FORMULATION OF MOMORDICA CHARANTIA FRUIT AND SYZYGIUM POLYANTHUM LEAF EXTRACTS BASED ON IN VITRO ANTIOXIDANT AND INHIBITORY ACTIVITY OF α- AMYLASE AND α-GLUCOSIDASE
MUHAMMAD JIHAD SANDIKAPURA Malaya
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FACULTY OF MEDICINE UNIVERSITY OF MALAYA UniversityKUALA LUMPUR
2018 FORMULATION OF MOMORDICA CHARANTIA FRUIT AND SYZYGIUM POLYANTHUM LEAF EXTRACTS BASED ON IN VITRO ANTIOXIDANT AND INHIBITORY ACTIVITY OF α- AMYLASE AND α-GLUCOSIDASE
MUHAMMAD JIHAD SANDIKAPURA
DISSERTATION SUBMITTED IN FULFILMENTMalaya OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF MEDICAL SCIENCEof
FACULTY OF MEDICINE UNIVERSITY OF MALAYA UniversityKUALA LUMPUR
2018 UNIVERSITY MALAYA ORIGINAL LITERARY WORK DECLARATION
Name of candidate: Muhammad Jihad Sandikapura Matrix No: MGN120049 Name of Degree: Master of Medical Science Title of Project Paper/Research Report/Dissertation/Thesis (“this Work”): “Formulation of Momordica charantia fruit and Syzygium Polyanthum Leaf Extracts Based On In Vitro Antioxidant And Inhibitory Activity Of α- Amylase And α- Glucosidase” Field of Study: Pharmacy
I do solemnly and sincerely declare that: (1) I am the sole author/writer of this Work; (2) This Work is original; (3) Any use of any work in which copyright exists was done by way of fair dealing and for permitted purposes and any excerpt or extract from, or reference to or reproduction of any copyright work has been disclosed expressly and sufficiently and the title of the Work and its authorship have been acknowledged in this Work; (4) I do not have any actual knowledge nor Malayado I ought reasonably to know that the making of this work constitutes an infringement of any copyright work; (5) I hereby assign all and every rights in the copyright to this Work to the University of Malaya (“UM”), who henceforth shall be owner of the copyright in this Work and that anyof reproduction or use in any form or by any means whatsoever is prohibited without the written consent of UM having been first had and obtained; (6) I am fully aware that if in the course of making this Work I have infringed any copyright whether intentionally or otherwise, I may be subject to legal action or any other action as may be determined by UM.
Candidate’s Signature Date:
Subscribed and solemnly declared before,
UniversityWitness’s Signature Date:
Name: Designation:
ii ABSTRACT
Natural products are rich in flavonoids, tannins and other polyphenolics with free radical scavenging potential. Free radicals have been claimed to play an important role in affecting human health by causing several diseases including cancer, hypertension, cardiovascular diseases and diabetes. The primary aim of this study is to extract Momordica charantia fruit, Syzygium polyanthum leaf with maceration, soxhlet, sonication and fresh juice methods, further evaluate the extracts either alone or in combinations for antioxidant (DPPH & FRAP) and enzyme inhibitory effect on α– amylase and α–glucosidase. Other objectives of the study include LC-MS, GC-MS qualitative profiling of extracts and conversion of Malaya the best antioxidant and enzyme inhibitory extracts into herbal formulation. Syzygium polyanthum demonstrated better free radical scavenging ability than Momordicaof charantia . It was observed that the % inhibition of DPPH by S. polyanthum (64.93 %) is comparable to standard quercetin
(69.21%). Interestingly the FRAP value of fresh juice of S. polyanthum (69.05 %) was better (p > 0.05) than the quercetin (63.27 %). The fresh juice of S. polyanthum demonstrated predominant inhibitory action against α-amylase (92.21%) and
α–glucosidase (96.06 %) than the standard acarbose (88.51 %). Among 28 different combinations of Momordica charantia (MC) and Syzygium polyanthum (SP) extracts in DPPHUniversity and FRAP analysis, Maceration MC -Sonication SP (65.35 %; DPPH; p < 0.05) and Soxhlet MC-Fresh Juice SP (59.16 %; FRAP; p < 0.05) have shown significant activities. However, 14 selective combinations were tested for enzyme inhibitory studies and found that Soxhlet MC-Fresh Juice SP (90.86 %; α-amylase; p < 0.05) and Soxhlet
MC-Fresh Juice SP (95.52 %; α–glucosidase; p < 0.05) have excellent inhibitory activity against α-amylase and α–glucosidase indicating their ability in controlling postprandial
iii hyperglycaemia. GC-MS and LC-MS qualitative analysis identified several polyphenolics, steroids, glycosides, fatty acids and flavonoids in extracts. A combination extract of Soxhlet (MC)-Fresh juice (SP) was formulated as a phytomedicine. The prepared solid herbal formulations (Tablets-550 mg) were evaluated for pharmacopeial tests and found to have good weight variation (554.5 ± 1.45 mg), hardness (56.8 ± 4.3 N), thickness (3.57 ± 0.17 mm), friability (0.72 < 1 %) and disintegration (< 15 min.). In conclusion, the fresh juice of S. polyanthum has superior FRAP scavenging, α-amylase and also α-glucosidase inhibitory activities than standards. Exogenous intake of antioxidants in the form of herbal extracts can help the body scavenge free radicals effectively and can control hyperglycaemia by α-amylase and α-glucosidase inhibition.
As these two plants are of dietary importance rich in antioxidants can prevent oxidative damage and improve quality of life in diabetic patientsMalaya upon inclusion in diet.
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(Formulasi Ekstrak-Ekstrak Buah Momordica Charantia Dan Daun Syzygium
Polyanthum Berdasarkan Aktiviti Antioksida Dan Perencatan α – Amilase Dan
α- Glukosidase)
ABSTRAK
Produk semula jadi kaya dengan flavonoid, tanin dan polyphenolik lain dengan potensi pengarkaan radikal bebas. Radikal bebas telah dituntut memainkan peranan penting dalam mempengaruhi kesihatan manusia dengan menyebabkan beberapa penyakit termasuk kanser, hipertensi, penyakit kardiovaskular dan diabetes. Tujuan utama kajian ini adalah untuk mengekstrak buah Momordica charantia, daun Syzygium polyanthum dengan maceration, soxhlet, sonication dan kaedah jus segar, selanjutnya menilai ekstrak sama ada secara bersendirian atau dalamMalaya gabungan antioksidan (DPPH & FRAP) dan kesan penghambatan enzim pada α- amilase dan α-glucosidase. Objektif lain kajian ini ialah penyiasatan kualitatifof LC- MS, GC-MS ekstrak dan penukaran ekstrak antioksida dan enzim yang terbaik kepada perumusan herba. Syzygium polyanthum menunjukkan keupayaan pemotongan radikal bebas yang lebih baik daripada Momordica charantia. Telah diperhatikan bahawa perencatan % DPPH oleh S. polyanthum (64.93 %) adalah sebanding dengan quercetin standard (69.21 %).
Menariknya nilai FRAP jus segar S. polyanthum (69.05 %) lebih baik (p> 0.05) daripada quercetin (63.27 %). Jus segar S. polyanthum menunjukkan tindakan penghambatanUniversity utama terhadap α-amylase (92.21 %) dan α-glucosidase (96.06 %) daripada acarbose standard (88.51 %). Antara 28 kombinasi yang berbeza dari ekstrak ekstrak dalam sampel DPPH dan FRAP, Maceration MC-Sonication SP (65.35 %;
DPPH; p <0.05) dan Soxhlet MC-Fresh Juice SP (59.16 %; FRAP; p <0.05) telah menunjukkan aktiviti penting. Walau bagaimanapun, 14 kombinasi selektif telah diuji untuk kajian penghambatan enzim dan mendapati Soxhlet MC-Fresh Juice SP (90.86 %;
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α-amylase; p <0.05) dan Soxhlet MC-Fresh Juice SP (95.52 %; α-glucosidase; p <0.05 ) mempunyai aktiviti perencatan yang sangat baik terhadap α-amilase dan α-glucosidase yang menunjukkan keupayaan mereka dalam mengawal hiperglikemia postprandial.
Analisis kualitatif GC-MS dan LC-MS mengenal pasti beberapa polifenolik, steroid, glikosida, asid lemak dan flavonoid dalam ekstrak. Ekstrak gabungan Soxhlet (MC)-
Fresh juice (SP) dirumuskan sebagai phytomedicine. Formulasi herba pepejal yang disediakan (Tablets-550 mg) telah dinilai untuk ujian farmakope dan didapati mempunyai variasi berat yang baik (554.5 ± 1.45 mg), kekerasan (56.8 ± 4.3 N), ketebalan (3.57 ± 0.17 mm), kebolehpercayaan (0.72 < 1%) dan perpecahan (<15 min.).
Sebagai kesimpulan, jus segar S. polyanthum mempunyai pengambilan FRAP unggul,
α-amilase dan juga aktiviti penghambatan α-glucosidase daripada piawaian. Pengambilan antioksidan luar biasa dalam bentuk ekstrakMalaya herba dapat membantu tubuh membuang radikal bebas secara efektif dan dapat mengendalikan hiperglikemia dengan perencatan α-amilase dan α-glukosidase. Olehof kerana kedua-dua tumbuhan ini penting dalam diet kaya dengan antioksidan dapat mencegah kerosakan oksidatif dan meningkatkan kualiti hidup pesakit diabetes apabila dimasukkan ke dalam diet.
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ACKNOWLEDGEMENTS
It is a great pleasure to thank everyone who helped me towards successful
completion of this thesis. My sincere and deepest gratitude goes first and
foremost to my supervisors, Associate Professor Dr. Mohamed Ibrahim Noordin
and Dr. Shaik Nyamathulla for their untiring guidance, encouragements, intuitive
comments, stimulating discussions and patience from the beginning till the
concluding level of my research. I am sure this significant accomplishment in my
life would have not been possible without their continuous support and belief in
me.
My heartfelt gratitude also goes out to the Head of the Department of Pharmacy, Faculty of Medicine, University ofMalaya Malaya, Kuala Lumpur and The Director of IPharm, Malaysian Institute of Pharmaceuticals and nutraceuticals, Pulau Pinang for providing conduciveof acad emic environment, all laboratory necessities and facilities to carry out and complete this project.
I am heartily thankful to all the academic and non-academic staff,
labmates and friends especially Yasir Osman Ali in the Department of
Pharmacy, Faculty of Medicine, University of Malaya. Special thanks to Dr.
Riyanto Teguh Widodo, Dr. Leong Kok Hoong, and Dr. Aditya Arya for their
additional guidance and advice in helping me to complete my research. UniversityFinally, the project would not have been possible without the unfailing support of my family members, especially my parents, Mr. Edy Supriyadi and
Mrs. Lily Yurida. I am truly indebted and thankful for their unconditional love
and confidence in me as well as their endless patience and encouragements to
help me to achieve my educational goals and career aims. I am grateful to my
wife, Tisnania Walyuni Wijiastuti, other family members Fermita Chelsyana and
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Muhammad Tesar Sandikapura and well-wishers for their words of
encouragement and support to complete my research successfully.
Thank you
Muhammad Jihad Sandikapura
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TABLE OF CONTENTS
PAGE
ABSTRACT ...... iii
ABSTRAK ...... v
ACKNOWLEDGEMENTS ...... vii
TABLE OF CONTENTS ...... ix
LIST OF FIGURES ...... xiv
LIST OF TABLES ...... xvi
LIST OF ABBREVIATIONS ...... xvii
CHAPTER 1: INTRODUCTION ...... 1
1.1 Introduction ...... 1
1.2 Problem statement and justification of the work...... Malaya...... 6 1.3 Aim and objectives ...... of...... 8 1.3.1 Aim ...... 8
1.3.2 Objectives ...... 8
CHAPTER 2: LITERATURE REVIEW ...... 9
2.1 Diabetes mellitus ...... 9
2.2 Classification of diabetes mellitus ...... 10
2.3 Mechanism of action of insulin ...... 11 2.4University Current available treatments for diabetes mellitus ...... 13 2.5 Antidiabetic key enzyme ...... 15
2.5.1 α-amylase ...... 15
2.5.2 α-glucosidase ...... 16
2.6 Free radicals and their association to diseases ...... 18
2.6.1 Free radicals ...... 18
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2.6.2 Types of free radicals ...... 19
2.6.3 Oxidatives stress and its role in diseases...... 21
2.6.3.1 Oxidatives stress and diabetes mellitus ...... 22
2.6.4 Role of antioxidants against free radicals ...... 27
2.6.4.1 Natural antioxidants ...... 27
2.7 Rationale behind selection of S. polyanthum and M. charantia in the
current study ...... 30
2.7.1 Syzygium polyanthum ...... 31
2.7.1.1 Habit, habitat and macroscopy of S. polyanthum ...... 31
2.7.1.2 Chemical constituents of S. polyanthum ...... 31
2.7.1.3 Pharmacological actions of S. polyanthum ...... 31 2.7.1.4 Taxonomic classification of S. polyanthumMalaya ...... 32 2.7.2 Momordica charantia ...... 33 2.7.2.1 Habit, habitat and macroscopyof of M. charantia ...... 33 2.7.2.2 Chemical constituents of M. charantia ...... 33
2.7.2.3 Pharmacological actions of M. charantia ...... 34
2.7.2.4 Taxonomic classification of M. charantia ...... 35
2.8 Extraction, profiling of phytoconstituents from selected plants ...... 35
2.8.1 Maceration ...... 38
2.8.2 Hot continous extraction (Soxhlet extraction) ...... 38 University2.8.3 Sonication ...... 39 2.8.4 Fresh juice ...... 40
2.9 Gas Chromatography-Mass Spectrometry (GC-MS) ...... 41
2.10 Liquid Chromatography-Mass Spectroimetry (LC-MS) ...... 43
2.11 Herbal formulations and alternative system of medicine ...... 45
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2.11.1 Standardization of herbal medicine...... 46
2.11.2 Herbal formulations ...... 47
CHAPTER 3: MATERIAL AND METHODS ...... 51
3.1 Materials ...... 51
3.1.1 Plant Materials ...... 51
3.1.2 Chemicals and reagents ...... 52
3.2 Methods ...... 52
3.2.1 Microscopic evaluation of plant samples ...... 52
3.2.2 Extraction methods applied to plant powders ...... 53
3.2.3 GC-MS and LC-MS profiling of extracts ...... 55
3.2.4 Antioxidant activity and free radical scavenging activity of the extracts ...... Malaya 56 3.2.5 Antidiabetic enzyme inhibitory activity of the extracts ...... 58 3.2.6 Formulation and evaluation of herbalof tablet dosage forms containing the best extracts ...... 60
3.2.6.1 Evaluation of granular flow properties ...... 63
3.2.6.2 Evaluation of prepared herbal tablet formulations ...... 64
3.2.7 Statistical analysis ...... 66
CHAPTER 4: RESULTS ...... 68
4.1 Identification of selected plants for the study ...... 68 University4.1.1 Macro and microscopy of S. polyanthum leaf...... 68 4.1.2 Macro and microscopy of M. charantia fruit ...... 69
4.2 Extraction of plants materials using different extraction methods ...... 72
4.3 GC-MS and LC-MS profiling of the M. charantia and S. polyanthum
extracts ...... 72
4.3.1 GC-MS data analysis ...... 73
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4.3.2 LC-MS data analysis ...... 82
4.4 Antioxidant activities of the extract ...... 88
4.4.1 DPPH radical scavenging activity (RSA) of the extracts ...... 88
4.4.1.1 DPPH-RSA for M. charantia single extract ...... 88
4.4.1.2 DPPH-RSA for S. polyanthum single extract ...... 88
4.4.1.3 DPPH-RSA for M. charantia and S. polyanthum
combination extracts ...... 89
4.4.2 FRAP activity of the extracts ...... 93
4.4.2.1 FRAP of M. charantia single extract ...... 93
4.4.2.2 FRAP of S. polyanthum single extract ...... 93
4.4.2.3 FRAP of M. charantia and S. polyanthum combination extracts ...... Malaya 94 4.5 Antidiabetic enzyme inhibitory activity of the extracts ...... 97 4.5.1 In vitro α-amylase inhibitory activityof of the extracts ...... 97 4.5.1.1 In vitro α-amylase inhibitory activity of M. charantia
single extracts ...... 97
4.5.1.2 In vitro α-amylase inhibitory activity of S. polyanthum
single extracts ...... 97
4.5.1.3 In vitro α-amylase inhibitory activity of M. charantia
and S. polyanthum combination extracts ...... 98 University4.5.2 In vitro α-glucosidase inhibitory activity of the extracts ...... 100 4.5.2.1 In vitro α-glucosidase inhibitory activity of M. charantia
single extracts ...... 100
4.5.2.2 In vitro α-glucosidase inhibitory activity of S. polyanthum
single extracts ...... 100
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4.5.2.3 In vitro α-glucosidase inhibitory activity of M. charantia
and S. polyanthum combination extracts ...... 100
4.6 Evaluation of herbal tablet formulation ...... 103
CHAPTER 5: DISCUSSION ...... 105
5.1 Identification of selected plants for the study ...... 105
5.2 Extraction of plants materials using different extraction method ...... 107
5.3 GC-MS and LC-MS profiling of the M. charantia and
S. polyanthum extracts ...... 108
5.4 Antioxidants activities of the extracts ...... 110
5.5 Antidiabetic enzyme inhibitory activity of the extracts ...... 117
5.6 Evaluation of prepared herbal tablet formulations ...... 122 CHAPTER 6: SUMMARY AND CONCLUSIONMalaya ...... 125 REFERENCES ...... 129 APPENDIX ...... of 151
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LIST OF FIGURES
Figure 2.1: Summary of signalling pathways involved in microvascular
diabetic complications ...... 26
Figure 2.2: Syzygium polyanthum ...... 32
Figure 2.3: Momordica charantia ...... 35
Figure 2.4: Typical GC-MS instrumentation ...... 43
Figure 2.5: Typical LC-MS instrumentation ...... 45
Figure 3.1: Individual percentage of tablet components ...... 62
Figure 3.2: List of instruments and equipments used in the study ...... 67
Figure 4.1: Macroscopy and microscopy of S. polyanthum ...... 71 Figure 4.2: Macroscopy and microscopy of M. charantiaMalaya ...... 71 Figure 4.3: The percent yields of different auqeous extracts of selected M. charantia and S. polyanthumof plants ...... 72 Figure 4.4: GC-MS Chromatogram showing peaks representing volatile
components detected in aqueous extracts of M. charantia ...... 74
Figure 4.5: GC-MS Chromatogram showing peaks representing volatile
components detected in aqueous extracts of S. polyanthum ...... 76
Figure 4.6: LC-MS profiles of different extracts of two selected plants ...... 82
Figure 4.7: DPPH radical scavenging activity of different aqueous extracts University of M. charantia and S. polyanthum ...... 92 Figure 4.8: FRAP radical scavenging activity of different aqueous extracts
of M. charantia and S. polyanthum ...... 96
Figure 4.9: α-amylase inhibitory effects of different aqueous extracts
of M. charantia and S. polyanthum ...... 99
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Figure 4.10: α-glucosidase inhibitory effects of different aqueous extracts
of M. charantia and S. polyanthum ...... 102
Figure 4.11: Prepared herbal tablet formulations of the best extracts of
M. charantia and S. polyanthum ...... 104
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LIST OF TABLES
Table 2.1: List of antidiabetic drugs for type 2 diabetes mellitus ...... 14
Table 2.2: Types of free radical reactions of some free radicals ...... 20
Table 2.3: List of few natural antioxidants reported for their antioxidant potential
in literature ...... 29
Table 2.4: List of few herbal tablet formulation described in the literature ...... 50
Table 3.1: Prepared extract and their combinations tested for antioxidant and
antidiabetic assays ...... 57
Table 3.2: The components of herbal tablet dosage forms ...... 62
Table 3.3: Relationship between angle of repose and powder flow ...... 63 Table 3.4: Scale of flowability of granules ...... Malaya 64 Table 4.1: List of volatile phytoconstituents identified in the aqueous extracts of the leaf of S. polyanthum by ofGC-MS ...... 78 Table 4.2: List of volatile phytoconstituents identified in the aqueous extracts
of the leaf of M. charantia by GC-MS ...... 80
Table 4.3: List identified phytoconstituents in the fruit aqueous extracts
of M. charantia by LC-MS ...... 83
Table 4.4: List identified phytoconstituents in the fruit aqueous extracts
of S. polyanthum by LC-MS ...... 86 TableUniversity 4.5: Evaluation results of herbal tablet formulations ...... 104
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LIST OF ABBREVIATIONS
WHO World Health Organization
IDF International Diabetes Federation
NADPH Nicotinamide Adenine Dinucleotide Phosphate
ROS Reactive Oxygen Species
IDDM Insulin Dependent Diabetes Mellitus
NIDDM Non-Insulin Dependent Diabetes Mellitus
T&CM Traditional and Complementary Medicine
NKEA National Key Economic Area
GC-MS Gas Chromatography Mass Spectrophotometry LC-MS Liquid ChromatographyMalaya Mass Spectrophotometry DPPH 1,1-Diphenyl-2-picryl-hydrazyl FRAP Ferric Reducingof Antioxidant Power cAMP cyclic Adenosine Monophosphate
AGEs Advanced Glycation End Products
DPP-4 Dipeptidylpeptidase-4
ODS Oxygen Derived Species
LDL Low Density Lipoprotein
ETC Electron Transport Chance UniversityDAG Diacylglycerol PKC Protein Kinase C
OP Oxidative Phosphorylation
GFAT Glutamine Fructose 6-phosphate aminotransferase
TGF Transforming Growth Factor
GLUT 4 Glucose Transporter 4
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RNS Reactive Nitrogen Species
O2 Superoxide
OH Hydroxyl
NO Nitric Oxide
ROO Peroxyl
RO Alkoxyl
O Oxygen
H2O2 Hydrogen Peroxide
O3 Ozone
HClO Hypochlorous Acid
ROOH Organic Peroxide HCOR Adehydes Malaya H+ Proton MnSOD Manganese Superoxideof Dismutase GADPH Glyceraldehyde-3-Phosphate Dehydrogenase
UDP-GlcNAc Uridinediphosphate N-acetylglucosamine
VEGF Vascular Endothelial Growth Factor
ICAM-1 Intercellular Adhesion Molecule 1
VCAM-1 Vascular Cell Adhesion Molecule 1
MCP-1 Monocyte Chemoattractant Protein-1 UniversityGC Gas Chromatography MS Mass Spectrometry
LC Liquid Chromatography
UV-Vis Ultraviolet-Visible
EI Electrospray Ionisation
APCI Atmospheric Pressure Chemical Ionisation
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MCP Multi Channel Plate
API Atmospheric Pressure Ion
MDIs Metered Dose Inhalers
DPIs Dry Powder Inhalers
V0 Initial Volume
Vf Final Volume
Fe3+-TPTZ Ferric Tripyridyltriazine
MC Momordica charantia
SP Syzygium polyanthum
MOS Mild Oxidative Stress
TOS Temperature Oxidative Stress SOS Strong Oxidative StressMalaya
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CHAPTER 1: INTRODUCTION
1.1 Introduction
Diabetes mellitus is a chronic metabolic disease or disorder with multiple aetiologies, is characterized by high blood sugar levels accompanied by impaired metabolism of carbohydrates, lipids, and proteins as a result of insufficiency of insulin function. This disease is chronic and can include all ages, and does not distinguish social status. In 2014, the International Diabetes Federation (IDF) estimated that 8.2 % of adults aged 20 - 79 (387 million people) were living with diabetes; this in comparison with 382 million people in 2013, and the number of people with the disease was projected to rise beyond 592 million in 2035 (Da et al., 2016), and more than 60 % of the people with diabetes live in Asia, with almostMalaya one-half in China and India combined. In Malaysia, the reported prevalence of diabetes was 11.6 % in 2006, 15.2 % in the 2011 national study, and 22.9 % in 2013.of The age distribution of the study groups was similar (Nanditha et al., 2016). In the elderly, the disease is usually asymptomatic and can only be known when there is a routine inspection. Common symptoms include thirst, polydipsia, polyphagia, weight loss, polyuria, itching, and weakness. Several pathogenic processes are involved in the development of diabetes. These range from autoimmune destruction of the β-cells of the pancreas with consequent insulin deficiency to abnormalities that result in resistance to insulin action (American Diabetes Association,University 2006). Environmental factors, diet and free radicals play a major role in the development of diabetes.
