[30] J. Bolsinger, A. Pronczuk, R. Sambanthamurthi, K.C. Hayes, Anti-diabetic effects of palm fruit juice in the Nile rat (Arvicanthis niloticus), Journal of Nutritional

Science, 3 (2014) e5. DOI: 10.1017/jns.2014.3.

[31] M. Gruca, T.R. van Andel, H. Balslev, Ritual uses of palms in traditional medicine in sub-Saharan Africa: a review, Journal of Ethnobiology and

Ethnomedicine, 10 (2014) 60. DOI: 10.1186/1746-4269-10-60.

[32] G. Frausin, R.B.S. Lima, A.F. Hidalgo, L.C. Ming, A.M. Pohlit, Plants of the

Araceae family for malaria and related diseases: a review, Revista Brasileira de

Plantas Medicinais, 17 (2015) 657-666. DOI: 10.1590/1983-084X/14_024.

[33] J. Dransfield, N.W. Uhl, C.B.A. Lange, W.J. Baker, M.M. Harley, C.E. Lewis,

Genera palmarum: the evolution and classification of palms, Kew Publishing, UK

2008.

[34] A.A. Starkov, The role of mitochondria in reactive oxygen species metabolism and signaling, Annals of the New York Academy of Sciences, 1147 (2008) 37-52.

10.1196/annals.1427.015.

[35] E. Wright, Jr., J.L. Scism-Bacon, L.C. Glass, Oxidative stress in type 2 diabetes: the role of fasting and postprandial glycaemia, International journal of clinical practice, 60 (2006) 308-314. DOI: 10.1111/j.1368-5031.2006.00825.x.

[36] M. Fehresti Sani, S. Montasser Kouhsari, L. Moradabadi, Effects of Three

Medicinal Plants Extracts in Experimental Diabetes: Antioxidant Enzymes Activities and Plasma Lipids Profiles in Comparison with Metformin, Iranian Journal of

Pharmaceutical Research, 11 (2012) 897-903.

[37] M.R. Elgindi, A.E.-N.B. Singab, S.H. Aly, I.I. Mahmoud, Phytochemical investigation and antioxidant activity of Hyophorbe verschaffeltii (Arecaceae),

Journal of Pharmacognosy and Phytochemistry, 5 (2016) 39-46.

121

REFERENCES

[38] S.M. Firdous, Phytochemicals for treatment of diabetes, EXCLI Journal, 13

(2014) 451-453.

[39] A. Yessoufou, J. Gbenou, O. Grissa, A. Hichami, A.-M. Simonin, Z. Tabka, M.

Moudachirou, K. Moutairou, N.A. Khan, Anti-hyperglycemic effects of three medicinal plants in diabetic pregnancy: modulation of T cell proliferation, BMC

Complementary and Alternative Medicine, 13 (2013) 77-77. DOI: 10.1186/1472-

6882-13-77.

[40] B.K. Tiwari, D. Kumar, A.B. Abidi, S.I. Rizvi, Efficacy of Composite Extract from Leaves and Fruits of Medicinal Plants Used in Traditional Diabetic Therapy against Oxidative Stress in Alloxan-Induced Diabetic Rats, ISRN Pharmacology,

2014 (2014). DOI: 10.1155/2014/608590.

[41] S. Ahmad, R. Rehman, S. Haider, Z. Batool, F. Ahmed, S.B. Ahmed, T. Perveen,

S. Rafiq, S. Sadir, S. Shahzad, Quantitative and qualitative assessment of additives present in broiler chicken feed and meat and their implications for human health,

Journal of Pakistan Medical Association, 68 (2018) 876-881.

[42] H. Agnaniet, E.J. Mbot, O. Keita, J.-A. Fehrentz, A. Ankli, A. Gallud, M. Garcia,

M. Gary-Bobo, J. Lebibi, T. Cresteil, C. Menut, Antidiabetic potential of two medicinal plants used in Gabonese folk medicine, BMC Complementary and

Alternative Medicine, 16 (2016) 71. DOI: 10.1186/s12906-016-1052-x.

[43] A.I. Prihantini, S. Tachibana, K. Itoh, Evaluation of antioxidant and alpha- glucosidase inhibitory activities of some subtropical plants, Pakistan Journal of

Biological Sciences, 17 (2014) 1106-1114. DOI: 10.3923/pjbs.2014.1106.1114.