Free radicals are atoms, molecules, or ions with unpaired electrons with open shell configuration. Free radicals may have positive, negative or zero charge. Many free radicals are unstable and highly reactive in their present state, which can either accept one electron or donate from other molecules. The free radicals are believed to be
1 generated as by-products of metabolism, cellular respiration, synthesized by enzyme systems (nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, myeloperoxidases), by exposure to ionizing radiation, smoking, herbicides, pesticides, pollution and fried foods (Randhir & Sushma, 2014). The most important free radicals in many diseased states are oxygen derived, particularly superoxide and the hydroxyl radical (Young & Woodside, 2001). Free radicals including reactive oxygen species
(ROS) and reactive nitrogen species generated by the human body by various endogenous systems, exposure to different physiochemical conditions, or pathological states, and have been implicated in the pathogenesis of many diseases. Therefore, ROS produced by vascular cells are implicated as possible underlying pathogenic mechanisms in the progression of cardiovascular diseases including ischemic heart disease, atherosclerosis, cardiac arrhythmia, hypertension,Malaya and cancer. These free radicals are also involved in pancreatic damage, lead to diabetic complication, neuropathy, nephropathy, and cardiopathyof (Randhir & Sushma, 2014; Yang & Omaye, 2009). There is substantial evidence that people with diabetes tend to have increased generation of reactive oxygen species, decreased antioxidant protection, and therefore increased oxidative damage.
As a defence against oxidative damage, the body normally maintains a variety of mechanisms to prevent such damage while allowing the use of oxygen for normal functions. Such “antioxidant protection” derives from sources both inside the body (endUniversityogenous) and outside the body (exogenous). Endogenous antioxidants include molecules and enzymes. The activities of key antioxidant enzymes are also found to be abnormal in people with diabetes (Young & Woodside, 2001). These enzyme activities are seen to be lower than normal, suggesting a compromised antioxidant defence, while other studies have shown higher activity, suggesting an increased response to oxidative stress. Oxidative damage is greater in people with type 2 diabetes compared to those
2 with type 1, especially people with type 2 diabetes and the metabolic syndrome, which involves central obesity, hypertension (high blood pressure), and high blood fat levels along with insulin resistance (decreased effectiveness of insulin in metabolizing blood glucose) (Jacob, 2007).
Generally, diabetes mellitus can be handled in several ways, dietary adjustments and regular exercise, the use of oral antidiabetic drugs such as sulfonylureas and biguanides, as well as insulin injections. However, the drugs in the market have considerable side effects and are expensive. Therefore, many patients of developing countries are looking for alternative treatments, such as complementary or alternative medicine. The use of traditional medicine is based on the knowledge gained from inheritance. Effects of herbal medicine are influenced by the form of presentation of herbal drugs that we consume (Kalichevsky, Knorr, &Malaya Lillford, 1995). The quality of herbal raw materials fluctuates greatly due to geographical location, soil environment and mode of collection,of diversity in climatic conditions, their habit and habitats. Standardization of herbal medicinal plants is therefore recommended to overcome disparity in recent times. Authenticity of the sample can be done either by macroscopy or microscopy or by chemical evaluation or by genotypic analysis
(Folashade, Omoregie, & Ochogu, 2012). It is estimated that the side effects associated with herbal medicines are actually more often due to improper identification, failure in distinguishing the beneficial herbs from their toxic counterparts (Sanders, Moran, Shi, Paul,University & Greenlee, 2016; Verma & Singh, 2008). Syzygium polyanthum, commonly referred as Indonesian bay leaf is reported to have antiinflammatory, antipyretic and detoxificant properties against various poisons.
The leaves of the plant are fragrant due to the presence of citral, eugenol in its volatile oil. In addition, several phenolic compounds such as tannins, flavonoids and phenolic acids were reported. Studies on hydroalcoholic extracts of the leaf over animal models
3 have indicated its antidiabetic potential. A preparation called “Jamu” is a traditional herbal medicine that has been practised for many centuries in the Indonesian community to maintain good health and to treat diseases and usually prepared by decoction. There are several plants used in “Jamu” based on the required therapeutic response, the plants that are used in Jamu for antidiabetic use are Syzygium polyanthum leaf and Momordica charantia fruit (Elfahmi, Herman, & Oliver, 2014). M. charantia also known as bitter gourd or bitter melon is the most frequently used plant based antidiabetic medicine. For many indigenous people in the world, this is the only available remedy to control diabetes mellitus. The plant is rich in several phytoconstituents like glycosides, saponins, phenolics, fixed oils, resins and alkaloids. Among them major antidiabetic alkaloids detected were charantins, momordicins, cucurbitacins and few glycosides such as momordicosides, goyaglycosides. Several studiesMalaya have reported antidiabetic, anticancer, antiinflammatory, antiviral and hypocholesterolemic activities for the fruits of the plant. Few mechanisms establishedof so far are, stimulation of glucose uptake by the cells, inhibition of glucose absorption, inhibition of gluconeogenetic enzymes, protection of pancreatic β-cells and others (Kumar, Saravanan, Kumar, & Jayakumar,
2014; Pal & Shukla, 2003; Posadzki, Watson, & Ernst, 2013; Jha & Rathi, 2008). Both
M. charantia and S. polyanthum are rich in polyphenolic flavonoids, tannins and alkaloids as mentioned in the literature, therefore dietary intake of these herbs can play an important role in the body’s antioxidant defence which most likely mediate their beneficialUniversity health effects by scavenging free radicals that cause oxidative stress associated with cancer, diabetes, hypertension, cardiovascular and neurodegenerative diseases, longevity and aging (Hui, Yixi, & Xiaoqing, 2015; Robert, 2014; Hugel,
Jackson, May, Zhang, & Xue, 2016; Erawati, 2012).
In view of the above, a study evaluating the effect of S. polyanthum leaf extract and M. charantia fruit extract produced by different extraction methods such as
4 maceration, sonication, soxhlet, fresh juice and their antioxidant and antidiabetic effects in vitro were examined. In addition, the study focused in developing a herbal supplement that can offer exogenous antioxidants as a nutraceutical. It is known that choosing the right food with controlled sugar levels and the exogenous antioxidant content becomes the perfect combination for diabetes to be in check. Since, good food is an important part of leading a healthy lifestyle.
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1.2 Problem statement & justification of the work
Diabetes mellitus is a major healthcare issue throughout the world. Based on
WHO data, the statistics show the number of cases of diabetes will double by 2030.
When free radicals overwhelm the body’s ability to regulate them, a condition known as oxidative stress will occur. Diabetes is one of the outcomes of oxidative stress. Some of the obstacles in the treatment of diabetes are, limited number of drugs, side effects of existing drugs, development of resistance, and as well as socioeconomical problems and poor accessibility to healthcare systems and facilities.
Therefore, many people seek the alternative treatments. At present nearly 70–80
% of the world's population still dependents on traditional & complementary medicine
(T&CM), however, as recent studies have shown, in addition to many benefits there are few risks associated with T&CM. Therefore, WHOMalaya endorsed T&CM within the health system given their safety and efficacy is established by proper standardization. The quality of herbal raw materials depends onof place of collection of raw material, soil, several external factors like light, temperature, rainfall and altitude.
Standardization of herbal medicinal plants is therefore recommended to overcome disparity in their quality. The standardization of herbal drugs starts from raw material identification, followed by evaluation of organoleptic, microscopical, chemical, physical and pharmacological characters of the samples to get assurance of quality, efficacy, safety and reproducibility. Hence, in the current study proper identification and characterizationUniversity of the extracts was carried out to the herbal formulations prepared. In the global phytomedicine market, Malaysia stands in the 11th position and has great potential to capture the T&CM market. Based on the NKEA (National Key
Economic Area) of Malaysia, agriculture and health care are sectors identified that can boost economy. No work has been reported on M. charantia and S. polyanthum in combination for antioxidant activity, there is also no available data of in vitro studies on
6 enzymatic assays of α-amylase, α-glucosidase using combination of these two plants, M. charantia and S. polyanthum.
Malaya
of
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7
1.3 Aim & Objectives
1.3.1 Aim
To determine the best extraction method for S. polyanthum and M. charantia and to determine the best combination of extracts that gives maximum α–amylase and
α–glucosidase inhibitory activity and antioxidant activity in vitro and preparation of herbal formulation.
1.3.2 Objectives
a. To identify M. charantia and S. polyanthum by microscopical evaluation.
b. To determine the best extraction method that gives maximum yield and profiling
of extract by GC-MS & LC-MS. c. To evaluate single and combined extractsMalaya for in vitro DPPH and FRAP inhibitory activity. d. To evaluate single and combinedof extracts for in vitro α–amylase and α– glucosidase inhibitory activity.
e. To formulate and evaluate the best combination extracts of S. polyanthum and
M. charantia as a tablet dosage form.
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8
CHAPTER 2: LITERATURE REVIEW
Chronic disease is a disease that persists for a long period of time. Chronic diseases generally cannot be prevented by vaccines or cured by medication, nor do they just disappear spontaneously. Chronic diseases are long-lasting conditions that usually can be controlled but not cured. People living with chronic illnesses often must manage daily symptoms that affect their quality of life, and experience acute health problems and complications that can shorten their life expectancy. Chronic diseases, such as heart disease, stroke, cancer, chronic respiratory diseases and diabetes, are by far the leading cause of mortality in the world, representing 60 % of all deaths (WHO, 2017). The literature review chapter mainly consists of a detailed review on chronic disease like diabetes mellitus, classification of diabetes, origin ofMalaya free radicals, type of free radicals, role of natural antioxidants to counter chronic diseases, and development of herbal formulations. of
2.1 Diabetes mellitus
Diabetes mellitus is a metabolic disorder characterized by the presence of hyperglycaemia and accompanied with a range of metabolic disorders as a result of hormonal disorders, due to defective insulin secretion, defective insulin action or both by the pancreatic β-cells giving rise to abnormalities in the metabolism of carbohydrates,University proteins and fats. The chronic hyperglycaemia of diabetes is associated with relatively specific long-term micro vascular complications affecting the eyes, kidneys and nerves, as well as an increased risk for cardiovascular disease (Ronald &
Zubin, 2013). The symptoms of the disease are polydipsia, polyphagia, polyuria, hyperglycaemia, and glycosuria to ketosis, acidosis and coma. Other symptoms that can be felt by patient are spasms on legs, calf muscles due to lack of fluid and electrolytes.
9
Diabetes mellitus is defined as a symptomatic or asymptomatic state of altered carbohydrate metabolism characterized by two or more fasting plasma glucose levels of
126 mg/dL (7.0 mmol/L) or greater or a value of 200 mg/dL (11.1 mmol/L), or greater, at 2 hours on an oral glucose tolerance test. Diagnosis of diabetes can also be made with a random blood glucose value of 200 mg/dL (11.1 mmol/L) or greater (American
Diabetes Association, 2006). After meal, the blood glucose level is high so insulin is produced by β-cells in the pancreas to normalize the glucose level. It increases plasma membrane glucose transporter of glucose from bloodstream into the muscle, liver and adipose tissue. In addition, it converts glucose to glycogen in the muscle and the liver for storage of the nutrients. Finally, the level of glucose in the blood will decrease, insulin secretion will slow down or stop, resulting in the body to come to homeostasis. In patients with diabetes, the absence or insufficientMalaya production of insulin causes high blood glucose levels or hyperglycaemia (Guthrie & Guthrie, 2009). of 2.2 Classification of diabetes mellitus
Diabetes mellitus can be classified into three categories. Type 1 diabetes mellitus or insulin dependent diabetes mellitus (IDDM), type 2 diabetes mellitus or non- insulin dependent diabetes mellitus (NIDDM) and Gestational diabetes mellitus. Type 1 diabetes mellitus (IDDM) is known as juvenile-onset begins at the young ages. The sufferer lack insulin hormone depends on insulin injections to control blood glucose. ThisUniversity form includes cases due to an autoimmune process and those for which the aetiology of β-cell destruction is unknown. Symptoms arising are ketoacidosis and can even cause fainting. This type of diabetics does not react to drug therapy. Type 2 diabetes mellitus (NIDDM) is known as adult-onset symptoms that appear in elderly, but it can also appear at the age of adolescence. Patients with type 2 diabetes mellitus sometimes does not show early symptoms, characterized by frequent thirst, hunger, and
10 increased water consumption, excessive urine volume and frequency. Patients with type
2 diabetes mellitus do not depend on the hormone insulin and can be treated with oral medications. Type 2 diabetes may range from predominant insulin resistance with relative insulin deficiency to a predominant secretory defect with insulin resistance.