[44] S. Lee, A. Mediani, A. Nur Ashikin, A. Azliana, F. Abas, Antioxidant and α- glucosidase inhibitory activities of the leaf and stem of selected traditional medicinal plants, International Food Research Journal, 21 (2014) 165-172.

122

REFERENCES

[45] G. Rehman, M. Hamayun, A. Iqbal, S. Ul Islam, S. Arshad, K. Zaman, A.

Ahmad, A. Shehzad, A. Hussain, I. Lee, In vitro antidiabetic effects and antioxidant potential of Cassia nemophila Pods, BioMed Research International, 2018 (2018) 1-6.

DOI: 10.1155/2018/1824790.

[46] F.T. Bothon, E. Debiton, F. Avlessi, C. Forestier, J.C. Teulade, D.K.

Sohounhloue, In vitro biological effects of two anti-diabetic medicinal plants used in

Benin as folk medicine, BMC Complementary Alternative Medicine, 13 (2013) 51.

DOI: 10.1186/1472-6882-13-51.

[47] R. Murugan, J. Prabu, R. Chandran, T. Sajeesh, M. Iniyavan, T. Parimelazhagan,

Nutritional composition, in vitro antioxidant and anti-diabetic potentials of Breynia retusa (Dennst.) Alston, Food Science and Human Wellness, 5 (2016) 30-38. DOI:

10.1016/j.fshw.2015.12.001.

[48] S. Chatterji, D. Fogel, Study of the effect of the herbal composition SR2004 on hemoglobin A1c, fasting blood glucose, and lipids in patients with type 2 diabetes mellitus, Integr Med Res, 7 (2018) 248-256. DOI: 10.1016/j.imr.2018.04.002.

[49] A. Konsue, C. Picheansoonthon, C. Talubmook, Fasting blood glucose levels and hematological values in normal and streptozotocin-induced diabetic rats of Mimosa pudica L. extracts, Pharmacognosy Journal, 9 (2017) 315-322. DOI:

10.5530/pj.2017.3.54.

[50] S.A. Mard, K. Jalalvand, M. Jafarinejad, H. Balochi, M.K.G. Naseri, Evaluation of the antidiabetic and antilipaemic activities of the hydroalcoholic extract of phoenix dactylifera palm leaves and its fractions in alloxan-induced diabetic rats, Malaysian

Journal of Medical Sciences, 17 (2010) 4-13.

123

REFERENCES

[51] M. Saadullah, B.A. Chaudary, M. Uzair, K. Afzal, Anti-diabetic potential of

Conocarpus lancifolius, Bangladesh Journal of Pharmacology 9(2014) 244-249. DOI:

10.3329/bjp.v9i2.18556.

[52] R. Riyanto, C. Wariyah, Hypoglycemic Effect Of Instant Aloe Vera on Deabetic

Rats Food Research, 2 (2018) 46-50. DOI: 10.26656/fr.2017.2(1).119.

[53] A.S. Abu-Zaiton, Anti-diabetic activity of Ferula assafoetida extract in normal and alloxan-induced diabetic rats, Pakistan Journal of Biological Sciences, 13 (2010)

97-100.

[54] K. Hafeez, S. Andleeb, T. Ghousa, R. G Mustafa, A. Naseer, I. Shafique, K.

Akhter, Phytochemical screening, alpha-glucosidase inhibition, antibacterial and antioxidant potential of Ajuga bracteosa extracts, Current Pharmaceutical

Biotechnology, 18 (2017) 336-342. DOI: 10.2174/1389201018666170313095033.

[55] A.B. Sassi, S. Amroussi, M. Besbes, M. Aouni, F. Skhiri, Antioxidant and α- glucosidase activities and phytochemical constituents of Chrysanthoglossum trifurcatum (Desf.), Asian Pacific Journal of Tropical Medicine 11 (2018) 285-291.

DOI: 10.4103/1995-7645.231469.

[56] D. Russo, P. Valentão, P.B. Andrade, E.C. Fernandez, L. Milella, Evaluation of

Antioxidant, Antidiabetic and Anticholinesterase Activities of Smallanthus sonchifolius Landraces and Correlation with Their Phytochemical Profiles,

International Journal of Molecular Sciences, 16 (2015) 17696-17718. DOI:

10.3390/ijms160817696.