People with diabetes mellitus often have altered glucose metabolism, a process with impaired glucose uptake in which glucose cannot get into the cells and energy is only retrieved from the metabolism of proteins and fats. Gestational diabetes mellitus refers to glucose intolerance with onset or first recognition during pregnancy. Because high blood sugar levels in a mother are circulated through the placenta to the baby, gestational diabetes must be controlled to protect the baby's growth and development (Olagbuji et al.,
2015). Diabetes mellitus can occur with no symptoms in children, and teenagers. While in older people who have symptoms of these diseasesMalaya very often endup with diabetes without being noticed by the patient if not inspected on a regular basis. of 2.3 Mechanism of action of insulin
Insulin affects glucose, lipid, and protein metabolisms in all tissues. In fat cells, insulin promotes the uptake and enhances triglyceride stores. In muscle cells, glucose enters via the cell membrane made permeable by insulin, and is converted to glycogen stores or used for energy. In liver cells, glucose is stored as glycogen. The intracellular effects of hormones are accomplished by second messengers, which are activated by receptorsUniversity on cell membranes that determine whether or not the cell responds to the hormones. Specific enzymes then allow the cell to perform its functions in response to hormones and second messengers. The second messenger for most hormones is cyclic adenosine monophosphate (cAMP), which is activated by the enzyme adenylcyclase in the cell membrane. Insulin suppresses adenylcyclase and cAMP, activates other second messengers. These messenger enzymes are activated by closure of the potassium
11 channels and opening of the calcium channels so that calcium can flow into the cell and activate formation of cAMP. One enzymatic mechanism that insulin-responsive cells have is the phosphorylasekinase system. Insulin stimulates the cells by interaction with a specific receptor on the cell surface, and this stimulates a series of enzymatic phosphorylation reactions within the cell. Finally, this phosphorylase cascade activates the PPAR-γ enzyme in the cell nucleus. This enzyme activates the gene for the formation of the RNA, which synthesizes a protein called a glucose transporter to facilitate the uptake of glucose by the cell. Glucose transporter proteins modify the cell membrane to absorb the glucose into the interior of the cell for utilization. These transporters are manufactured inside the cell and carried to the cell membrane under the control of insulin and the subsequent enzyme reactions within the cell. Insulin also controls the reabsorption and degradation of the Malaya transporters that are numbered to differentiate the proteins of different cells-GLUT 4 (Glucose Transporter 4 is found within muscle cells). A hexokinase enzymeof inside the cell is also stimulated to facilitate the glycolytic process for the metabolism of glucose to CO2, water, and energy (the kreb’s cycle). This hexokinase enzyme is the only enzyme of the glycolytic pathway activated by insulin. It catalyses the initial step of this process, the phosphorylation of glucose to form glucose 6-phosphate. This catalysis is a vital step in the metabolism of glucose for energy, and insulin deficiency will result in blockage of the entire glycolytic pathway for energy production. The enzymatic system for lipogenesis is specific to the fat Universitycell, whereas the enzymatic system for conversion of glucose to energy occurs in all cells. By a separate set of enzymes, the fat can, of course, also convert glucose to energy for its own metabolic processes, because the conversion of glucose to fat is an energy- requiring process. All of these specifics are probably mediated by a second messenger through an activation of the sodium potassium pump and by the calcium flux (Guthrie
& Guthrie, 2009).
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2.4 Current available treatments for diabetes mellitus
Type 2 diabetes mellitus is a progressive and complex disorder that is difficult to treat effectively in the long term. The majority of patients are overweight or obese at diagnosis and will be unable to achieve or sustain near normoglycaemia without oral and parenteral antidiabetic agents. Today’s clinicians are presented with an extensive range of oral antidiabetic drugs for type 2 diabetes. Administration of antidiabetic drug generally begins with monotherapy and slowly progresses into multidrug therapy if the initial treatments were not effective. Various drugs are available which works in different ways to control hyperglycaemic condition in diabetic patients. The main classes are heterogeneous in their modes of action, safety profiles and tolerability. These main classes include agents that stimulate insulin secretion, reduce hepatic glucose production, delay digestion and absorption of intestinalMalaya carbohydrate or improve insulin action. Currently, there are six types of commercially available oral antidiabetic drugs for type 2 diabetes mellitus including of Biguanides (e.g.Metformin), α-glucosidase inhibitors (e.g. Acarbose), Sulfonylureas (e.g. Chlorpropamide), Calcium channel blockers (e.g. Repaglinide), Thiazolidinediones (e.g. Rosiglitazone), and
Dipeptidylpeptidase-4 (DPP-4) inhibitors (e.g. Vildagliptin). Each type has a different mechanism of action in controlling blood glucose level of type 2 diabetic patients and side effects as shown in Table 2.1 (Carl, 2007; Andrew & Clifford, 2005). The drugs offer many advantages as well as common side effects such as hypoglycaemia, gastrointestinalUniversity problems and weight gain. Insulin is used in injectable form as a sole therapy (particularly for type 1 diabetes mellitus patients) or in combination with other oral drugs. Insulin therapy in a long acting form possesses a lower risk of hypoglycaemia. However, the therapy generally does not mimic physiological functions of insulin completely in the human body (Katzung & Trevor, 1982).
13
Table 2.1: List of antidiabetic drugs for type 2 diabetes mellitus
Drug Class Mechanism of action Side Effects Drug Names Marketed Names
Reduce glucose production Abdominal pain, nausea Glucophage® (Merck) Biguanides Metformin from the liver and diarrhoea Glufor®(Pyridam) Amaryl® (Aventis) Chlorpropamide ® Stimulate the beta cells of Gluconic (Nicholas) Hypoglycaemia, weight Glibenclamide ® Sulfonylureas pancreas to release more Diamicron MR (Darya gain Gliclazide insulin Varia) Glipizide Minidiab®(Kalbe)
® Weight gain, swelling Avandaryl Increase glucose uptake by Rosiglitazone Thiazolidinediones (edema), increased riskMalaya of (GlaxoSmithKline) the skeleton muscle cells Pioglitazone ® congestive heart failure Actos (Takeda)
Inhibit carbohydrate Acarbose ® α-glucosidase Abdominal pain, Glucobay (Bayer) absorption by intestinal of Voglibose inhibitors diarrhoea, flatulence mucosa Miglitol Stimulate the beta cells of Novonorm®(Dexa Calcium channel Hypoglycaemia, weight Repaglinide pancreas to release more Medica) blockers gain Nateglinide insulin Starlix®(Novartis) Inhibit DPP-4 enzyme not to Galvus®(Novartis) Dipeptidyl breakdown GLP-1 (increase Rash (Stevens-Johnson Vildagliptin Januvia®(MSD) peptidase-4 (DPP-4) GLP-1 release) resulting to syndrome), acute Sitagliptin Tradjenta®(Boehringer) inhibitors increase insulin secretion and pancreatitis Linagliptin decrease glucagon secretion Lantus®(Aventis) Inconsistent absorption Insulin glargine ® Insulin analogues Mimics insulin action Novomix 30 (Novo Hypoglycaemia Insulin aspart Nordisk)
University (Source: Katzung & Trevor, 1982; Carl, 2007, www.mims.com)
14 1
2.5 Antidiabetic key enzymes
Inhibition of enzymes involved in the metabolism of carbohydrates such as α- amylase and α-glucosidase are an important therapeutic approach for reducing postprandial hyperglycaemia (Shobana, Sreerama, & Malleshi, 2009). In fact, several synthetic drugs such as acarbose widely used as inhibitors of these enzymes in patients with type 2 diabetes mellitus and obesity (Yee & Fong, 1996; Padwal & Majumdar,
2007). A largest number of medicinal plants are used in managing diabetes mellitus and its related complications, due to their phytochemical active contents such as phenolics and flavonoids with strong antioxidant properties. These compounds have been reported to be effective inhibitors of α-amylase, α-glucosidase enzymes and lipase (Henda, Kais, Khaled, Mohamed, Abdelfattah & Noureddin, 2014;Malaya Kook, 2007).
2.5.1 α-amylase of α-amylase is the enzyme secreted by salivary glands and pancreas in humans, which can hydrolyse starch at α-1,4 glycosidic bond into oligosaccharides and maltose.
In humans the digestion of starch involves several stages. Initially, partial digestion by the salivary amylase results in the degradation of polymeric substrates into shorter oligomers. Later in the gut these are further hydrolyzed by pancreatic α-amylases into maltose, maltotriose and small malto-oligosaccharides. The digestive enzyme (α- amylase)University is responsible for hydrolysing dietary starch (glucose), which breaks down into glucose prior to absorption. Inhibition of α-amylase can lead to reduction in post prandial hyperglycaemia in diabetic condition (Kook, 2007). α-amylase inhibitors can be proteins and non-proteins. α-amylase inhibitors are found in plants and animal source are mainly present in cereals such as wheat (Triticuma estivum) (Feng, Richardson,
Chen, Kramer, Morgan & Reeck, 1996; Singh & Blundel, 2001), barley (Hordeum
15 vulgareum) (Weselake, Macgregor, Hill & Duckworth, 1983; Pekkarinen & Jones,
2003), sorghum (Sorghum bicolor) (Bloch & Richardson, 1991), rye (Secale cereal)
(Garcia, Sanchez, Lopez, & Salcedo, 1994; Lulek et al., 2000) and rice (Oryza sativa)
(Yamagata, Kunimatsu, Kamasaka, Kuramoto, & Iwasaki, 1998) but also in
Leguminosae such as pigeon pea (Cajanus cajan) (Giri & Kachole, 1998), cowpea (Vigna unguiculata) (Melo, Sales, Pereira, Bloch, Franco, & Ary, 1999) and bean (Phaseolus vulgaris) (Grossi, Mirkov, Ishimoto, Colucci, Bateman, Chrispeel,
1997; Young, Thibault, Watson, & Chrispeels, 1999; Marshal & Lauda, 1997). α- amylase activity can be measured in vitro by hydrolysis of starch in presence of α- amylase enzyme. This process can be quantified by its ability to reduce 3,5- dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid, which gives red-orange colour with starch. The reduced intensity of red-orange colMalayaour indicates the enzyme induced hydrolysis of starch into monosaccharides. If the substance or extract possesses α- amylase inhibitory activity, the intensity of red- orange colour will be more. In other words, the intensity of red-orange colour in test sample is directly proportional to α- amylase inhibitory activity (Bernfeld, 1955).
2.5.2 α-glucosidase
α-glucosidase is the enzyme that hydrolyses α-1,4 glycosidic bond in carbohydrate digestion (disaccharides such as maltose, sucrose and lactose into monosaccharidesUniversity or glucose), is often locat ed in the brush-border surface membrane of intestinal cells in human, catalysing the cleavage of disaccharides to form glucose (Kim,
Nam, Kurihara, Kim, 2008). The development of the α-glucosidase inhibitor acarbose provided a new approach in the management of diabetes. By competitive and reversible inhibition of intestinal α-glucosidases, acarbose delays carbohydrate digestion, prolongs the overall carbohydrate digestion time, and thus reduces the rate of glucose absorption.
16
After oral administration of acarbose, the postprandial rise in blood glucose is dose- dependently decreased, and glucose-induced insulin secretion is attenuated. Because of diminished postprandial hyperglycaemia and hyper-insulinemia by acarbose, the triglyceride uptake into adipose tissue, hepatic lipogenesis, and triglyceride content are reduced. Therefore, acarbose treatment not only flattens postprandial glycaemia, due to the primary and secondary pharmacodynamic effects, but also ameliorates the metabolic state in general. In diabetic animals, acarbose reduced urinary glucose loss, the blood glucose area under the curve, and prevented the decrease in skeletal muscle GLUT4 glucose transporters. As a consequence of the reduced mean blood glucose area under the curve, the formation of advanced glycation end-products (AGEs) was decreased.
The prevention of basement membrane glycation and thickening in various tissues indicated that acarbose treatment of diabetic animalsMalaya produced beneficial effects against the development of nephropathy, neuropathy, and retinopathy. Thus, the α-glucosidase inhibitor acarbose may have the potential ofto delay or possibly prevent the development of diabetic complications (Kook, 2007). This process can be quantified under specific conditions (pH = 6.9; T = 37 °C), α -glucosidase will catalyze the conversion of the substrate 4-nitrophenyl-α-D-glucopyranoside to α-D-glucopyranoside and p- nitrophenol. The yellow colour of the later product is measured by spectrophotometer
(Wehmeier & Piepersberg, 2004). Hence, natural products with acarbose like activity can be a potential source of remedy to alleviate postprandial hyperglycaemia. University
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2.6 Free radicals and their association to diseases
2.6.1 Free radicals
A free radical can be defined as any molecular species capable of independent existence that contains an unpaired electron in an atomic orbital. The presence of unpaired electrons in free radicals results in certain common properties. Free radicals are weakly attracted to a magnetic field and are said to be paramagnetic. Many free radicals are highly reactive and can either donate an electron to or extract an electron from other molecules, therefore behaving as oxidants or reductants (Young &
Woodside, 2001). Free radicals are highly reactive species, capable in the nucleus, and in the membranes of cells of damaging biologically relevant molecules such as DNA, proteins, carbohydrates, and lipids. Free radicals attack important macromolecules leading to cell damage and homeostatic disruption.Malaya Targets of free radicals include all kinds of molecules in the body. Among them, lipids, nucleic acids, and proteins are the major targets. Free radicals and other reactiveof oxygen species are derived either from normal essential metabolic processes in the human body or from external sources such as exposure to ultraviolet, (UV-A, UV-B, UV-C), γ-radiation, X-rays, ozone, cigarette smoking, air pollutants, and industrial chemicals (Bagchi & Puri, 1998). Free radical formation occurs continuously in the cells as a consequence of both enzymatic and non- enzymatic reactions. Enzymatic reactions, which serve as source of free radicals, include those involved in the respiratory chain, in phagocytosis, in prostaglandin synthesis,University and in the cytochrome P-450 system (Liu, Stern, Robert, Morrow, 1999).
Free radicals can also be formed in non-enzymatic reactions of oxygen with organic compounds as well as those initiated by ionizing reactions. Some internally generated sources of free radicals are mitochondria, xanthine oxidase, inflammation, peroxisomes, phagocytosis, and arachidonate pathways. The oxidative damage occurs when the critical balance between free radical generation and antioxidant defences is
18 unfavourable. Oxidative stress, arising as a result of an imbalance between free radical production and antioxidant defences, is associated with damage to a wide range of molecular species including lipids, proteins, and nucleic acids. Short-term oxidative stress may occur in tissues injured by trauma, infection, heat injury, hypertoxia, toxins, and excessive exercise. These injured tissues produce increased radical generating enzymes (e.g. xanthine oxidase, lipogenase, cyclooxygenase) activation of phagocytes, release of free iron, copper ions, or a disruption of the electron transport chains of oxidative phosphorylation, producing excess ROS (Lobo, Patil, Phatak, & Chandra,
2010).
2.6.2 Types of free radicals The most important free radicals in many diseasedMalaya states are oxygen derivatives, particularly superoxide and the hydroxyl radicals (Cheesman & Slater, 1993). Free radical formation in the body occurs of by several mechanisms, involving both endogenous and environmental factors (Young & Woodside, 2001). Oxygen-derived prooxidants or generally known as reactive oxygen species (ROS) (other terms include oxygen-derived species, ODS) and reactive nitrogen species (RNS) can be grouped as radicals and non-radicals. Radicals include superoxide (O2), hydroxyl (OH), peroxyl
(ROO), alkoxyl (RO) and one form of singlet oxygen (O) and non-radicals include hydrogen peroxide (H2O2), hypochlorous acid (HClO), organic peroxide (ROOH), singletUniversity oxygen and peroxynitrite (ONOOH) as shown in Table 2.2 (Kohen, Moor, & Oron, 2003). Prooxidants such as HClO, H2O2, HCOR, ROOH and O3 are commonly found in living system (Klaunig, Kamendulis, & Hocevar, 2010; Kohen & Nyska, 2002;
Lodovici & Bigagli, 2011; Poljsak & Fink, 2014; Rahal et al., 2014).
19
Table 2.2: Types of free radical reactions of some free radicals
Free radicals Description
One-electron reduction state O2, formed in many autoxidation reactions and by the electron transport chain. 2+ O2ˉ, superoxide anion Rather unreactive but can release Fe from iron sulphur proteins and ferritin. Undergoes dismutation to form H2O2 spontaneously or by enzymatic catalysis and is a precursor for metal-catalyzed OH formation.
H2O2, hydrogen Two-electron reduction states, formed by dismutation of O2ˉ or by direct reduction of O2. Lipid soluble and thus peroxide able to diffuse across membranes. Three-electron reduction state, formed by Fenton Malaya reaction and decomposition of peroxynitrite. Extremely OH, hydroxyl radical reactive, will attack across membranes. ROOH, organic Formed by radical reactions with cellular components such as lipids and nucleobases. hydroperoxide of RO, alkoxy and ROO, Oxygen centered organic radicals. Lipid forms participate in lipid peroxidation reactions. Produced in the peroxy radicals presence of oxygen by radical addition to double bonds or hydrogen abstraction.
HOCl, hypochlorous Fomed from H2O2 by myeloperoxidase. Lipid soluble and highly reactive. Will readily oxidize protein acid constituents, including thiol groups, amino groups, and methionine.
Formed in a rapid reaction between O2ˉ and NO. Lipid soluble and similar in reactivity to hypochlorous acid. ONOOˉ, peroxynitrite Protonation forms peroxynitrous acid, which can undergo homolytic cleavage to form hydroxyl radical and nitrogen dioxide. (Source: Sies, 1985; Docampo, 1995; Rice & Gopinathan, 1995) University
20 2.6.3 Oxidative stress and its role in diseases
Role of oxidative stress has been postulated in many conditions, including atherosclerosis, inflammatory condition, (Rosenfeld, 1998) certain cancers (Hecht,
1999), and in the process of aging (Ashok & Ali, 1999). Oxidative stress is now thought to make a significant contribution to all inflammatory diseases (e.g. arthritis, vasculitis, glomerulonephritis, lupus erythematous, adult respiratory disease syndrome), ischemic diseases (e.g. heart diseases, stroke, intestinal ischemia), hemochromatosis, acquired immunodeficiency syndrome, emphysema, organ transplantation, gastric ulcers, hypertension and preeclampsia, neurological disorder (e.g. alzheimer's disease,
Parkinson's disease, muscular dystrophy), alcoholism, smoking-related diseases, and cancer as well as the side-effects of radiation and chemotherapy, have been linked to the imbalance between ROS and the antioxidant defenceMalaya system. ROS have been implicated in the induction and complications of diabetesof me llitus, age-related eye disease, and neurodegenerative diseases. An excess of oxidative stress can lead to the oxidation of lipids and proteins, which is associated with changes in their structure and functions
(Young & Woodside, 2001). Heart diseases continue to be the biggest killer, responsible for about half of all the deaths. The oxidative events may affect cardiovascular diseases.
Poly unsaturated fatty acids occur as a major part of the low density lipoproteins (LDL) in blood and oxidation of these lipid components in LDL play a vital role in atherosclerosisUniversity (Esterbauer, Puhl, Dieber , Waeg, & Rabl, 1991). The three most important cell types in the vessel wall are endothelial cells, smooth muscle cell and macrophages, can release free radical, which affect lipid peroxidation. With continued high level of oxidized lipids, blood vessel damage to the reaction process continues and can lead to generation of foam cells and plaque, the symptoms of atherosclerosis.
Oxidized LDL is atherogenic and thought to be important in the formation of
21 atherosclerosis plaques. Furthermore, oxidized LDL is cytotoxic and can directly damage endothelial cells (Neuzil, Thomas, & Stocker, 1997).
2.6.3.1 Oxidative stress and diabetes mellitus
There is substantial evidence that people with diabetes tend to have increased generation of reactive oxygen species, decreased antioxidant protection, and therefore increased oxidative damage. Hyperglycaemia or a high blood glucose level has been shown to increase reactive oxygen species and end products of oxidative damage in isolated cell cultures, in animals with diabetes, and in humans with diabetes.
Measurement of the end products of oxidative damage to body fat, proteins, and DNA are commonly used to assess the degree of oxidative damage to body cells and tissues. The oxidative damage is greater in type 2 diabetic patientsMalaya compared to those with type 1, especially people with type 2 diabetes and the metabolic syndrome, which involves central obesity, hypertension (high blood pressure),of and high blood fat levels along with insulin resistance (decreased effectiveness of insulin in metabolizing blood glucose).
The antioxidant protection is decreased and oxidative stress increased in some people even before the onset of diabetes. For instance, increased levels of oxidative stress have been found in people who have impaired glucose tolerance, or prediabetes (Jacob,
2007).
Diabetic complications can be divided into two major groups, macrovascular (injuryUniversity of the arteries) which lead to complications such as cardiovascular disease and stroke and microvascular injury of blood capillaries which lead to complications such as retinopathy (eye disease), nephropathy (kidney disease) and neuropathy (neural disease). Other diabetic complications are peripheral neuropathy, amputation, dementia, sexual dysfunction and depression (Forbes & Cooper, 2013). Oxidative stress has been highlighted as the major contributor towards both macrovascular and microvascular
22 complications of diabetes (Giacco & Brownlee, 2010; Matough, Budin, Hamid,
Alwahaibi, & Mohamed, 2012; Tiwari, Pandey, Abidi, & Rizvi, 2013). Prolonged exposure to high glucose level in hyperglycaemic condition has been believed to induce over production of ROS from mitochondria of cells. Influx of glucose initiates glycolysis (oxidation of glucose), followed by downstream reactions in the Krebs cycle and eventually the electron transport chain (ETC) to produce energy in aerobic respiration (oxygen serves as the final electron acceptor). Proton (H+) gradient develops between inner mitochondrial membranes and intermembrane space when electron donors enter and cross the ETC. Heavy flux in proton gradient due to increased entry of electron donors into ETC causes overproduction of superoxides from oxidation of oxygen. Superoxides are degraded by manganese superoxide dismutase (MnSOD) into H2O2 which is removed by catalase or peroxidase. However,Malaya failure in this action results in production of more free radicals such as the highly reactive OH (Nishikawa et al., 2000; Wallace, 1992). Generation of ROSof causes oxidative damage as well as tissue injury, particularly in microvascular complications through four major mechanisms, namely stimulation of protein kinase C pathway, polyol pathway, hexosamine pathway and increased formation of intracellular AGE and AGE receptor (RAGE). In macrovascular complications, effect of hyperglycaemia is through pathway-specific insulin resistance that causes increased fatty acid oxidation (Brownlee, 2005; Du,
Edelstein, Dimmeler, Ju, Sui, & Brownlee, 2001; Giacco & Brownlee, 2010; Singh, Bali,University Singh, & Jaggi 2014; Tarr, Kaul, Chopra, Kohner, & Cibber, 2013; Tiwari, Pandey, Abidi, & Rizvi, 2013).
The activity of protein kinase C (PKC) isoforms is triggered by diacylglycerol
(DAG). Activation of the pathway has been reported in diabetic atherosclerosis, cardiomyopathy, smooth muscle cells and glomerulus of diabetic rats (Ganz & Seftel,
2000; Kikkawa, Haneda, Uzu, Koya, Sugimoto, & Shigeta, 1994). PKC also mediates
23 changes in blood flow, vascular permeability, release of angiogenic factors and neovascularisation in diabetic retinopathy (Frank, 2004). On the other hand, polyol pathway involves conversion of glucose into sorbitol (sugar alcohol or polyol) by the aldose reductase enzyme. The enzyme is commonly found in lens, retina, glomerulus and nerve. The conversion of glucose to sorbitol requires nicotinic acid adenine dinucleotide phosphate (NADPH) as a cofactor. NADPH is also needed as the cofactor for the generation of GSH, a potent scavenger of ROS. Hence, the consumption of
NADPH reduces production of GSH and enhances oxidative stress in the cells.
Increased expression of aldose reductase gene and reduced level of GSH in lens of transgenic mice have been reported previously (Snow et al., 2015; Vikramadithyan et al., 2005). Hexosamine pathway, increased hyperglycaemia-induced O-GlcNAcylation activity has been observed in smooth muscle and cardiomyoMalayacyte calcium cycling, which inhibits the influx of calcium (Akimoto, Kreppel, Hirano, & Hart, 2001; Clark et al., 2003; Weigert, Friess, Brodbeck, Haring, &of Schleicher, 2003). AGE is a product of non-enzymatic reaction between reducing sugar (glucose or related compounds such as methylglyoxal, glyoxal and 3-deoxyglucosone derived from glucose or fatty acid oxidation) and macromolecules particularly proteins (candido et al., 2003; Wautier & Schmidt, 2004). Endogenous factors such as increased oxidative stress, insulin resistance and improper utilization of glucose and exogenous sources like cigarette smoke, thermolyzed food and beverages accelerated formation of AGE rapidly (Kandarakis,University Piperi, Topouzis, & Papavassiliou, 2014). The compounds are commonly found in the extracellular matrix in cells affected by high glucose (diabetes) and could modify functions of intracellular proteins, plasma proteins and other matrix components or receptors. High level of AGE in serum of type 2 diabetes mellitus patients has been reported previously (Kilhovd, Berg, Birkeland, Thorsby, & Hanssen, 1999). AGE-
RAGE (AGE receptor) complex significantly induces production of ROS, NFkB (an
24 inflammatory marker) and alters gene expression (Goldin, Beckman, Schmidt, &
Creager, 2006). The combination of RAGE-NFkB has shown a damaging effect in diabetic neuropathy (Bierhaus et al., 2004). Activation of NFkB also triggers transcription of vascular endothelial growth factor (VEGF), intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), TNF-α, monocyte chemoattractant protein-1 (MCP-1) and interleukins that lead to the development of atherosclerosis (Goldin, Beckman, Schmidt, & Creager, 2006; Schiekofer et al., 2003).
Methylglyoxal in hyperglycaemic condition has been shown to enhance expression of angiopoietin-2 which promotes the release of various proinflammatory markers such as
TNF-α, ICAM-1 and VCAM-1 in renal cells (Yao et al., 2007). Inhibition of RAGE was shown to reduce progression of atherosclerosis due to reduced expression of VCAM-1, NFkB, MCP1 and oxidative stress (Soro Malayaet al., 2008). Other theories related to the contribution of oxidative stress towards hyperglycaemia and related complications include auto-oxidation of of glucose, which results in the formation of radicals (Wolff, 1993), glycation of antioxidant enzyme which prevents its ability to remove radicals (Giugliano, Ceriollo, & Paolisso, 1995) and ketosis particularly in type
1 diabetes mellitus patients that contributes towards the formation of ROS as shown in
Figure 2.1 (Jain, Kannan, & Lim, 1998).
University
25
Hyperglycaemia : High Glucose Levels
Reactive oxygen intermediate pathway Protein Kinase C Polyol Pathway Hexosamine pathway Advanced glycation (PKC) pathway endproduct pathway Aldose NADPH à NADP reductase Fructose-6- Nonenzymatic Glycolysis phosphate glycation Oxidative Sorbitol Glycoxidation phosporylation Proteins Glyceraldehyde- Redox GFAT 3-phosphate imbalance Osmotic stress AGE Reactive OP Glucosamine-6- (extracellular/ oxidative stress phosphate intracellular) (ROS) ROS/AGE Reactive species AGE/RAGE DAG UDP-GlcNAc binding Oxidative stress Oxidative stressMalaya AGE Activation of TGF-β1 PKC of ROS
Activation of cell signaling molecules à change in genes expression and proteins function
Oxidative damage, tissue injury and dysfunction
Microvascular diabetic complication : retinopathy, neuropathy, nephropathy
Figure 2.1: Summary of signalling pathways involved in microvascular diabetic complications (Source: Sheetz & King, 2002) (DAG: Diacylglycerol; AGE: Advanced glycation endproduct; OP: Oxidative phosphorylation; GFAT: Glutamine fructose 6-phosphate aminotransferase; TGF: TransformingUniversity growth factor)
26 2.6.4 Role of antioxidants against free radicals
An antioxidant is a molecule stable enough to donate an electron to a rampaging free radical and neutralize it, thus reducing its capacity to damage. These antioxidants delay or inhibit cellular damage mainly through their free radical scavenging property
(Halliwell, 1997). Antioxidants act as radical scavengers, hydrogen donors, electron donors, peroxide decomposers, singlet oxygen quenchers, enzyme inhibitors, synergists, and metal-chelating agents. Both enzymatic and non-enzymatic antioxidants exist in the intracellular and extracellular environment to detoxify ROS (Young & Woodside, 2001;
Frie, Stocker, & Ames, 1988). Two principle mechanisms of action have been proposed for antioxidants (Rice & Diplock, 1993). The first is a chain-breaking mechanism by which the primary antioxidant donates an electron to the free radical present in the systems. The second mechanism involves removal ofMalaya ROS or reactive nitrogen species initiators (secondary antioxidants) by quenching chain-initiating catalyst. Antioxidants may exert their effect on biological systemsof by different mechanisms including electron donation, metal ion chelation, co-antioxidants, or by gene expression regulation
(Krinsky, 1992). Research suggests that free radicals have a significant influence on aging as well, that free radical damage can be controlled with adequate antioxidant defence, and that optimal intake of antioxidant nutrient may contribute to enhanced quality of life. Recent research indicates that antioxidants may even positively influence life span (Lobo, Patil, Phatak, & Chandra, 2010). University 2.6.4.1 Natural antioxidants
Natural food antioxidants are used routinely in foods and medicine especially those containing oils and fats to protect the food against oxidation. The use of natural antioxidants in food, cosmetic, and therapeutic industry would be promising alternative for synthetic antioxidants in respect of low cost, highly compatible with dietary intake
27 and no harmful effects inside the human body. Many antioxidant compounds, naturally occurring in plant sources have been identified as free radical or active oxygen scavengers (Brown & Rice, 1998). Natural antioxidants can decrease oxidative stress induced carcinogenesis by direct scavenging of ROS and/or by inhibiting cell proliferation secondary to the protein phosphorylation. β-carotene may be protective against cancer through its antioxidant function, because oxidative products can cause genetic damage. Thus, the photo protective properties of β-carotene may protect against ultraviolet light induced carcinogenesis (Levine, Ramsey, & Daruwara, 1991). Vitamin
C may be helpful in preventing cancer. The possible mechanisms by which vitamin C may affect carcinogenesis include antioxidant effects, blocking of formation of nitrosanines, enhancement of the immune response, and acceleration of detoxification of liver enzymes. Vitamin E, an important antioxidant, Malayaplays a role in immune competence by increasing humoral antibody protection, resistance to bacterial infections, cell- mediated immunity, the T-lymphocytes tumourof necrosis factor production, inhibition of mutagen formation, repair of membranes in DNA, and blocking micro cell line formation. Hence vitamin E may be useful in cancer prevention and inhibit carcinogenesis by the stimulation of the immune system. Antioxidants like β-carotene or vitamin E also play a vital role in the prevention of various cardiovascular diseases
(Jacob, 1996; Knight, 1998). Attempts have been made to study the antioxidant potential of a wide variety of vegetables like potato, spinach, tomatoes, and legumes (FurutaUniversity, Nishiba, & Suda, 1997). There are several reports showing antioxidant potential of fruits (Wang, Cao, & Prior, 1996). Strong antioxidant activities have been found in berries, cherries, citrus, prunes, and olives. Green and black teas have been extensively studied in the past for antioxidant properties since they contain up to 30 % of the dry weight as phenolic compounds (Lin, Lin, Ling, Lin, & Juan, 1998). Table 2.3 shows the summary of natural antioxidants reported in the literature.
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Table 2.3: List of few natural antioxidants reported for their antioxidant potential in literature
Class of No. Natural antioxidant Part of plant used Mechanism of its action References phytoconstituents 1 Vitamin C - Hydroxyl radical scavenging activity Vitamin Wang et al., 2016 Hydroxyl and superoxide radical 2 Vitamin E - Vitamin Chen et al., 2016 scavenging activity Hydroxyl, superoxide and nitric oxide 3 Dregea volubilis ext. Flowers Flavonoids Das et al., 2017 radical scavenging activity 4 Leucaena leucochepala ext. Seeds DPPH radical scavenging activity Phenolic compounds Chowtivannakul et al., 2016 Schinsandra sphenanthera Hydroxyl and superoxide radical 5 Fruits Lignan and Triterpenoids Niu et al., 2017 ext. scavenging activity Hydroxyl and superoxide radicalMalaya 6 Arcangelisia flava ext. Leaves Flavonoids Wahyudi et al., 2016 scavenging activity Hydroxyl, superoxide and nitric oxide 7 Swertia corymbosa ext. Aerial parts Phenolic compounds Mahendran et al., 2015 radical scavengingof activity Hydrogen peroxide radical 8 Acacia nilotica ext. Bark Phenolic compounds Barapatre et al., 2015 scavenging activity 9 Eugeissona insignis ext. Vegetable palm heart Peroxyl radical scavenging activity Phenolic compounds Zabidah et al., 2014
10 Vigna radiata L ext. Beans Hydroxyl radical scavenging activity Phenolic compounds Yang et al., 2013
11 Syzygium malaccense ext. Fruits and leaves Peroxyl radical scavenging activity Flavonoids Batista et al., 2017 12 Capsicum ext. Fruits DPPH radical scavenging activity Phenolic compounds Sricharoen et al., 2016 13 Lonicera japonica ext. Fresh buds and flowers Hydroxyl radical scavenging activity Phenolic compounds Kong et al., 2017 Superoxide radical scavenging 14 Brassica oleracea ext. Flowers Phenolic compounds Wei et al., 2010 activity 15 Psidium Guajava ext. Fruits Peroxyl radical scavenging activity Phenolic compound Thaipong et al., 2006 University
29 2.7 Rationale behind selection of S. polyanthum and M. charantia in the current study
Based on Indonesian folklore data “Jamu” is traditional herbal medicine that has been practised for many centuries in the Indonesian community to maintain good health and to treat diseases and usually prepared by decoction. Although modern
(conventional) medicine is becoming increasingly important in Indonesia, jamu is still very popular in rural as well as in urban areas. Based on its traditional use jamu is being developed into a rational form of therapy, by herbal practitioners and in the form of phytopharmaceuticals. Jamu has acquired a potential benefit, both economically and clinically. We believe in Indonesia most research activity into natural products is limited to the inventory of folkloric information and utilization of various plants and trees, scientific proof for their biological activity isMalaya still challenging. M. charantia and S. polyanthum are two popular medicinal plants used in Indonesia. The leaves of S. polyanthum, is used as a culinary additiveof and also used for diabetes, diarrhoea, and skin infections (Kusuma et al., 2011; Dalimartha, 2007; Lelono et al., 2009). In other findings, S. polyanthum leaf extracts were proven to possess antibacterial activity against Staphylococcus aureus (Grosvenor, Supriono, & Gray, 1995), antitumor promoting activity (Ali, Mooi, & Yih, 2000), and antioxidant activity (Wong, Leong, &
Koh, 2006; Perumal, Mahmud, Piaru, Cai, & Ramanathan, 2012). The fruits of M. charantia are used as an antidiabetic remedy (Elfahmi, Herman, & Oliver, 2014), M. charantiaUniversity juice prepared by crushing and straining the unripe fruit added with water is taken once or twice a day (Bailey, Day, & Leatherdalc, 1986). Despite the extensive use by Indonesian people, there have been only limited attempts to explore the biological properties of these plants in relation to their medicinal uses. Therefore, evaluation of their scientific authenticity and proof of concept was felt appropriate on the above plants.
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2.7.1 Syzygium polyanthum
2.7.1.1 Habit, habitat and macroscopy of S. polyanthum
S. polyanthum grows wild in the forests and mountains or planted in the garden and around the house. This tree can be found in low lands up to an altitude of 1400 m above sea level. It is a medium-sized tree up to 30 m tall with dense crown, bole up to
60 cm in diameter; bark surface fissured and scaly, grey, leaf is opposite, simple, petiole up to 12 mm long; blade oblong-elliptical, narrowly elliptical or lanceolate, 5-16 cm x
2.5-7 cm, with 6-11 pairs of secondary veins distinct below and a distinct intramarginal vein, the apex is blunt and the base of the leaf stretches along length with presence of small schizogenous oil glands. The dried brown leaves are aromatic, slightly sour and astringent. The small flowers are in loose bunches that arise from twigs behind the leaf; the flowers are creamy and later turn pink or red Malaya and have fragrance with the fruits around. Fruit is a one-seeded berry, up to 12 mm in diameter, it is green and turns red to brown when mature; the ripe fruits are ofsweet mixed sour as shown in Figure 2.2 (Amalina, 2014).
2.7.1.2 Chemical constituents of S. polyanthum
S. polyanthum contains glycosides, fatty acids, terpenoids, citral, eugenol, tannins, and flavonoids (Ratna, Ferawati, Wahyu, Lucia, Iwan, & Elisabeth, 2015;
Widyawati, Nor, Mohd, & Mariam, 2015). Other major phytochemical constituents of the essential oil from S. polyanthum leaves include cis-4-decenal, octanal, 훼-pinene, farnesol,University 훽-ocimene, and nonanal (Ismail, Mohamed, Sulaiman, & Ahmad, 2013). A review by Kusuma et al. (2011) on phytochemical screening of the S. polyanthum revealed triterpenoids, steroids and alkaloids.
2.7.1.3 Pharmacological actions of S. polyanthum
Saponins showed hypocholesterolemic and antidiabetic properties, while steroids and triterpenoids displayed analgesic properties (Rupasinghe, Jackson, Poysa,
31
Di, Bewley, & Jenkinson, 2003; Sayyah, Hadidi, & Kamalinejad, 2004; Malairajan,
Geetha, Narasimhan, & Jessi, 2006). Previously, it has been proven that the administration of tannin fractions for 30 days significantly reversed the increase of the blood glucose levels of an STZ-induced hypercholesterolemia-associated diabetic rat model (Velayutham, Sankaradoss, & Ahamed, 2012). Eugenol, a phenolic compound abundantly found in Syzygium family, has reputed ability as a vasorelaxant compound that causes vasodilation in vitro and reduces blood pressure and heart rate of rats in vivo
(Ismail, Mohamed, Sulaiman, & Ahmad, 2013). Furthermore, some flavonoids e.g. quercetin, glycosides and phytols have been reported to have antihyperglycaemic activity through various mechanisms of action, such as the inhibition of α-glucosidase, the increase of blood insulin levels, regeneration of pancreatic β-cells, antibacterial, antioxidant, anti-inflammatory, anti-allergic, anti-mutagenic,Malaya and vasodilatory activities (Miller, 1996; Widyawati, Nor, Mohd, & Mariam, 2015; Sharma & Balomajumder, 2008; Singab, El, Yonekawa, Nomura, &of Fukai, 2005; Tapas, Sakarkar, & Kakde, 2008; Jananie, Priya, & Vijayalakshmi, 2011)
2.7.1.4 Taxonomic classification of S. polyanthum
Based on the taxonomy of plants, S. polyanthum can be classified as follows:
Kingdom : Plantae Sub-Kingdom : Tracheobionta Super-Division: Spermatophyta Division : Magnoliophyta Sub-DivisionUniversity : Angiospermae Class : Magnoliopsida Order : Myrtales Family : Myrtaceae
Genus : Syzygium Figure 2.2: Syzygium polyanthum Species : Syzygium polyanthum Walp. (Source: www.florafauna.web.nparks.gov.sg) Local common names: Salam (Indonesia); Serai kayu (Malaysia); Bay leaf (England);
Dokmaeo (Thailand); San thuyen (Vietnam).