[57] M.H. Al-Zuaidy, A.A. Hamid, A. Ismail, S. Mohamed, A.F. Abdul Razis, M.W.

Mumtaz, S.Z. Salleh, Potent Antidiabetic Activity and Metabolite Profiling of

Melicope Lunu-ankenda Leaves, Journal of Food Science, 81 (2016) C1080-1090.

DOI: 10.1111/1750-3841.13293.

124

REFERENCES

[58] M.H. Al-Zuaidy, M.W. Mumtaz, A.A. Hamid, A. Ismail, S. Mohamed, A.F.A.

Razis, Biochemical characterization and (1)H NMR based metabolomics revealed

Melicope lunu-ankenda leaf extract a potent anti-diabetic agent in rats, BMC

Complementary Alternative Medicine 17 (2017) 359. DOI: 10.1186/s12906-017-

1849-2.

[59] Y.N.G. Quispe, S.H. Hwang, Z. Wang, G. Zuo, S.S. Lim, Screening In Vitro

Targets Related to Diabetes in Herbal Extracts from Peru: Identification of Active

Compounds in Hypericum laricifolium Juss. by Offline High-Performance Liquid

Chromatography, International Journal of Molecular Sciences, 18 (2017) 2512. DOI:

10.3390/ijms18122512.

[60] K.V. Jabir, B. Jayalakshmi, K. Hashim, Research, In-vitro anti diabetic studies and phytochemical evaluation of Heracleum candolleanum, Asian Journal of Plant

Science and Research 4(2014) 31-36.

[61] M. Yilmazer-Musa, A.M. Griffith, A.J. Michels, E. Schneider, B. Frei, Grape seed and tea extracts and catechin 3-gallates are potent inhibitors of α-amylase and α- glucosidase activity, J Agric Food Chem, 60 (2012) 8924-8929. DOI:

10.1021/jf301147n.

[62] M.W. Mumtaz, M.H. Al-Zuaidy, A. Abdul Hamid, M. Danish, M.T. Akhtar, H.

Mukhtar, Metabolite profiling and inhibitory properties of leaf extracts of Ficus benjamina towards α-glucosidase and α-amylase, International Journal of Food

Properties, 21 (2018) 1560-1574. DOI: 10.1080/10942912.2018.1499112.

[63] J. Montes, J. Rodriguez, J. Vaca, C. Guzmán, E. Calandri, Characterization of

Jatropha curcas L. seed and its oil, from Argentina and Paraguay, The Journal of the

Argentine Chemical Society, 98 (2011) 1-9.

125

REFERENCES

[64] A. Iqbal, F. Anwar, R. Nadeem, B. Sultana, M. Mushtaq, Proximate composition and minerals profile of fruit and flower of Karir (Capparis decidua) from Different regions of Punjab (Pakistan), Asian Journal of Chemistry, 26 (2014) 360. DOI:

10.14233/ajchem.2014.15394.

[65] M. Kashif, S. Ullah, Chemical composition and minerals analysis of Hippophae rhamnoides, Azadirachta indica, Punica granatu and Ocimum sanctum leaves, World

Journal of Dairy Food Science, 8 (2013) 67-73. DOI:

10.5829/idosi.wjdfs.2013.8.1.1119.

[66] G. Zengin, Y.S. Cakmak, G.O. Guler, A. Aktumsek, In vitro antioxidant capacities and fatty acid compositions of three Centaurea species collected from

Central Anatolia region of Turkey, Food and Chemical Toxicology, 48 (2010) 2638-

2641. DOI: 10.1016/j.fct.2010.06.033.

[67] J. Zhishen, T. Mengcheng, W. Jianming, The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals, Food Chemistry, 64

(1999) 555-559. DOI: 10.1016/S0308-8146(98)00102-2.

[68] I. Fki, N. Allouche, S. Sayadi, The use of polyphenolic extract, purified hydroxytyrosol and 3, 4-dihydroxyphenyl acetic acid from olive mill wastewater for the stabilization of refined oils: a potential alternative to synthetic antioxidants, Food

Chemistry, 93 (2005) 197-204. DOI: 10.1016/j.foodchem.2004.09.014.

[69] P. Prieto, M. Pineda, M. Aguilar, Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E, Analytical Biochemistry, 269 (1999)

337-341. DOI: 10.1006/abio.1999.4019.