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2.7.2 Momordica charantia
2.7.2.1 Habit, habitat and macroscopy of M. charantia
M. charantia is an annual creeper plant. Leaves are simple and alternate, borne on a long channeled petiole, down from 1.5 to 7 cm at the base of which is a simple tendril. The leaves are palmate and deeply lobed, the general shape suborbicular, 3-12 cm wide. Lobes are deeply cut up to half the length of the limb or more. The base is widely cordate, the apex is acute. The margin is irregularly toothed, both sides are almost hairless with a few scattered hairs long the nerves on the lower surface. The stem is slender, slightly pubescent, grooved and light green, it branches at the base. The sepals are lanceolate, 4 to 6 mm long and 2 mm wide, glabrous. The petals are yellow, obovate and smooth, 10 to 20 mm long and 715 mmMalaya wide, and two of them carry a scale at their base. Fruit is fleshy, broadly ovoid oblong to fusiform, 4 to 20 cm long and 2.5 to 4 cm wide, dehiscent at the top by 3 valves.of It is yellow orange to scarlet, it contain many seeds. Seeds are oval-elliptic and covered by red mucilage, almost toothed at the top, 10 to 16 mm long and 7-9 mm wide and 2-3 mm thick, as shown in Figure 2.3
(Kendrick, Xia, Mei, & Nirmal, 2004).
2.7.2.2 Chemical constituents of M. charantia
M. charantia contains biologically active chemicals that include glycosides, saponins,University alkaloids, fixed oils, triterpenes, proteins and steroids. The immature fruits are a good source of vitamin C and also provide vitamin A, phosphorus, and iron. Several phytochemicals such as momorcharins, momordenol, momordicilin, momordicins, momordicinin, momordin, momordolol, charantin, charine, cryptoxanthin, cucurbitins, cucurbitacins, cucurbitanes, cycloartenols, diosgenin, elaeostearic acids, erythrodiol, galacturonic acids, gentisic acid, goyaglycosides, goyasaponins, multi-florenol, have
33 been isolated (Husain, Tickle, & Wood, 1994; Xie, Huang, Deng, Wu, & Ji, 1998;
Yuan, He, Xiong, & Xia, 1999; Parkash, Ng, & Tso, 2002). These are reported in all parts of the plant (Murakami, Emoto, Matsuda, & Yoshikawa, 2001). The hypoglycaemic chemicals of M. charantia are a mixture of steroidal saponins known as charantins, insulin-like peptides and alkaloids and these chemicals are concentrated in fruits of M. charantia. (Grover &Yadav, 2004).
2.7.2.3 Pharmacological actions of M. charantia
M. charantia contains bitter chemicals like, charantin, vicine, glycosides and karavilosides along with polypeptide-p, which are hypoglycaemic in action and improve blood sugar levels by increasing glucose uptake and glycogen synthesis in the liver, muscles and fat cells. Reports indicate that they Malaya also improve insulin release from pancreatic beta cells, and repair or promote new growth of insulin-secreting beta cells. P-Insulin, a polypeptide from the fruits andof seeds rapidly decreased and normalized the blood sugar level in rats (Singh, Kumar, Giri, Bhuvaneshwari, & Pandey, 2012). M. charantia contains another bioactive compound i.e. lectin that has insulin like activity.
The insulin-like bioactivity of lectin is due to its binding on two insulin receptors. This lectin lowers blood glucose concentrations by acting on peripheral tissues and, similar to insulin's effects in the brain, suppressing appetite. This lectin is a major contributor to the hypoglycaemic effect that develops after eating M. charantia. Charantin, an alcohol solubleUniversity potent hypoglycaemic agent composed of mixed steroids is used in the treatment of diabetes to lower the blood sugar levels (Khan & Flier, 2000; Shetty,
Kumar, Sambaiah, & Salimath, 2005; Gupta, Sharma, Gautam, & Bhadauria, 2011).
2.7.2.4 Taxonomic classification of M. charantia
Based on the taxonomy of plants, M. charantia can be classified as follows:
Kingdom : Plantae
34
Sub-Kingdom : Tracheonbionta Super-Division : Spermatophyta Division : Magnoliophyta Sub-Division : Angiospermae Class : Dicotyledoneae Order : Cucurbitales Family : Cucurbitaceae Figure 2.3: Momordica charantia Genus : Momordica (Source: www.florafauna.web.nparks.gov.sg) Species : Momordica charantia L. Local common names: Pare (Indonesia); Peria (Malaysia); Bitter melon (England);
Mara, Phakha (Thailand); Kho qua (Vietnam).
2.8 Extraction, profiling of phytoconstituents from selected plants
The history of the extraction of natural products dates back to Mesopotamian and Egyptian times, where production of perfumes Malayaor pharmaceutically active oils and waxes was a major business. In archeologicalof excavations 250 km south of Baghdad extraction pots from about 3500 BC were found, made from a hard, sandy material presumably air-dried brick earth. Also, well-documented recipes to obtain creams and perfumes, from the time of the Assyrian king Tukulti-Ninurta I, 1120 BC. The natural feedstock was crushed in a mortar, and then leached in boiled water for one day. New feed was then added gaining higher concentrations. After percolation, oil was added while increasing the temperature. After cooling, the top oil extract can be removed, and theUniversity use of demisters (sieves of clay filled with wool or hair) is also report (Levey, 1959).
Extraction (as the term is pharmaceutically used) is the separation of medicinally active portions of plant (and animal) tissues using selective solvents through standard procedures. Such extraction techniques separate the soluble plant metabolites and leave behind the insoluble cellular marc. The products so obtained from plants are relatively complex mixtures of metabolites, in liquid or semisolid state or (after removing the
35 solvent) in dry powder form, and are intended for oral or external use. These include classes of preparations known as decoctions, infusions, fluid extracts, tinctures, pilular
(semisolid) extracts or powdered extracts. Such preparations have been popularly called galenicals, named after Galen, the second century Greek physician. The purpose of standardized extraction procedures for crude drugs (medicinal plant parts) is to attain the therapeutically desired portions and to eliminate unwanted material by treatment with a selective solvent known as menstruum. The extract thus obtained, after standardization, may be used as medicinal agent as such in the form of tinctures or fluid extracts or further processed to be incorporated in any dosage form such as tablets and capsules. These products contain complex mixtures of many medicinal plant metabolites, such as alkaloids, glycosides, terpenoids, flavonoids and lignans. In order to be used as a modern drug, an extract may be Malaya further processed through various techniques of fractionation to isolate individual chemical entities. The industrial processing of medicinalof and aromatic plants primarily starts with the extraction of the active components using various technologies. The general techniques of medicinal plant extraction include maceration, infusion, percolation, digestion, decoction, hot continuous extraction (soxhlet), aqueous-alcoholic extraction by fermentation, counter-current extraction, microwave-assisted extraction, ultrasound extraction (sonication), supercritical fluid extraction, and phytonic extraction (with hydrofluorocarbon solvents). For aromatic plants, hydrodistillation techniques (water distillation,University steam distillation, water and steam distillation), hydrolytic maceration followed by distillation, expression and enfleurage (cold fat extraction) may be employed. Some of the latest extraction methods for aromatic plants include headspace trapping, solid phase micro-extraction, protoplast extraction, microdistillation, thermomicrodistillation and molecular distillation. With the increasing demand for herbal medicinal products, nutraceuticals, and natural products for health care all over
36 the world, medicinal plant extract manufacturers and essential oil producers have started using the most appropriate extraction technologies in order to produce extracts and essential oils of defined quality with the least variations from batch to batch. Such approach has to be adopted by medicinal and aromatic plants-rich developing countries in order to meet the increasing requirement of good quality extracts and essential oils for better efficacy generation within the country, as well as for capturing this market in developed countries. The basic parameters influencing the quality of an extract are the plant parts used as starting material, the solvent used for extraction, the manufacturing process (extraction technology) used with the type of equipment employed, and the crude-drug : extract ratio. The use of appropriate extraction technology, plant material, manufacturing equipment, extraction method and solvent and the adherence to good manufacturing practices certainly help to produceMalaya a good quality extract. From laboratory scale to pilot scale, all the conditions and parameters can be modelled using process simulation for successful industrial-scaleof production. With the advances in extraction technologies and better knowledge for maintaining quality parameters, it has become absolutely necessary to disseminate such information to emerging and developing countries with a rich medicinal and aromatic plants biodiversity for the best industrial utilization of medicinal and aromatic plant resources (Sukhdev, Suman,
Gennaro, & Dutt, 2008).
2.8.1 Maceration UniversityIn this process, the whole or coarsely powdered crude plant is placed in a stoppered container with the solvent and allowed to stand at room temperature for a period of 3 days with frequent agitation until the soluble matter has dissolved. The process is intended to soften and break the plant’s cell wall to release the soluble phytochemicals. The mixture then is strained, the marc (the damp solid material) is pressed, and the combined liquids are clarified by filtration or decantation after standing
37
(Sukhdev, Suman, Gennaro, & Dutt, 2008). In this conventional method the choice of solvents will determine the type of compound extracted from the samples. The strength and limitation of this technique are that it is the easiest and simple method. However, organic waste come into an issue as large volume of solvents is used and proper management of the waste is needed. Alteration in temperature and choice of solvents enhance the extraction process, reduce the volume needed for extraction and can be introduced in the maceration technique, when such alteration is not objectionable
(Azwanida, 2015). There are so many studies in natural product using maceration as an extraction method. Maceration extraction for pyrethrum flowers (Chrysanthemum cinerariifolium) was done by Gallo et al. (2017), using hexane as solvent and kept in room temperature carried out for 4 days. The result was with a high yield of extract it is possible to obtain pyrethrin extracts for use inMalaya the production of low toxicity insecticides (Albuquerque et al., 2017). of 2.8.2 Hot continuous extraction (soxhlet extraction)
This technique uses continuous extraction by solvent of increasing polarity. The extract is placed in thimble constructed of muslin or cellulose, through which solvent is continuously refluxed. The soxhlet apparatus will empty its content into round bottomed flask once the solvent reaches a certain level. As fresh solvent enters the apparatus by a reflux condenser, extraction is very efficient and compounds are effectively drawn into theUniversity solvent from the extract due to their low initial concentration in the solvent. The method suffers from the same drawback as other hot extraction methods (possible degradation in the solvent), but it is the best extraction method for the recovery of big yields of extract. Moreover, providing biological activity is not lost on heating, the technique can be used in drug lead discovery (Sukhdev, Suman, Gennaro, & Dutt,
2008). This method requires a smaller quantity of solvent compared to maceration.
38
However, the soxhlet extraction comes with disadvantage such as exposure to hazardous and flammable liquid organic solvents, with potential toxic emissions during extraction. Solvents used in the extraction system need to be of high-purity that might add to cost (Azwanida, 2015). The ideal sample for soxhlet extraction is also limited to a dry and finely divided solid and many factors such as temperature, solvent-sample ratio and agitation speed need to be considered for this method. Soxhlet extraction of sweet passion fruit (Passiflora alata Curtis) extraction yield was 28.33 % using n- hexane as solvent because the heating during the soxhlet extraction could be contributed to the partial degradation of some of these compounds compared with the sonication extraction (Pereitra, Fabiane, Eriel, Agnes, & Marcos, 2017).
2.8.3 Sonication Malaya The procedure involves the use of ultrasound with frequencies ranging from 20 kHz to 2000 kHz; the mechanical effectof of acoustic cavitation from the ultrasound increases the surface contact between solvents and samples and permeability of cell walls. Physical and chemical properties of the materials subjected to ultrasound are altered and disrupt the plant cell wall; facilitating release of compounds and enhancing mass transport of the solvents into the plant cells (Azwanida, 2015). The benefits of sonication extraction are mainly due to reduction in extraction time and solvent consumption and the procedure is simple and relatively low cost technology that can be usedUniversity in both small and larger scale of phytochemical extraction. However, the use of ultrasound energy more than 20 kHz may have an effect on the active phytochemicals through the formation of free radicals and consequently undesirable changes in the drug molecules and large-scale application is limited due to the higher costs (Sukhdev,
Suman, Gennaro, & Dutt, 2008). An investigation by Safdar et al. (2016) was carried out to extract polyphenols from the peel of kinnow (Citrus reticulate Linn.) by
39 sonication extraction techniques. The highest extraction yield was obtained through the solvent ethanol at 80 % concentration level. Sonication was a more efficient technique and yielded comparatively higher polyphenol contents extract using sonication extraction.
2.8.4 Fresh juice
In the current study, the fresh fruits were cut open to remove the seeds and fresh leaves chopped into small pieces and then homogenised with water in a commercial blender. The fresh juice was then centrifuged and the supernatant was lyophilized. The benefits of fresh juice extraction are mainly reduction in extraction time and solvent consumption and the procedure is simple and relatively low cost technology using the household mixer (Kumar, Balaji, Um, & Sehgal, 2009).Malaya A study by Torti et al. (1995) on Acomastylis rossi and Ouratea lucens, fresh juices were extracted by using the homogenizer for 60 seconds at maximumof speed, it proved to be both efficient and consistent in extracting phenolics from tender, as well as tough, leaves. That adoption of the fresh juice extraction will increase phenolic yield and efficiency. The fresh juice has its efficacy in breaking down cell walls. The joint action of the partial vacuum created by the homogenizer and the tearing by the saw tooth generator serve to break the cell wall of plant.
2.9 Gas chromatography - Mass spectrometry (GC-MS)
UniversityGas chromatography (GC), is a type of chromatography in which the mobile phase is a carrier gas, usually an inert gas such as helium or an un-reactive gas such as nitrogen, and the stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside glass or metal tubing, called a column. The capillary column contains a stationary phase; a fine solid support coated with a non-volatile liquid.
40
The solid can itself be the stationary phase. The sample is swept through the column by a stream of helium gas. Components in a sample are separated from each other because some take longer to pass through the column than others. Mass spectrometry (MS), the detector for the GC is the mass spectrometer. As the sample exits the end of the GC column it is fragmented by ionization and the fragments are sorted by mass to form a fragmentation pattern. Like the retention time (RT), the fragmentation pattern for a given component of sample is unique and therefore is an identifying characteristic of that component. It is so specific that it is often referred to as the molecular fingerprint.
Gas chromatography mass spectrometry (GC-MS) is an analytical method that combines the features of gas-liquid chromatography and mass spectrometry to identify different substances within a test sample. GC can separate volatile and semi-volatile compounds with great resolution, but it cannot identifyMalaya them. MS can provide detailed structural information on most compounds such that they can be exactly identified, but it cannot readily separate them. GC-MS of is a combination of two different analytical techniques, gas chromatography (GC) and mass spectrometry (MS), is used to analyse complex organic and biochemical mixtures. Spectra of compounds are collected as they exit a chromatographic column by the mass spectrometer, which identifies and quantifies the chemicals according to their mass to-charge ratio (m/z). These spectra can then be stored on the computer and analysed. Carrier gas is fed from the cylinders through the regulators and tubing to the instrument. It is usual to purify the gases to ensureUniversity high gas purity and gas supply pressure. The sample is volatilized and the resulting gas entrained into the carrier stream entering the GC column. Capillary GC columns are usually several meters long (10-120 m is typical) with an internal diameter of 0.10-0.50 mm, whilst packed GC columns tend to be 1-5 meters in length with either
2 or 4 mm internal diameter. Gas chromatography have ovens that are temperature programmable, the temperature of the gas chromatographic ovens typically range from
41
5 to 400 oC but can go as low as -25 oC with cryogenic cooling. There are several very popular types of mass analyser associated with routine GC-MS analysis and all differ in the fundamental way in which they separate species on a mass-to-charge basis. Mass analysers require high levels of vacuum in order to operate in a predictable and efficient way. The ion beam that emerges from the mass analyser, have to be detected and transformed into a usable signal. The detector is an important element of the mass spectrometer that generates a signal from incident ions by either generating secondary electrons, which are further amplified, or by inducing a current (generated by moving charges) as shown in Figure 2.4 (Hussain & Maqbool, 2014). Earlier report by Singh,
Kumar, Giri, Bhuvaneshwari, & Pandi, (2012), the volatile components of the fruits of vegetable M. charantia were analysed using Varian 450GC, 240MS (VF-5 MS column). The results of the GC-MS analysis identifiedMalaya the various compounds present in the methanolic extract of this plant fruit showed the presence of important bioactive compounds especially, gentisic acid which ofhas antioxidant activity.
University
Figure 2.4: Typical GC-MS instrumentation (Source: www.chromacademy.com)
42
2.10 Liquid chromatography - Mass spectrometry (LC-MS)
Liquid chromatography is a fundamental separation technique in the life sciences and related fields of chemistry. Unlike gas chromatography, which is unsuitable for non-volatile and thermally fragile molecules, liquid chromatography can safely separate a very wide range of organic compounds, from small-molecule drug metabolites to peptides and proteins. Traditional detectors for liquid chromatography include refractive index, electrochemical, fluorescence, and ultraviolet-visible (UV-Vis) detectors. Some of these generate two dimensional data; that is, data representing signal strength as a function of time. Others, including fluorescence and diode array UV-Vis detectors, generate three-dimensional data. Three-dimensional data include not only signal strength but spectral data for each point in time. Mass spectrometers also generate three-dimensional data. In addition to signal strength,Malaya they generate mass spectral data that can provide valuable information about the molecular weight, structure, identity, quantity, and purity of a sample. Mass of spectral data add specificity that increases confidence in the results of both qualitative and quantitative analyses. LC-MS is a technique, which combines the separating power of high performance liquid chromatography (HPLC), with the detection power of mass spectrometry. Mass spectrometry is a wide ranging analytical technique, which involves the production and subsequent separation and identification of charged species. Mass spectrometer involves the separation of charged species which are produced by a variety of ionisation methods in University LC-MS. These include electrospray ionisation (EI) and atmospheric pressure chemical ionisation (APCI) in all cases the charged species are produced as gas phase ions under atmospheric pressure conditions. In addition to the analyser, the mass spectrometer also includes an atmospheric ionisation chamber, a vacuum system and detector. Ion source: the HPLC eluent is sprayed into the atmospheric pressure region; skimmer cone: A cone with a sampling orifice of reduced the gas load entering the
43 vacuum system of the mass analyser device; quadrupole: device that uses electric fields in order to separate ion according to their mass to charge ratio (m/z) as they pass along the central axis of four parallel equidistant rods; collision cell: ion emerging from the first mass analyser are accelerated using a potential difference and collide with neutral gas molecules such as H2, N2 or Ar, causing analyte fragmentation; detector: once produced and separated, the ions need to be detected and transformed into a usable signal. Electron multiplier, dynode, photodiode, and multi-channel plate (MCP) ion detection system are widely employed in most modern mass spectrometer system; vacuum system: mass analysers require high level of vacuum in order to operate in a predictable and efficient way. The vacuum system of most modern LC-MS systems consist of two or more differentially pumped vacuum chambers, separated by baffles or orifice plates of varying design depending upon the instrumentMalaya manufacture. There are several discrete stages in LC-MS analysis, typically these include: separation of the sample components usingof an HPLC column where the analytes are differentially partitioned between the mobile phase (eluent) and the stationary phase
(coated onto a support material and packed into the column). The mechanism of retention and separation will depend on the mode of chromatography but may include hydrophobic interaction, ion exchange, ion-pair, surface localisation, etc. The separated sample species are then sprayed into an atmospheric pressure ion source (API) where they are converted to ions in the gas phase and the majority of the eluent is pumped to waste.University Most popular analyser includes quadrupole, time of flight, ion trap and magnetic sector. The mass analyser may be used to isolate ions of specific mass to charge ratio to scan over all ion m/z values present. All mass analysis and detection is carried out under high vacuum established using a combination of foreline (roughing) and turbomoleculer pumps as shown in Figure 2.5 (Niessen & Tinke, 1995; Hoffmann,
Charatte, & Stroobant, 1996; Catalin, Charatte, Roland, & William, 2004).