[70] T.C. Dinis, V.M. Maderia, L.M. Almeida, Action of phenolic derivatives

(acetaminophen, salicylate, and 5-aminosalicylate) as inhibitors of membrane lipid

126

REFERENCES peroxidation and as peroxyl radical scavengers, Archives of biochemistry and biophysics, 315 (1994) 161-169. DOI: 10.1006/abbi.1994.1485.

[71] B. Jabeen, N. Riaz, M. Saleem, M.A. Naveed, M. Ashraf, U. Alam, H.M. Rafiq,

R.B. Tareen, A. Jabbar, Isolation of natural compounds from Phlomis stewartii showing alpha-glucosidase inhibitory activity, Phytochemistry, 96 (2013) 443-448.

DOI: 10.1016/j.phytochem.2013.09.015.

[72] M.I. Kazeem, J.O. Adamson, I.A. Ogunwande, Modes of inhibition of alpha - amylase and alpha -glucosidase by aqueous extract of Morinda lucida Benth leaf,

Biomedical Research International, 2013 (2013). DOI: 10.1155/2013/527570.

[73] A. Mediani, F. Abas, A. Khatib, C.P. Tan, I.S. Ismail, K. Shaari, A. Ismail, N.

Lajis, Phytochemical and biological features of Phyllanthus niruri and Phyllanthus urinaria harvested at different growth stages revealed by 1H NMR-based metabolomics, Industrial Crops and Products, 77 (2015) 602-613. DOI:

10.1016/j.indcrop.2015.09.036.

[74] M. Ali, S. Ali, M. Khan, U. Rashid, M. Ahmad, A. Khan, A. Al-Harrasi, F.

Ullah, A. Latif, Synthesis, biological activities, and molecular docking studies of 2- mercaptobenzimidazole based derivatives, Bioorganic Chemistry, 80 (2018) 472-479.

DOI: 10.1016/j.bioorg.2018.06.032.

[75] G. Kumar, L. Karthik, K.V.B. Rao, Hemolytic activity of Indian medicinal plants towards human erythrocytes: an in vitro study, Elixir Appl Botany, 40 (2011) 5534-

5537.

[76] S. Bonner-Weir, Morphological evidence for pancreatic polarity of beta-cell within islets of Langerhans, Diabetes, 37 (1988) 616-621. DOI:

10.2337/diab.37.5.616.

127

REFERENCES

[77] J.E. Emordi, E.O. Agbaje, I.A. Oreagba, O.I. Iribhogbe, Antidiabetic and hypolipidemic activities of hydroethanolic root extract of Uvaria chamae in streptozotocin induced diabetic albino rats, BMC Complementary and Alternative

Medicine, 16 (2016) 468. DOI: 10.1186/s12906-016-1450-0.

[78] D. Syiem, G. Syngai, P.Z. Khup, B.S. Khongwir, B. Kharbuli, H. Kayang,

Hypoglycemic effects of Potentilla fulgens L in normal and alloxan-induced diabetic mice, Journal of ethnopharmacology, 83 (2002) 55-61. 10.1016/s0378-

8741(02)00190-3.

[79] X. Han, Y.L. Tao, Y.P. Deng, J.W. Yu, J. Cai, G.F. Ren, Y.N. Sun, G.J. Jiang,

Metformin ameliorates insulitis in STZ-induced diabetic mice, PeerJ, 5 (2017) e3155.

10.7717/peerj.3155.

[80] T. Himoto, H. Yoneyama, A. Deguch, K. Kurokohchi, M. Inukai, H. Masugata,

F. Goda, S. Senda, S. Watanabe, S. Kubota, S. Kuriyama, T. Masaki, Insulin resistance derived from zinc deficiency in non-diabetic patients with chronic hepatitis

C, Experimental and Therapeutic Medicine, 1 (2010) 707-711. DOI:

10.3892/etm_00000109.

[81] N. Azwanida, A review on the extraction methods use in medicinal plants, principle, strength and limitation, Med Aromatic Plants, 4 (2015) 3-8. DOI:

10.4172/2167-0412.1000196.

[82] P.S. Widyawati, T.D.W. Budianta, F.A. Kusuma, E.L. Wijaya, Difference of solvent polarity to phytochemical content and antioxidant activity of Pluchea indicia less leaves extracts, International Journal of Pharmacognosy Phytochemical Research,

6 (2014) 850-855.