44
Figure 2.5: Typical LC-MS instrumentation (Source: www.chromacademy.com)
2.11 Herbal formulations and alternative systems of medicine Herbs and products containing herb(s) have beenMalaya in trade and commerce and are currently used for a variety of purposes. The WHO defined herb as fresh or dried, fragmented or powdered plant material, whichof can be used in crude state or further processed and formulated to final herbal product. Treatment of herbs by squeezing, steaming, roasting, decocting or infusing in water, extracting with alcohol, or sweetening and baking with honey can create “herbal products” such as juices, tinctures, decoctions, infusions, gums, fixed oils, essential oils, and resins. These may be used medicinally or as the starting material for additional processing and as food ingredients.
Depending on the sophistication of the herbal preparation, these products may be subjectUniversityed to any number of physical, chemical, or biological processes, including pulverization, extraction, distillation, expression, fractionation, purification, concentration, or fermentation. Formulation of the final product may require mixing one or more plant preparations with minerals or animal products and constituents isolated from herbal materials or synthetic compounds. These phytomedicine formulations may also be referred to as drugs or botanicals, or when taken orally to provide health
45 benefits, they may be called dietary supplements or even food ingredients in some cases. Herbal medicines are plant derived materials and preparations with therapeutic or other human health benefits, which contain either raw or processed ingredients from one or more plants, inorganic materials or animal origin (WHO, 1996)
2.11.1 Standardization of herbal medicine
In many developing countries, a large proportion of the population relies on traditional practitioners and their armamentarium of medicinal plants in order to meet health care needs. Although modern medicine may exists side-by-side with such traditional practices, herbal medicines have often maintained their popularity for historical and cultural reasons. Such products have become more widely available commercially, especially in developed countries. In Malayathis modern setting, ingredients are sometimes marketed for uses that were never contemplated in the traditional healing systems from which they emerged. An exampleof is the use of ephedra (= Ma huang) for weight loss or athletic performance enhancement (Shaw, 1998). While in some countries, herbal medicines are subjected to rigorous manufacturing standards, this is not so everywhere. Herbal products are sold as ‘phytomedicines’, they are subject to the same criteria for efficacy, safety and quality as are other drug products (Carmona &
Pereira, 2013). Based on the WHO Traditional Medicine Strategy 2014-2023 the strategy has two key goals: to support Member States in harnessing the potential contributionUniversity of traditional and complementary medicine (T&CM) to health, wellness and people centred health care and to promote the safe and effective use of T&CM through the regulation of products, practices and practitioners. These goals will be reached by implementing three strategic objectives: building the knowledge base and formulating national policies; strengthening safety, quality and effectiveness through regulation; and promoting universal health coverage (WHO, 2014). Standardization of
46 herbal formulations is essential in order to assess the quality of drugs, based on the concentration of their active principles, physical, chemical and phytochemical standardization. The quality assessment of herbal formulations is of paramount importance in order to justify their acceptability in modern system of medicine. One of the major problems faced by the herbal industry is the unavailability of rigid quality control profiles for herbal materials and their formulations (Sahoo, Padmavati, &
Satyahari, 2010).
2.11.2 Herbal formulations
Dosage form is a drug delivery system designed to deliver the active ingredient to the body and, upon administration should deliver the drug at a rate and amount that assures the desired pharmacological effect. Such dosageMalaya forms are manufactured under current good manufacturing procedures (cGMP), using equipment and packaging to ensure product stability. The dosage form ofmust produce the same therapeutic response each time it is administered. To maintain this reproducibility between and within batches, manufacturing procedures are validated under a specific quality assurance program. Non-parenteral dosage forms can be categorized based on the route of administration or physical form. Based on physical form they can be classified as solids, liquids (homogenous and heterogeneous systems), semisolids, and aerosols. Dosage forms can also be categorized based on the route of administration. Solid dosage forms includeUniversity different types of compressed tablets, granules, troches, lozenges, coated dosage forms, and hard and soft gelatin capsules. Liquid dosage forms include solutions, suspensions, emulsions, and buccal and sublingual sprays. Topical dosage forms are applied to the skin and include ointments, pastes, creams, lotions, liniments, and transdermal patches. Some dosage forms are formulated for application to body cavities, viz. rectal and urethral suppositories and vaginal pessaries. Inhalation aerosols, using
47 metered dose inhalers (MDIs), dry powder inhalers (DPIs) and nebulizers, are used to deliver drugs to the respiratory tract. Nasal route uses solution and suspension dosage forms. Occular route is used to administer solutions and suspensions to the eye for local and systemic effects (Swarbrick, 2007). Formulation studies involve developing a preparation of the drug which is both stable and acceptable to the patient. For orally administered drugs, this usually involves incorporating the drug into a tablet or a capsule. It is important to make the distinction that a tablet contains a variety of other potentially inert substances apart from the drug itself, and studies have to be carried out to ensure that the encapsulated drug is compatible with these other substances in a way that does not cause harm, whether direct or indirect. Preformulation involves the characterization of a drug's physical, chemical, and mechanical properties in order to choose what other ingredients (excipients) shouldMalaya be used in the preparation. Formulation studies then consider such factors as particle size, polymorphism, pH, and solubility, as all of these can influence bioavailabilityof and hence the activity of a drug. The drug must be combined with inactive ingredients by a method which ensures that the quantity of drug present is consistent in each dosage unit e.g. each tablet. The dosage should have a uniform appearance, with an acceptable taste, tablet hardness, or capsule disintegration (USP 34, 2009; BP, 2013). Many drugs commonly used today are of herbal origin. A study of herbal formulation of tuberous roots of Ipomoea digitata as a potent antidiabetic drug was developed and investigated for its pharmacognostical studies,University in vitro evaluation of the tablets and finally its pharmacological evaluation for the antidiabetic activity (Margret & Jayakar, 2010). A detailed list of formulations developed and reported in literature was shown in the Table 2.4.
48
Table 2.4: List of few herbal tablet formulations described in the literature Plants Phytoconstituents Activity References
Azadirachta indica, Camellia sinensis and Catechin, terpenoids, tannins Antidiabetic Mishra et al., 2014 Asparagus racemosus
Gymnema sylvestre Gymnemic acids Antidiabetic Devi & Nimisha, 2015
Tinospora cordifolia, Gymnema sylvestre, Gymnemic acids, tannins, Antidiabetic Suman et al., 2015 Pterocarpus marsupium and Acacia arabica flavonids, epicathechin
Harpagophytum procumbent Iridoid glycoside harpagoside Rheumatic pain Chrubasic et al., 2000
Osteoporosis and cardiovascular Soybean Isoflavones Oliveira et al., 2013 diseases Echinacea purpurea and Echinacea TreatmentMalaya of colds and minor Alkylamides Matthias et al., 2007 angustifolia infections Schisantherin A, schisandrin A, Hepatitis, inflammation and Schisandra sphenanthera schisandrin B, schisandrin C, Jin et al., 2015 of cancer schisandrol A and schisandrol B Hedera helix Saponin Respiratory disorders Stauss et al., 2011
Peumus boldus Cathecin Antioxidant Palma et al., 2002 Memory impairment, Crocus sativus Safranal and Picrocrocin antidepressant, anticonvulsant and Modaghegh et al ., 2008 antitumor effect
Syzygium cumini Polyphenol Antidiabetic Peixoto & Freitas, 2013
Begonia laciniata, Cusuta epithymum and Phenolic compounds, alkaloids, Hepatoprotective Seru et al., 2013 Dendrobium ovatum flavonoids University
50 CHAPTER 3: MATERIAL AND METHODS
The research methodology includes the determination of the standardization of plant materials by macroscopic and microscopic evaluation. Firstly, extraction methods were used like maceration, fresh juice, soxhlet and sonication. Secondly, examination and profiling of extracts by using GC-MS and LC-MS was carried out. In vitro antioxidant tests for DPPH, FRAP and antidiabetic enzyme inhibitory activity for α- amylase and α-glucosidase were carried out. Finally, herbal tablet formulation was formulated using best extract by conventional granulation and compression. This section represents the detailed methodology used in the study.
3.1 Materials Malaya 3.1.1 Plant materials Leaves of S. polyanthum were collectedof from Equine Park, Seri Kembangan, Selangor, Malaysia in the month of June, 2014. Whereas, the fruits of M. charantia were purchased from the traditional market (pasar rakyat gelugor taman tun sardon) in
Pulau Pinang, Malaysia in the same month and year. Both the plants were authenticated by a taxonomist, Dr. Sugumaran Manickam at Rimba Ilmu facility available within
University Malaya campus, Kuala Lumpur, Malaysia. A herbarium specimen was deposited and voucher specimens of the samples S. polyanthum (KLU 49084) and M. charantiaUniversity (KLU 49083) were kept in the Rimba Ilmu. Plant materials were washed separately with fresh water to remove dirt, processed to exclude inner pulp and seeds of
M. charantia fruit and were dried in oven for 2 days at temperature 50 °C. The dried materials were grinded into powder by commercial blender (National, Malaysia) and the powders were stored in airtight polyethylene plastic containers at room temperature (25
°C) for future use.
51
3.1.2 Chemicals and reagents
Phloroglucinol, glycerol, were purchased from Merck (Germany), HPLC grade methanol, 1,1-diphenyl-2-picryl-hydrazyl, quercetin, 2,4,6-tripyridyl-s-triazine, FeCl3,
FeSO4, glacial acetic acid, sodium acetate trihydrate, potato starch, sodium chloride, monobasic sodium phosphate, 3,5-dinitrosalicylic acid, potassium tartrate tetrahydrate, sodium hydroxide, sodium carbonate, 4-nitrophenyl-β-D-glucopyranoside, α-amylase enzyme and α-glucosidase enzyme, were procured from Sigma-Aldrich (St Louis,
USA), monobasic potassium phosphate and acarbose 95 % were obtained from Acros
Organic (USA). Sodium starch glycolate, PEG 4000, HPMC (E15LV), lactose, magnesium stearate, talc, microcrystalline cellulose, were purchased from R&M Chemicals (UK). All other chemicals and reagents wereMalaya of analytical quality grade and were used as received. of 3.2 Methods
3.2.1 Microscopic evaluation of plant samples
Freshly collected leaves of S. polyanthum were evaluated for their length and width. A transverse section of the lamina and midrib region of fresh leaf was taken to evaluate microscopic characters of the leaf for microscopical identification. Similarly freshly collected fruits of M. charantia were evaluated for its length and width, a thin sectionUniversity of the rind of the fruit of M. charantia was taken to evaluate microscopic characters of the fruit for further evaluation of its identity. A thin section was taken by placing a piece of leaf midrib of S. polyanthum and a piece of rind of M. charantia in potato pith to ensure manual sectioning. The section was immediately treated with phloroglucinol and concentrated HCl for identification of lignified tissues and for tissue differentiation. Glycerol was added as mounting solution, and as humectant to prevent
52 tissue dehydration before placing a coverslip for microscopical examination. In the same manner finely powdered leaf powder, fruit rind powder were treated chemically and both powders, transverse sections were observed under light microscope at a magnification of 4x, 10x and 40x using Olympus CH30 (Olympus, Japan), photographs were taken using a digital camera and Scanning Electron Microscope (SEM, Quanta
FEG 450, USA).
3.2.2 Extraction methods applied to plant powders a. Extraction by maceration
The maceration method was adopted from Montanez et al. (2014). Briefly, 250 g of dried coarse fruit powder of M. charantia and dried fine leaf powder of S. polyanthum was mixed with 5000 mL and 1500 mL of water respectively, both powders were agitated using mechanical stirrer (1500 rpm)Malaya for 30 minutes to ensure uniform powder and solvent mix. The difference in the volumes is primarily due to the difference in their absorption capacitiesof to water. The beaker was covered with aluminium foil and was kept for 3 days under refrigeration at 5-8 oC. The macerated mixtures were filtered through a muslin cloth and subsequently filtered by vacuum filtration method. The final filtrates were freeze dried using Labconco freezone freeze dryer, USA.
b. Fresh juice extraction UniversityThe modified method of Kumar et al. (2009) was followed in the current study. About 1 kg of fresh M. charantia fruit devoid of pulp, seeds and fresh S. polyanthum leaves were cut into small pieces with the help of kitchen knife. The chopped pieces were homogenized in a commercial blender (National, Malaysia) such that water to M. charantia (1:2) and water to S. polyanthum (1:4) ratios were maintained. The homogenized mixtures were then stirred at 1500 rpm for 15 min. The fresh juice was
53 filtered through a muslin cloth and subsequently filtered by vacuum filtration method.
The final filtrates were freeze dried using Labconco freezone freeze dryer, USA.
c. Extraction by sonication
The modified method of Montanez et al. (2014) was followed for extraction by sonication. About 250 g dried coarse fruit powder of M. charantia and dried fine powder of S. polyanthum leaf were mixed with 5000 mL and 1500 mL of water respectively, both powders were stirred at 1500 rpm for 15 min. A bath sonicator
(Fisher scientific FB15057, USA) was used for sonication of the sample for 30 minutes at a constant frequency of 37 kHz at a temperature of 30 oC. The sonicated mixtures were filtered through a muslin cloth and subsequently filtered by vacuum filtration method. The final filtrates were freeze dried usingMalaya Labconco freezone freeze dryer, USA. of d. Soxhlet extraction
The method of Montanez et al. (2014) was adopted to extract plant materials by soxhlet. The dried coarse fruit powder of M. charantia and dried fine leaf powder of S. polyanthum weighing 250 g was placed in a “thimble” made of muslin placed in the chamber of the soxhlet apparatus. About 5000 mL of water was used as a solvent in a 5
L round bottomed flask to its maximum capacity with temperature 100oC. The condensedUniversity vapours of solvent drip into the thimble containing the sample and ensure hot percolation of solvent to produce an efficient extraction. This process was carried out until the residue completely exhausted. The soxhlet extracts of both powders were filtered through a muslin cloth and subsequently filtered by vacuum filtration method.
The final filtrates were freeze dried using Labconco freezone freeze dryer, USA.
54
3.2.3 GC-MS and LC-MS profiling of extracts a. GC-MS profiling
The GC-MS profiling was carried out as per the modified method described by
Udayaprakash et al. (2015). Shimadzu GC-MS QP2010 PLUS Japan, 30 m x 0.25 mm x
0.25 μm of capillary column (Agilent J&W GC columns, USA) was used for the analysis. Injection temperature was maintained at 250 °C, helium flow rate was 0.85 mL/min and ion source temperature of 230 °C. Injection was performed in the splitless mode and the volume was 2 μL of sample (1 mg/10 mL in methanol, HPLC grade
Merck, Germany). The instrument was set to an initial temperature of 70 oC and later programmed to an increase of 10 oC/min to 300 oC. The mass spectra (MS) of compounds in samples were obtained by electron ionization (EI) and the detector operated in scan mode from 50 – 1000 m/z. The startMalaya time of MS was 4 minutes; end time was 71 minutes. Identifications were made based on mass spectral matching with standard compounds in NIST08 library. Theof relative amounts of individual components were expressed as percent peak areas relative to the total peak area.
b. LC-MS profiling
LC-MS profiling was carried out as per the modified method described by
Terpinc et al. (2016). An Agilent 6550 iFunnel Q-TOF LC-MS instrument was used.
The sample was analysed upon injecting 10 µL sample (1 mg/10 mL in methanol, HPLCUniversity grade Merck, Germany). The profile of the sample was acquired using a C-18 column at a flow rate of 0.200 mL/min. The solvent gradient for HPLC, phase A consisted of 0.1 % formic acid in water, phase B consisted of 0.1 % formic acid in methanol: 19 % A, 81 % B from 0 to 10 min, 21 % A, 79 % B from 10 to 15 min, 28 %
A, 72 % B from 15 to 35 min. Positive ion electrospray ionization (ESI) was used for the detection of the eluents without solvent splitting.
55
3.2.4 Antioxidant activity and free radical scavenging activity of the extracts a. DPPH assay
Antioxidant activity test using DPPH or 1,1-Diphenyl-2-picryl-hydrazyl (Sigma,
Germany) was carried out as per the modified method described by Brand et al. (1995).
The test was performed for individual extracts and combination extracts as shown in the
Table 3.1. The reaction mixture was prepared in a 96-well microplate (solid clear F- bottom, Greiner Bio One, Austria) adding 20 µL of sample (1 mg/mL extract) and 120
µL of 100 µM DPPH in methanol, and incubated in dark at 25 °C for 20 min. The absorbance of the solutions was measured using UV-Visible spectrophotometry (infinite
M 200 Tecan, Switzerland) at 517 nm. Free radical scavenging activity of the samples was estimated by the colour change from deep purpleMalaya to yellow and decrease in the absorbance value in comparison to the blank as an indication of antioxidant activity. The free radical scavenging activity percentageof (RSA %) of the samples were evaluated with Eq. 1. The mixture of methanol (20 µL) and DPPH (120 µL) served as blank, quercetin (Sigma-aldrich, USA) mixture of DMSO (20 µL) and DPPH radical solution
(120 µL) served as control. Where, A0 is the blank and as is sample absorbances.
퐴 −퐴 RSA % = 표 푠 x 100 (Eq. 1) 퐴표
University
56
Table 3.1: Prepared extracts and their combinations tested for antioxidant and antidiabetic assays
No Sample No Sample
1 Extract M. charantia Maceration §Ψ 19 Combination extract 2 and extract 6 §
2 Extract M. charantia Fresh Juice §Ψ 20 Combination extract 2 and extract 7 §Ψ
3 Extract M. charantia Sonication §Ψ 21 Combination extract 2 and extract 8 §Ψ
4 Extract M. charantia Soxhlet §Ψ 22 Combination extract 3 and extract 4 §
5 Extract S. polyanthum Maceration §Ψ 23 Combination extract 3 and extract 5 §Ψ
6 Extract S. polyanthum Fresh Juice §Ψ 24 Combination extract 3 and extract 6 §Ψ
7 Extract S. polyanthum Sonication §Ψ 25 Combination extract 3 and extract 7 §Ψ 8 Extract S. polyanthum Soxhlet §Ψ 26 CombiMalayanation extract 3 and extract 8 §Ψ 9 Combination extract 1 and extract 2§ 27 Combination extract 4 and extract 5 §Ψ
10 Combination extract 1 and extract 3 § of28 Combination extract 4 and extract 6 §Ψ
11 Combination extract 1 and extract 4 § 29 Combination extract 4 and extract 7 §Ψ
12 Combination extract 1 and extract 5 § 30 Combination extract 4 and extract 8 §Ψ
13 Combination extract 1 and extract 6 §Ψ 31 Combination extract 5 and extract 6 §
14 Combination extract 1 and extract 7 §Ψ 32 Combination extract 5 and extract 7 §
15 Combination extract 1 and extract 8 §Ψ 33 Combination extract 5 and extract 8 § 16University Combination extract 2 and extract 3 § 34 Combination extract 6 and extract 7 § 17 Combination extract 2 and extract 4 § 35 Combination extract 6 and extract 8 §
18 Combination extract 2 and extract 5 §Ψ 36 Combination extract 7 and extract 8 §
Notes: (§) = Samples for DPPH and FRAP assays (Ψ) = Samples for α-amylase and α-glucosidase
57 b. FRAP assay
The FRAP assay or ferric reducing antioxidant power was performed as per the previous method described by Udayaprakash et al. (2015). The test was performed for individual extracts and combination extracts as shown in the Table 3.1. A fresh working solution was prepared by mixing 25 mL acetate buffer 300 mM, pH 3.6, 2.5 mL TPTZ
10 mM (2,4,6-tripyridyl-s-triazine) in 40 mM HCl, and 2.5 mL FeCl3.6H2O 20 mM.