[83] O. Alara, N. Abdurahman, O. Olalere, Ethanolic extraction of flavonoids, phenolics and antioxidants from Vernonia amygdalina leaf using two-level factorial

128

REFERENCES design, Journal of King Saud University-Science, (2017). DOI:

10.1016/j.jksus.2017.08.001.

[84] R. Vardanega, D.T. Santos, M.A. Meireles, Intensification of bioactive compounds extraction from medicinal plants using ultrasonic irradiation,

Pharmacognosy Reviews, 8 (2014) 88-95. DOI: 10.4103/0973-7847.134231.

[85] A. Alkhatib, C. Tsang, A. Tiss, T. Bahorun, H. Arefanian, R. Barake, A. Khadir,

J. Tuomilehto, Functional Foods and Lifestyle Approaches for Diabetes Prevention and Management, Nutrients, 9 (2017) 1310. DOI: 10.3390/nu9121310.

[86] G.K. Jayaprakasha, B.S. Jena, P.S. Negi, K.K. Sakariah, Evaluation of antioxidant activities and antimutagenicity of turmeric oil: a byproduct from curcumin production, Zeitschrift für Naturforschung C, 57 (2002) 828-835. DOI: 10.1515/znc-

2002-9-1013.

[87] C.C. Denardin, G.E. Hirsch, R.F. da Rocha, M. Vizzotto, A.T. Henriques, J.C.F.

Moreira, F. Guma, T. Emanuelli, Antioxidant capacity and bioactive compounds of four Brazilian native fruits, Journal of Food and Drug Analysis, 23 (2015) 387-398.

DOI: 10.1016/j.jfda.2015.01.006.

[88] F.-T. Ni, L.-Y. Chu, H.-B. Shao, Z.-H. Liu, Gene expression and regulation of higher plants under soil water stress, Current Genomics, 10 (2009) 269-280. DOI:

10.2174/138920209788488535.

[89] A.B. Aliyu, M.A. Ibrahim, A.M. Musa, A.O. Musa, J.J. Kiplimo, A.O. Oyewale,

Free radical scavenging and total antioxidant capacity of root extracts of Anchomanes difformis Engl. (Araceae), Acta Poloniae Pharmaceutica, 70 (2013) 115-121.

[90] D. Ahmed, M.M. Khan, R. Saeed, Comparative Analysis of Phenolics,

Flavonoids, and Antioxidant and Antibacterial Potential of Methanolic, Hexanic and

129

REFERENCES

Aqueous Extracts from Adiantum caudatum Leaves, Antioxidants (Basel,

Switzerland), 4 (2015) 394-409. DOI: 10.3390/antiox4020394.

[91] Z. Huyut, Ş. Beydemir, İ. Gülçin, Antioxidant and Antiradical Properties of

Selected Flavonoids and Phenolic Compounds, Biochemistry Research International,

2017 (2017). DOI: 10.1155/2017/7616791.

[92] R. Sudan, M. Bhagat, S. Gupta, J. Singh, A. Koul, Iron (FeII) chelation, ferric reducing antioxidant power, and immune modulating potential of Arisaema jacquemontii (Himalayan Cobra Lily), Biomed Research International, 2014 (2014).

DOI: 10.1155/2014/179865.

[93] H. Bischoff, The mechanism of alpha-glucosidase inhibition in the management of diabetes, Clinical and Investigative Medicine, 18 (1995) 303-311.

[94] S. Kalra, M. Chadha, S. Sharma, A. Unnikrishnan, Untapped diamonds for untamed diabetes: The α-glucosidase inhibitors Indian Journal of Endocrinology and

Metabolism, 18 (2014) 138-141. DOI: 10.4103/2230-8210.129102.

[95] M. Telagari, K. Hullatti, In-vitro alpha-amylase and alpha-glucosidase inhibitory activity of Adiantum caudatum Linn. and Celosia argentea Linn. extracts and fractions, Indian Journal of Pharmacology, 47 (2015) 425-429. DOI: 10.4103/0253-

7613.161270.

[96] B. Dineshkumar, A. Mitra, M. Manjunatha, A comparative study of alpha amylase inhibitory activities of common anti-diabetic plants at Kharagpur 1 block,

International Journal of Green Pharmacy, 4 (2010) 115-121. DOI: 10.4103/0973-

8258.63887.

[97] I.G. Tamil, B. Dineshkumar, M. Nandhakumar, M. Senthilkumar, A. Mitra, In vitro study on α-amylase inhibitory activity of an Indian medicinal plant, Phyllanthus

130

REFERENCES amarus, Indian Journal of Pharmacology, 42 (2010) 280-282. DOI: 10.4103/0253-

7613.70107.

[98] S.O. Oyedemi, B.O. Oyedemi, I.I. Ijeh, P.E. Ohanyerem, R.M. Coopoosamy,

O.A. Aiyegoro, Alpha-Amylase Inhibition and Antioxidative Capacity of Some

Antidiabetic Plants Used by the Traditional Healers in Southeastern Nigeria, The

Scientific World Journal, 2017 (2017). DOI: 10.1155/2017/3592491.

[99] K.M. Ramkumar, T. Balsamy, P. Thayumanavan, R. Palanisamy, Inhibitory effect of Gymnema Montanum leaves on α-glucosidase activity and α-amylase activity and their relationship with polyphenolic content, Medicinal Chemistry

Research, 19 (2010) 948-961. DOI: 10.1007/s00044-009-9241-5.

[100] S.Y. Liew, K.Y. Khaw, V. Murugaiyah, C.Y. Looi, Y.L. Wong, M.R. Mustafa,

M. Litaudon, K. Awang, Natural indole butyrylcholinesterase inhibitors from Nauclea officinalis, Phytomedicine, 22 (2015) 45-48. DOI: 10.1016/j.phymed.2014.11.003.

[101] R. Ben Said, A.I. Hamed, U.A. Mahalel, A.S. Al-Ayed, M. Kowalczyk, J.

Moldoch, W. Oleszek, A. Stochmal, Tentative Characterization of Polyphenolic

Compounds in the Male Flowers of Phoenix dactylifera by Liquid Chromatography

Coupled with Mass Spectrometry and DFT, International Journal of Molecular

Sciences, 18 (2017) 512. DOI: 10.3390/ijms18030512.

[102] L.G. Roy, A. Urooj, Antioxidant Potency, pH and Heat Stability of Selected

Plant Extracts, Journal of Food Biochemistry, 37 (2013) 336-342. DOI:

10.1111/j.1745-4514.2011.00638.x.

[103] M. Aadesariya, V. Ram, P. Dave, Evaluation of antioxidant activities by use of various extracts from Abutilon pannosum and Grewia tenax in the Kachchh Region,

MOJ Food Process Technology, 5 (2017) 00116. DOI:

10.15406/mojfpt.2017.05.00116.

131

REFERENCES

[104] D.F. Pereira, L.H. Cazarolli, C. Lavado, V. Mengatto, M.S. Figueiredo, A.

Guedes, M.G. Pizzolatti, F.R. Silva, Effects of flavonoids on alpha-glucosidase activity: potential targets for glucose homeostasis, Nutrition (Burbank, Los Angeles

County, Calif.), 27 (2011) 1161-1167. DOI: 10.1016/j.nut.2011.01.008.

[105] R.T. Dewi, F.J.P.C. Maryani, Antioxidant and α-glucosidase inhibitory compounds of Centella asiatica, Procedia Chemistry, 17 (2015) 147-152. DOI:

10.1016/j.proche.2015.12.130.

[106] S. Jo, E. Ka, H. Lee, Comparison of antioxidant potential and rat intestinal α- glucosidases inhibitory activities of quercetin, rutin, and isoquercetin, International journal of Applied Research in Natural Products, 2 (2009) 52-60.

[107] S. Habtemariam, G. Lentini, The therapeutic potential of rutin for diabetes: an update, Mini Reviews in Medicinal Chemistry, 15 (2015) 524-528. DOI:

10.2174/138955751507150424103721.

[108] J.S. Kim, C.S. Kwon, K.H. Son, Inhibition of alpha-glucosidase and amylase by luteolin, a flavonoid, Bioscience, Biotechnology, and Biochemistry, 64 (2000) 2458-

2461. DOI: 10.1271/bbb.64.2458.

[109] Z. Yin, W. Zhang, F. Feng, Y. Zhang, W. Kang, α-Glucosidase inhibitors isolated from medicinal plants, Food Science and Human Wellness, 3 (2014) 136-174.

DOI: 10.1016/j.fshw.2014.11.003.