The reagent was warmed at 37 °C before use, the assay was carried out in a 96-well microplate (solid clear F-bottom, Greiner Bio One, Austria) with 10 µL (1 mg/mL) of individual extract solution with 300 μL of FRAP solution incubated for 30 min in dark.
Absorbance of each well was measured at 593 nm using UV-Visible spectrophotometry
(Infinite M 200 Tecan, Switzerland). The experiment for all the extracts was repeated in triplicate. The percentage ferric (Fe3+) reduction toMalaya ferrous (Fe2+) was calculated by 2 FeSO4 standard curve (R = 0.9959) between 200 to 1000 µM using the equation below Eq. 2. of FRAP value = Absorbance (sample +FRAP reagent) - Absorbance (FRAP reagent)
FRAP Value of Sample % FRAP = x 100 (Eq.2) FRAP Value of 퐹푒푆푂4.7H2O
3.2.5 Antidiabetic enzyme inhibitory activity of the extracts a. Inhibitory activity of extracts against α-amylase
The α-amylase assay was evaluated using the method of Loizzo et al. (2007) withUniversity few modifications. The test was performed for individual extracts and combination extracts as shown in the Table 3.1. Briefly, 20 µL of aqueous sample solution (1 mg/mL extract) or standard acarbose 95 % (acros organic, USA) was mixed with 50 µL of phosphate buffer solution of α-amylase enzyme (porcine pancreas Amylase; 5 mg/10 mL; 10 Unit/mg; Sigma-aldrich, St Louis, USA) and was incubated at 37 °C for 10 min.
To this mixture 100 µL of starch solution (1 % w/v of potato starch {Sigma-aldrich, St
58
Louis, USA} in pH 6.9 phosphate buffer prepared by mixing 20 mM monobasic sodium phosphate and 6.7 mM of sodium chloride in 50 mL, heated at 65 °C for 15 min), was added with an incubation of 10 minutes at 37 °C. The reaction was terminated by adding 100 µL of 3,5-dinitrosalicylic acid 96 mM (prepared by mixing 15 g of sodium potassium tartrate tetrahydrate in 10 ml of 2 M NaOH and 0.5 mg 3,5-dinitrosalicylic acid solution) and was further incubated in water bath for 10 min. The colorimetric reagent was prepared mixing a sodium potassium tartrate solution and 0.5 mg 3,5- dinitrosalicylic acid solution. Control and sample extracts were added to starch solution and left to react with α-amylase solution under an alkaline condition at 25 °C. The reaction was measured over 3 min. The generation of maltose was quantified by the reduction of 3,5-dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid. The absorbance of the reaction was detected at 540 nm by using UV-VisibleMalaya spectrophotometry (infinite M 200 Tecan, Switzerland) the assay was carried out in 96-well microplates (solid clear F- bottom, Greiner Bio One, Austria). The percentageof of inhibition was calculated using the equation below Eq. 3.
퐴 −퐴 % Inhibition = 푏푙푎푛푘 푠푎푚푝푙푒 x 100 (Eq. 3) 퐴 푏푙푎푛푘
b. Inhibitory activity of extracts against α-glucosidase
The α-glucosidase assay was evaluated using the method of Ting et al. (2007) with few modifications. The test was performed for individual extracts and combination extractsUniversity as shown in the Table 3.1. The assay was determined in 96-well microplates
(Solid clear F-bottom, Greiner Bio One, Austria). Briefly, 40 µL of aqueous sample solution (1 mg/mL extract) or standard acarbose 95 % (acros organic, USA) was mixed with 100 µL of phosphate buffer solution of α-glucosidase enzyme (Saccharomyces cerevisiae; 2.2 mg/10 mL; 10 Unit/mg; Sigma-aldrich, St Louis, USA) and was incubated at 37 °C for 10 min. To the assay mixture 50 µL of 4-Nitrophenyl β-D-
59 glucopyranoside (PNPG) substrate solution (5 mM of PNPG {Sigma-aldrich, St Louis,
USA} in pH 6.9 phosphate buffer prepared by mixing 67 mM monobasic potassium phosphate), was added with an incubation of 10 min. at 37 °C. The reaction was stopped by adding 80 µL of 100mM Na2CO3. The absorbance of the reaction was measured on
UV-Visible spectrophotometry (infinite M 200 Tecan, Switzerland) at 405 nm. The percentage of inhibition was calculated using the equation below Eq. 4.
퐴 −퐴 % Inhibition = 푏푙푎푛푘 푠푎푚푝푙푒 x 100 (Eq. 4) 퐴 푏푙푎푛푘
3.2.6 Formulation and evaluation of herbal tablet dosage forms containing the best
extracts
The tablet formulations were made with weights of 550 mg per tablet, with a wet granulation method was use this method is theMalaya most commonly chosen method considering its normal use and its application. Tablet dosage was determined based on the in vitro antidiabetic activity of best combinationof extracts of M. charantia soxhlet and S. polyanthum fresh juice. About 300 mg of extract of both plants were incorperated into each tablet based on required concentration of extract in gastrointestinal environment and also as per the quatity of extract composition in single diet.
All the ingredients listed in Figure 3.1 and Table 3.2 were sieved using sieve no.100 (# 0.149 mm). The required quantities of the best extracts and other ingredients for 100 tablets were weighed on a digital balance (Mettler Toledo, Switzerland). Then, ingredientsUniversity as per the formula given in Table 3.2 were mixed using a mortar and pestle by geometrical dilution method. A wet mass was produced using 1% w/v HPMC as granulating solution, the granules were prepared by passing the wet mass of the blend through sieve no.10 (# 2 mm) The wet granules were dried in a hot air oven at 45 °C until constant weight of the granules was achieved. These granules were resized using sieve no.18 (# 1 mm), granules were analysed for flow properties. Just before
60 compression, talc, PEG 4000 and magnesium stearates were added as glidant and lubricants. Finally, the tablets were compressed by using single punch manual tablet compression machine equipped with flat-faced round punches of size 12 mm (Allen,
Popovich,Ansel,2011).
Malaya of
University
61
Table 3.2: The components of herbal tablet dosage forms
Quantity per tablet Ingredients (mg)
M. charantia extract 150 (Soxhlet)
S. polyanthum extract 150 (Fresh Juice)
HPMC (1%) 0.33 Lactose 153.42 Malaya Mg Stearate 2.75 Talc 27.5 of Sodium starch glycolate 22
PEG 4000 16.5
Microcrystalline cellulose 27.5
Total weight of the tablet 550 Figure 3.1: Individual percentage of tablet components
University
62 3.2.6.1 Evaluation of granular flow properties a. Determination of angle of repose (θ) of granules
As much as 25 g of granular powder was incorporated into funnel flow tester.
Funnel cover is opened so that the granules flow out and fit on top of the flat areas on the millimetre graph paper. Flow time is recorded with the stopwatch and the angle of repose (θ) was calculated using the formula:
h Angle of repose (Ɵ) = tan -1 ( ) (Eq. 5) r
Where, θ is the angle of tilt of the cone; h is the height of a cone; and r is the radius of the base of the cone. The relationship between the angle of repose and the flow properties was given in the Table 3.3. Wherein, the angle of repose between 20-40o and the flow time of more than 10 g per second exhibit good flow properties (USP 34, 2009). Malaya Table 3.3: Relationship between angle of repose and powder flow
Angle of repose (Ɵ) of Powder flow 25-30 Excellent 31-35 Good 36-40 Fair 41-45 Passable 46-55 Poor 56-65 Very poor > 66 Very very poor University
b. Determination of Carr index and Hausner ratio of granules
As much as 100 g granulate was weighed, transferred into a measuring cylinder of a tap density tester (Dr.Schleuniger, Switzerland) to record its initial volume (Vo).
The apparatus was switched on to ensure 500 taps then the final volume (Vf) was noted
63 at the end of the tapping (USP 34, 2009). Flow properties of granulate can be known indirectly by using the percentage of the compressibility as shown in the Table 3.4 with the formula given below Eq. 6 and Eq.7.
푉 −푉 Carr Index (%) = 0 f x 100 (Eq. 6) 푉0
푉 Hausner Ratio = 0 (Eq. 7) 푉f
Table 3.4: Scale of flowability of granules
Compressibility index (%) Flow character Hausner ratio ≤10 Excellent 1.00-1.11 11-15 Good Malaya1.12-1.18 16-20 Fair 1.19-1.25 21-25 ofPassable 1.26-1.34 26-31 Poor 1.35-1.45
32-37 Very poor 1.46-1.59
>38 Very very poor > 1.60
3.2.6.2 Evaluation of prepared herbal tablet formulations a. The uniformity of size and shape (n=10)
UniversityTen tablets were randomly selected, the thickness and diameter of each tablet was measured using a digital venier calliper. Unless otherwise stated, the diameter of the tablet should not be more than 3 times the thickness of tablets and should not be less than 1 1/3 of tablet thickness (Indonesian Pharmacopoeia, 1995).
64 b. The uniformity of weight among the herbal tablet formulations (n=20)
Twenty tablets were selected randomly and the average weight was calculated.
Then individual tablets were weighed to check for weight variation from the average weight calculated. According to USP 34, the deviation of individual masses from the average mass should not exceed more than 5 % for a tablet more than 324 mg weight.
Not more than 2 tablet weights should deviate from the allowable weight (USP 34,
2009).
c. The hardness test (n=10)
Hardness or crushing strength of tablets was estimated by using Dr.Schleuniger hardness tester to evaluate the ability of tablets to withstand mechanical shock and strength. Ten tablets were randomly selected and were placed into the jaws of hardness tester. The tablets were crushed by the two jaws ofMalaya the tester to evaluate the hardness. Hardness of 50 to 150 N was considered to be ideal as per Binega et al. (2013).
of d.The friability test (n=20)
The crispness of tablets was tested to know the ability of tablets prepared to withstand various mechanical shocks during the manufacturing, transportation process, packaging, packing and delivery to the consumer. The crispness of tablets includes abrasion and friability parameters.
Friability examination was carried out using USP 34 method, by weighing the initialUniversity weight of as many as 20 tablets (W1), later they were placed in a friabilator chamber, and the friability chamber was rotated for 100 revolutions with a speed of 25 rpm for 4 minutes. After the test the tablets were dedusted with a camel brush to remove any superficial adherents, final weight was measured (W2). The weight loss among the tablets was estimated using the formula given below:
65
The friability of the tablets (F) was expressed by the formula:
푊 −푊 F = 1 2 푥 100 (Eq. 8) 푊1
Where, F was the friability/crispness of tablet; W1 was the initial weight of 20 tablets and W2 was the final weight of the tablets after testing. The friability of pharmaceutically acceptable tablets should exhibit a value of 0.5-1 % (USP 34, 2009; Sarfaraz & Niazi, 2009).
e. The disintegration test (n=6)
USP method was employed in this study, six tablets were placed into the glass tubes of basket rack assembly of the disintegration tester,Malaya The basket rack was lowered into a simulated gastric medium of 1 L maintained at 37 ± 2 °C in an one litre beaker for 29-32 times per minute. The tablets were ofdeclared disintegrated completely if no parts are left over on gauze. As per USP 34, the uncoated tablets should be disintegrated within 15 minutes. If one or two tablets fail to disintegrate within 15 minutes then the test should be repeated on another 12 tablets. The test is considered as pass if 16 out of
18 tablets able to disintegrate by disappearing from the glass tubes during the test (USP
34, 2009; Sarfaraz & Niazi, 2009).
3.2.7University Statistical analysis
Statistical analysis was performed using SPSS 22 statistical package (IBM
Software, USA). The data were analysed by one way analysis of variance (ANOVA) followed by least square design (LSD) multiple comparisons. All the results were expressed as mean ± SD for triplicate determinations.
66
A B C
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D Eof F
University G H I Figure 3.2: List of instruments and equipments used in the study. A. Bath Sonicator, B. Soxhlet apparatus, C. GC-MS, D. LC-MS, E. Single punch manual compression machine, F. Digital calliper, G. Dr. Schleuniger hardness tester, H. Erweka TAR 10 friability tester, I. Electrolab ED-2 SAPO disintegration tester.
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CHAPTER 4: RESULTS
The results of this study are presented in this chapter. M. charantia and S. polyanthum plant materials were observed for macroscopy and microscopy. Later they were extracted by various extraction methods, yields of the extracts were recorded and the profiling of the extracts was also carried out by using the GC-MS and LC-MS.
Extract samples were tested for antioxidant and antidiabetic activity in both single and combination format to determine existence of synergism between the extracts of two plants. The best combination that produced good result was formulated as a herbal formulation and it was evaluated for pharmacopoeial standards.
4.1 Identification of selected plants for the study Malaya 4.1.1 Macro and microscopy of S. polyanthum leaf S. polyanthum grows wild in the forestsof and mountains or in the garden and in waste lands. This tree can be found in lowlands up to an altitude of 1400 m above sea level. Tree reaches 25 m height, with slippery surface, overgrown and single rooted.
Leaves are simple oval-shaped with elliptical tapered tip, base, tapered edge flat, fin shaped, upper surface is dark green, light green coloured lower surface, 5-15 cm long,
3-8 cm wide, fragrant. Flowers at the ends of the twigs, are white, smells fragrant
(Amalina, 2014). Microscopy of leaves of S. polyanthum shows the presence of cuticle on Universitythe outside of bifacial leaf, upper epidermis is corrugated and thin-walled, palisade layer contains 1-2 layers of columnar shaped cells, 8-16 layered loose spongy parenchyma of mesophyll (Soh & Parnell, 2011) and have spherical lysigenous oil cells, the midrib shows the lignified bicollateral closed vascular bundles with xylem at the centre and phloem on either sides, vascular bundles are completely encircled with sclerenchymatous pericyclic tissue. Above the lower epidermis in the midrib region
68 collenchyma can be seen which has a thick walled and tightly arranged cells. In addition the epidermis of the leaf shows a unique paracytic type of stomata, each stomata is surrounded by two subsidiary cells which are parallel to the longitudinal axis of the pore and guard cells as observed under scanning electron microscopy (Figure 4.1). The powder characteristics of S. polyanthum showed abundant lignified reticulate xylem vessels, pericyclic sclerenchymatous tissues with thick secondary walls having strong lignification, mesophyll region was observed as fragmented tissues scattered in the powder and pieces of epidermis showing paracytic stomata were also observed.
4.1.2 Macro and microscopy of M. charantia fruit
The fruit was elongated, ribbed, 8-30 cm long, bitter taste. Ripe fruit usually burst with 3 valves and expose red pulp embedded Malayawith seeds. Hard seeds with size 8- 13 mm, elongated flat shaped with irregular grooves and golden brown colour (Gupta, Sharma, Gautam, & Bhaduria, 2011). Microscopyof of fresh M. charantia fruit showed epidermis with thick epicarp covered with a thick striated cuticle and has prominent irregular large ridges and tapering outgrowths which are extensions of the pericarp.
Epidermis consists of relatively small rectangular parenchymal layer of cells rich in chlorophyll. The epidermis occasionally showed modified unisireate multicellular glandular trichomes, with multicellular head and multicellular stalk. Sub-epidermal tissue composed of several layers of round to oval cells enclosing chloroplasts. Aqueous mucilaginousUniversity mesocarp parenchyma consists of large oval cells almost without chlorophyll occasionally with strongly lignified groups of sclereids as discontinuous bands. Inner mesocarp mostly constituted by several layers of almost colourless isodiametric parenchyma cells followed by several layers of collenchyma traversed by lignified vascular branching strands some of which form a ring structure. The endocarp is represented by several layers of abundant continuous lignified irregular
69 parenchymatous cells. Microscopy of M. charantia fruit powder showed fragments of sclerieds in groups, lignified annular xylem and sclerenchyma (Figure 4.2) (Aswar &
Kuchekar, 2012).
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Figure 4.1: Macroscopy and microscopy of S. polyanthum : A. S. polyanthum fresh leaf; B. Transverse section of leaf at 4x magnification; C. Cuticle (CU), Upper epidermis (UE), Palisade (PL), Lysigenous oil cells (LY), Parenchyma (PR), Xylem (XL), Phloem (PH), Collenchyma (CL); D. Xylem and Phloem at 40x magnification; E & F. Paracytic stomata observed under Scanning Electron Microscope at 1500x Malaya and 12000x magnification; G. S. polyanthum powder; H. Paracytic type of stomata; I. Mesophyll fragment; J. Reticulate xylem vessels; K. Pericylic sclerenchymatous tissue. of
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Figure 4.2: Macroscopy and microscopy of M. charantia : A. M. charantia fresh fruit; B. Transverse section of fruit; C. Longitudinal section of fruit coat; Cuticle (CU), Outer mesocarp (OM), Middle mesocarp (MM), Vascular bundle (VB), Inner mesocarp (IM); Endocarp (ED) D. Epidermis showing glandular trichome (TR). E. M. charantia powder; F. Group of sclereids; G. Lignified parenchymatous tissue; H. Lignified xylem with annular thickenings.
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4.2 Extraction of plant materials using different extraction methods
The order of yield value obtained from various aqueous extracts of M. charantia were sonication > soxhlet > maceration > fresh juice. The best method with maximum percentage yield was observed in the sonication with 26.37 % (65.93 g).
Temperature influenced extraction method such as soxhlet extraction also significantly showed higher yield of 24.25 % (60.63 g) next to sonication. The yields of maceration and fresh juices were 12.98 % (32.44 g) and 3.06 % (30.63 g) respectively. On the contrary the order of yield value obtained from various extracts of S. polyanthum were fresh juice > soxhlet > sonication > maceration. The best method for S. polyanthum with maximum percentage yield was fresh juice having yield value of 10.07 % (100.73 g).
The yields of maceration, sonication and soxhlet were 7.44 % (18.6 g), 8.22 % (20.54 g) and 8.7 % (21.75 g) respectively. The yield values Malayaof S. polyanthum and M. charantia obtained by different extraction methods are shown in Figure 4.3.
Momordica charantiaofSyzygium polyanthum
26.37% 30% 24.25%
25%
20% 12.98% 15% 10.07% 8.70% 8.22% 7.44% % Yield value % Yield 10% 3.06% 5%
0% Sonication Maceration Soxhlet Fresh Juice FigureUniversity 4.3: The percent yields of different aqueous extracts of selected M. charantia and S. polyanthum plants
4.3 GC-MS and LC-MS profiling of the M. charantia and S. polyanthum extracts
The extracts were subjected for chemical profiling using GC-MS and LC-MS as described in section 3.2.3.