[110] F.A. van de Laar, Alpha-glucosidase inhibitors in the early treatment of type 2 diabetes, Vascular Health and Risk Management, 4 (2008) 1189-1195. DOI:

10.2147/vhrm.s3119.

[111] H. Zhang, G. Wang, T. Beta, J. Dong, Inhibitory properties of aqueous ethanol extracts of propolis on alpha-glucosidase, Evidence-based Complementary and

Alternative Medicine, 2015 (2015). DOI: 10.1155/2015/587383.

132

REFERENCES

[112] H.P. Sudasinghe, D.C. Peiris, Hypoglycemic and hypolipidemic activity of aqueous leaf extract of Passiflora suberosa L, PeerJ, 6 (2018) e4389. DOI:

10.7717/peerj.4389.

[113] A.I. Martinez-Gonzalez, E. Alvarez-Parrilla, A.G. Diaz-Sanchez, L.A. de la

Rosa, J.A. Nunez-Gastelum, A.A. Vazquez-Flores, G.A. Gonzalez-Aguilar, In vitro

Inhibition of Pancreatic Lipase by Polyphenols:A Kinetic, Fluorescence Spectroscopy and Molecular Docking Study, Food Technology and Biotechnology, 55 (2017) 519-

530. DOI: 10.17113/ftb.55.04.17.5138.

[114] N.Y. Puttaswamy, G.D. Rupini, F. Ahmed, A. Urooj, In vitro hypoglycemic potential of spices: Cinnamon and Cumi, Pakistan Journal of Pharmaceutical

Sciences, 31 (2018) 2367-2372.

[115] L.A. Dra, S. Sellami, H. Rais, F. Aziz, A. Aghraz, K. Bekkouche, M. Markouk,

M. Larhsini, Antidiabetic potential of Caralluma europaea against alloxan-induced diabetes in mice, Saudi Journal of Biological Sciences, 26 (2019) 1171-1178. DOI:

10.1016/j.sjbs.2018.05.028.

[116] H.F. Bunn, K.H. Gabbay, P.M. Gallop, The glycosylation of hemoglobin: relevance to diabetes mellitus, Science (New York, N.Y.), 200 (1978) 21-27. DOI:

10.1126/science.635569.

[117] N. Khandouzi, F. Shidfar, A. Rajab, T. Rahideh, P. Hosseini, M. Mir Taheri,

The effects of ginger on fasting blood sugar, hemoglobin a1c, apolipoprotein B, apolipoprotein a-I and malondialdehyde in type 2 diabetic patients, Iranian Journal of

Pharmaceutical Research, 14 (2015) 131-140.

[118] F. Francini-Pesenti, D. Beltramolli, S. Dall'acqua, F. Brocadello, Effect of sugar cane policosanol on lipid profile in primary hypercholesterolemia, Phytotherapy

Research, 22 (2008) 318-322. DOI: 10.1002/ptr.2315.

133

REFERENCES

[119] A.M. Al-Attar, T.A. Zari, Influences of crude extract of tea leaves, Camellia sinensis, on streptozotocin diabetic male albino mice, Saudi Journal of Biological

Sciences, 17 (2010) 295-301. DOI: 10.1016/j.sjbs.2010.05.007.

[120] M.R. Ben Khedher, M. Hammami, J.R.S. Arch, D.C. Hislop, D. Eze, E.T.

Wargent, M.A. Kepczynska, M.S. Zaibi, Preventive effects of Salvia officinalis leaf extract on insulin resistance and inflammation in a model of high fat diet-induced obesity in mice that responds to rosiglitazone, PeerJ, 6 (2018) e4166. DOI:

10.7717/peerj.4166.

[121] R. Uzzaman, M. Ghaffar, Anti-diabetic and hypolipidemic effects of extract from the seed of Gossypium herbaceum L. in Alloxan-induced diabetic rabbits,

Pakistan Journal of Pharmaceutical Sciences, 30 (2017) 75-86.

[122] Y.S. Jaiswal, P.A. Tatke, S.Y. Gabhe, A.B. Vaidya, Antidiabetic activity of extracts of Anacardium occidentale Linn. leaves on n-streptozotocin diabetic rats,

Journal of Traditional and Complementary Medicine, 7 (2017) 421-427. DOI:

10.1016/j.jtcme.2016.11.007.

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