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4.3.1 GC-MS data analysis
GC-MS profiling of the extracts was carried out to detect volatile phytoconstituents in aqueous leaf extracts of S. polyanthum and fruit extracts of M. charantia. Since each plant material was extracted by four different extraction procedures eight GC-MS chromatograms were obtained corresponding to each individual extract. A total of 8, 10, 10 and 11 peaks were observed in GC-MS chromatograms of S. polyanthum soxhlet, sonication, fresh juice and maceration extracts respectively. Similarly a total of 9, 10, 12 and 15 peaks were observed in GC-
MS chromatograms of M. charantia sonication, soxhlet, fresh juice and maceration extracts respectively. The details of the compounds detected in various extraction methods were shown in Table 4.1, Table 4.2. and Figure 4.4, Figure 4.5.
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A
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Figure 4.4 (a): GC-MS Chromatograms showing peaks representing volatile components detected in aqueous extracts of M. charantia A. Fresh Juice, B. Maceration University
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C
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Figure 4.4 (b): GC-MS Chromatograms showing peaks representing volatile components detected in aqueous extracts of M. charantia C. Sonication, D. Soxhlet University
62 75 A
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Figure 4.5 (a): GC-MS ChromatogramsUniversity showing peaks representing volatile components in aqueous extracts of S. polyanthum A. Fresh Juice, B. Maceration
63 76 C
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Figure 4.5 (b): GC-MS Chromatograms showing peaks representing volatile components in aqueous extracts of S. polyanthum C. Sonication, D. Soxhlet 64
77 Table 4.1: List of volatile phytoconstituents identified in the aqueous extracts of the leaf of S. polyanthum by GC-MS
Elemental Molecular No Compound name RT(min.) Area (%) CAS composition weight
2,2-diethoxy- ethanol/ Glycolaldehyde, diethyl acetal / 2-Hydroxyacetaldehyde 1 C6H14O3 134 6.958 (Maceration) 1.54 (Maceration) 621-63-6 diethylacetal / 2,2-Diethoxyethanol
10.233 (Maceration) 23.49(Maceration) 2 N-Methoxy-N-methylacetamide / N-Methyl-N-methoxyacetamide C4H9NO2 103 78191-00-1 10.175 (Soxhlet) 11.27(Soxhlet)
Benzoic acid / Benzenecarboxylic acid / Benzeneformic acid / 12.792 (Maceration) 3.35 (Maceration) 3 C7H6O2 122 65-85-0 Benzenemethanoic acid / Benzoesaeure GK / Benzoesaeure GV 12.850 (Sonication) 6.37(Sonication)
14.192 (Maceration) 1.31 (Maceration) 4 Cyclopentane, 1,1,3-trimethyl- / 1,1,3-Trimethylcyclopentane C8H16 112 4516-69-2 14.158 (Fresh juice) 1.63 (Fresh Juice)
17.642 (Maceration) 1.23 (Maceration) 5 3,3-Dimethylhexane C8H18 Malaya114 563-16-6 17.617 (Fresh juice) 1.04 (Fresh Juice)
22.617 (Maceration) 1.01 (Maceration) of 22.600 (Fresh juice) 1.34 (Fresh Juice) 6 Isooctanol / Isooctyl alcohol / 6-Methyl-1-heptanol C8H18O 130 26952-21-6 22.633 (Sonication) 1.87 (Sonication) 22.642 (Soxhlet) 2.03 (Soxhlet)
1,4-Benzenedicarboxylic acid, dimethyl ester / Terephthalic acid, dimethyl ester 7 C10H10O4 194 26.783 (Maceration) 3.21 (Maceration) 120-61-6 / Dimethyl p-phthalate / Dimethyl terephthalate
27.158 (Maceration) 12.35(Maceration) Phenol, 3,5-bis(1,1-dimethylethyl)- / Phenol, 3,5-di-tert-butyl- / 3,5-Di-tert- 8 C14H22O 206 27.142 (Fresh juice) 11.29 (Fresh Juice) 1138-52-9 butylphenol / Phenol, 3,5-bis(t-butyl) / 3,5-Di-t-butylphenol/ 27.183 (Sonication) 27.50 (Sonication)
42.450 (Maceration) 5.88 (Maceration) Hexadecanoic acid, methyl ester / Palmitic acid, methyl ester / n-Hexadecanoic 9 acid methyl ester / Metholene 2216 / Methyl hexadecanoate / Methyl n- C17H34O2 270 42.442 (Fresh juice) 7.16 (Fresh Juice) 112-39-0 hexadecanoate / Methyl palmitate University 42.475 (Sonication) 6.60 (Sonication)
65 78 Elemental Molecular No Compound name RT(min.) Area (%) CAS composition weight
48.775 (Maceration) 46.21(Maceration) Octadecanoic acid, methyl ester / Stearic acid, methyl ester / n-Octadecanoic 48.775 (Fresh juice) 61.55 (Fresh Juice) 10 C19H38O2 298 112-61-8 acid, methyl ester / Kemester 9718 / Methyl n-octadecanoat 48.808 (Sonication) 52.32 (Sonication) 48.825 (Soxhlet) 38.89 (Soxhlet)
3.05 (Fresh Juice) 11 2-fluoro-2-methyl-Propane / tert-Butyl Fluoride / 2-Fluoro-2-methylpropane C4H9F 76 6.425 (Fresh Juice) 353-61-7
1,2,3-Propanetriol, monoacetate / Acetin, mono- / Acetin / Acetoglyceride / 9.06 (Fresh Juice) 12 C5H10O4 134 10.200 (Fresh Juice) 26446-35-5 Acetyl monoglyceride / Glycerinmonoacetate / Glycerol
Benzene-1,4-dicarboxylic acid, monohydrazide, methyl ester / Methyl 4- 26.758 (Fresh juice) 2.63 (Fresh Juice) 13 C9H10N2O3 194 13188-55-1 (hydrazinocarbonyl)benzoate / Malaya26.800 (Sonication) 2.02 (Sonication) Methane, nitro- / Nitromethane / Nitrocarbol / CH3NO2 / Nitrometan / UN 14 CH3NO2 61 10.083 (Sonication) 0.17 (Sonication) 75-52-5 1261 / Nitrofuel / Nitroparaffin / NM / NM-55
2,4,5-Trihydroxypyrimidine / Isobarbituric acid / 2,4,5(3H)-Pyrimidinetrione, of 15 C4H4N2O3 128 13.925 (Sonication) 0.64 (Sonication) 496-76-4 dihydro- / Dihydro-2,4,5(3H)-pyrimidinetrione
16 2-Dodecene, (Z)- / (2Z)-2-Dodecene C12H24 168 14.183 (Sonication) 1.91 (Sonication) 7206-26-0
Butane, 2,2-dimethyl- / Neohexane / 2,2-Dimethylbutane / (CH3)3CCH2CH3 / 17 C6H14 86 26.525 (Sonication) 0.61 (Sonication) 75-83-2 UN 1208 /
N-Nitrosodimethylamine / Methanamine, N-methyl-N-nitroso- / 18 C2H6N2O 74 7.333 (Soxhlet) 11.99 (Soxhlet) 62-75-9 Dimethylamine, N-nitroso- / Dimethylnitrosamine / DMN / DMNA
19 1,2,3-Trimethyldiaziridine C4H10N2 86 17.650 (Soxhlet) 0.83 (Soxhlet) 113604-56-1
2,4-bis(1,1-dimethylethyl)-Phenol / 2,4-di-tert-butyl-Phenol / 2,4-Di-tert-butyl 20 C14H22O 206 27.192 (Soxhlet) 29.62 (Soxhlet) 96-76-4 phenol / 2,4-di-t-Butyl phenol
Tridecanoic acid, methyl ester / Methyl tridecanoate / n-Tridecanoic acid methyl 21 C14H28O2 228 42.492 (Soxhlet) 4.76 (Soxhlet) 1731-88-0 ester / Methyl ester Universityof tridecanoic acid /
66 79 Table 4.2: List of volatile phytoconstituents identified in the aqueous extracts of the fruit of M. charantia by GC-MS
Elemental Molecular No Compound name RT(min.) Area (%) CAS composition weight
2-fluoro-2-methyl- Propane/ tert-Butyl Fluoride / 2-Fluoro-2- 1 C4H9F 76 6.458 (Maceration) 1.51 (Maceration) 353-61-7 methylpropane
10.250 (Maceration); 8.17 (Maceration); 1,2,3-Propanetriol, monoacetate / Acetin, mono- / Acetin / Acetoglyceride / 10.225 (Fresh Juice); 5.68 (Fresh Juice); 2 C5H10O4 134 26446-35-5 Acetyl monoglyceride / Glycerinmonoacetate / Glycerol 10.200 (Sonication); 2.37 (Sonication); 10.217 (Soxhlet) 4.22 (Soxhlet)
2,4,5-Trihydroxypyrimidine / Isobarbituric acid / 2,4,5(3H)- 3 C4H4N2O3 128 13.950 (Maceration) 0.15 (Maceration) 496-76-4 Pyrimidinetrione, dihydro- / Dihydro-2,4,5(3H)-pyrimidinetrione 4 Cyclopropane, nonyl- / Nonylcyclopropane C12H24 Malaya168 14.208 (Maceration) 14.208 (Maceration) 74663-85-7 22.658 (Maceration); 0.99 (Maceration) 5 6-methyl 1-Heptanol / 6-Methyl-1-heptanol C8H18O 130 1653-40-3 22.633 (Fresh juice) 1.03 (Fresh Juice)
6 1,2,3-Trimethyldiaziridine ofC4H10N2 86 26.550 (Maceration) 0.28 (Maceration) 113604-56-1 Benzene-1,4-dicarboxylic acid, monohydrazide, methyl ester / Methyl 4- 7 C9H10N2O3 194 26.825 (Maceration) 2.12 (Maceration) 13188-55-1 (hydrazinocarbonyl)benzoate
27.200 (Maceration); 8.10 (Maceration); Phenol, 2,4-bis(1,1-dimethylethyl)- / Phenol, 2,4-di-tert-butyl- / 2,4-Di-tert- 8 C14H22O 206 27.175 (Fresh juice); 9.47 (Fresh Juice); 96-76-4 butylphenol / 2,4-di-t-Butylphenol 27.158 (Sonication) 7.65 (Sonication)
30.717 (Maceration); 0.30 (Maceration); 9 1,1,3-trimethyl Cyclopentane / 1,1,3-Trimethylcyclopentane C8H16 112 14.167 (Sonication); 0.56 (Sonication); 4516-69-2 22.642 (Soxhlet) 0.70 (Soxhlet)
39.117 (Maceration); 3.20 (Maceration); Hexadecanoic acid, methyl ester / Palmitic acid, methyl ester / n- 42.458 (Fresh juice); 6.76 (Fresh Juice); 112-39-0 10 C17H34O2 270 Hexadecanoic acid methylUniversity ester / Metholene 2216 / Methyl hexadecanoate 42.458 (Sonication); 7.65 (Sonication); 42.483 (Soxhlet) 6.23 (Soxhlet)
80 67
Elemental Molecular No Compound name RT(min.) Area (%) CAS composition weight
Hexadecanoic acid, methyl ester / Palmitic acid, methyl ester / n- 11 C17H34O2 270 42.500 (Maceration) 4.70 (Maceration) 112-39-0 Hexadecanoic acid methyl ester / Metholene 2216 / Methyl hexadecanoate
47.633 (Maceration); 27.52 (Maceration); Octadecanoic acid, methyl ester / Stearic acid, methyl ester / n- 48.792 (Fresh juice); 55.62 (Fresh Juice); 12 C19H38O2 298 112-61-8 Octadecanoic acid, methyl ester / Kemester 9718 / Methyl n-octadecanoate 48.800 (Sonication); 66.81(Sonication) 48.825 (Soxhlet) 54.39 (Soxhlet)
Octadecanoic acid, methyl ester / Stearic acid, methyl ester / n- 13 C19H38O2 298 48.833 (Maceration) 39.87 (Maceration) 112-61-8 Octadecanoic acid, methyl ester / Kemester 9718 / Methyl n-octadecanoate
Glycerin / 1,2,3-Propanetriol / Glycerol / Glycerine / Glyceritol / Glycyl 9.658 (Fresh juice); 4.73 (Fresh Juice); 14 C3H8O3 92 56-81-5 alcohol / Glyrol / Glysanin / Osmoglyn / Propanetriol 9.650 (Soxhlet) 20.83 (Soxhlet)
Glycerin / 1,2,3-Propanetriol / Glycerol / Glycerine / Glyceritol / Glycyl Malaya 15 C3H8O3 92 9.742 (Fresh juice) 9.47 (Fresh Juice) 56-81-5 alcohol / Glyrol / Glysanin / Osmoglyn / Propanetriol
16 2-Dodecene, (Z)- / (2Z)-2-Dodecene ofC12H24 168 14.192 (Fresh juice) 1.18 (Fresh Juice) 7206-26-0
17 Hexane, 3,3-dimethyl- / 3,3-Dimethylhexane C8H18 114 17.650 (Fresh juice) 0.59 (Fresh Juice) 563-16-6
9.47 (Fresh Juice); Phenol, 3,5-bis(1,1-dimethylethyl)- / Phenol, 3,5-di-tert-butyl- / 3,5-Di-tert- 27.175 (Fresh juice); 18 C14H22O 206 7.24 (Soxhlet) 1138-52-9 butylphenol / Phenol, 3,5-bis(t-butyl) / 3,5-Di-t-butylphenol 27.192 (Soxhlet)
Nonane, 1-iodo- / n-Nonyl iodide / Nonyl iodide / 1-n-Nonyl iodide / 1- 17.633 (Sonication) 19 C9H19I 254 1.09 (Sonication) 4282-42-2 Iodononane
22.608 (Sonication) 20 Isooctanol / Isooctyl alcohol / Exxal 8 / 6-Methyl-1-heptanol C8H18O 130 3.51 (Sonication) 26952-21-6
Ethoxyacetaldehydediethylacetal / 1,1,2-Triethoxyethane / Ethane, 1,1,2- 6.958 (Soxhlet) 21 C8H18O3 162 0.71 (Soxhlet) 4819-77-6 triethoxy- / UniversityEthoxyacetaldehyde diethyl acetal
81 68
4.3.2 LC-MS data analysis
The constituents identified for both plants were summarized in Table 4.3 and
Table 4.4. Figure 4.6 shows the LC-MS chromatograms of aqueous extracts of S. polyanthum and M. charantia. Results revealed that as many as 53 phytoconstituents were detected in M. charantia extracts. In comparison to M. charantia, the S. polyanthum extracts had lesser, 39 phytoconstituents. Some of the constituents are specific to the method applied during the extraction.
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University Figure 4.6: LC-MS profiles of different extracts of two selected plants A. S. polyanthum extracts; B. M. charantia extracts.
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Table 4.3: List of identified phytoconstituents in the fruit aqueous extracts of M. charantia by LC-MS
Elemental Mass m/z Fresh No Compound name RT(min.) Maceration Sonication Soxhlet composition (g/mol) Juice
1 5S-HETE di-endoperoxide C20H34O8 402.2251 441.1884 1.198 (Maceration) √ X X X
2 Tyr ArgSer C18H28N6O6 402.2251 425.2155 1.202 (Maceration) √ X X X
3 11-amino-undecanoic acid C11H23NO2 201.1731 202.1805 4.162 (Maceration) √ X X X
4 Nonactin C40H64O12 736.4367 605.3661 4.263 (Maceration) √ X X X 4.266 (Maceration); 5 2-Amino-3-methyl-1-butanol C5H13NO 103.0999 104.1072 4.233 (Fresh juice); √ √ √ X 4.185 (Sonication) 4.598 (Maceration); 6 Adenine C5H5N5 135.0502 136.0614 √ X X √ 4.706 (Soxhlet) 4.693 (Maceration); 7 Anthraquinone C14H8O2 208.053 209.0603 √ √ X X 5.11 (Fresh juice) 4.704 (Maceration);Malaya Cis-1,2-Dihydroxy-1,2- 8 C12H10O2S 218.0404 219.0473 4.748 (Fresh juice); √ √ √ X dihydrodibenzothiophene 4.824 (Sonication) 4.82 (Maceration); 9 Allo-inositol C6H12O6 180.064 203.0533of 4.826 (Fresh juice); √ √ √ X 4.798 (Sonication)
10 1,3,7-Trimethyluric acid C8H10N4O3 210.075 233.0642 4.838 (Maceration) √ X X X 4.861 (Maceration); 11 Sphinganine C16H35NO2 273.2669 274.2742 4.881 (Fresh juice); √ √ X √ 4.932 (Soxhlet) 4.876 Maceration); 4.854 (Fresh juice); 12 Phytosphingosine C18H39NO3 317.2925 318.2998 √ √ √ √ 4.893 (Sonication); 4.934 (Soxhlet)
13 y-Glu-Cys C8H14N2O5S 250.0637 251.0708 5.074 (Maceration) √ X X X 5.141 (Maceration); 14 3-propylmalic acid C7H12O5 176.0687 199.0579 √ √ X X 5.127(Fresh juice) 5.244 (Maceration); 15 δ-Valerolactam C5H9NO 99.0685 100.0757 5.235 (Fresh juice); √ √ X √ University 5.254 (Soxhlet)
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83 Elemental Mass m/z Fresh No Compound name RT(min.) Maceration Sonication Soxhlet composition (g/mol) Juice a-Methyl-3,4- 5.361 (Maceration); 16 C10H12O4 196.0739 219.0631 √ X √ X dihydroxyphenylpropionic 5.319(Sonication)
17 DiallylTrisulfide C6H10S3 177.9941 179.0013 5.777 (Maceration) √ X X X 6.339 (Maceration); 18 Burseran C22H26O6 386.1736 409.163 6.335 (Fresh juice); √ √ √ X 6.282 (Sonication) 6.492 (Maceration); 19 Apiole C12H14O4 222.0897 245.079 6.491 (Fresh juice); √ √ X √ 6.54 (Soxhlet) 7.283 (Maceration); Estra-1,3,5(10)-triene- 20 C24H30O6 414.2045 437.1938 7.273 (Fresh juice); √ √ X √ 3,6alpha,17beta-triol triacetate 7.373 (Soxhlet) 7.449 (Maceration); Catechin 3-O-(1-hydroxy-6-oxo-2- 21 C22H20O9 428.1099 451.101 7.415 (Fresh juice); √ √ √ X cyclohexene-1-carboxylate) 7.325 (Sonication)Malaya Apigenin 7-(3”-acetyl-6”-E-p- 22 C32H28O13 620.153 643.1424 11.009 (Maceration) √ X X X coumaroylglucoside) of12.924 (Maceration); 13.006 (Fresh juice); 23 Proanthocyanidin A2 C30H24O12 576.1272 599.1172 √ √ √ √ 12.515 (Sonication); 13.779 (Soxhlet) 9.202 (Fresh juice); 24 Elaidamide C18H35NO 281.272 304.2614 9.198 (Sonication); X √ √ √ 8.027 (Soxhlet) 4.564 (Fresh juice); 25 p-Aminobenzoic acid C7H7NO2 137.0479 138.0547 X √ X √ 4.779 (Soxhlet) 4.841 (Fresh juice); 26 Alpha-D-Mannoheptulopyranose C7H14O7 210.0748 233.0641 4.842 (Sonication); X √ √ √ 4.896 (Soxhlet)
27 1-Deoxy-D-xylulose C5H10O4 134.0583 157.0475 5.031 (Fresh juice) X √ X X
28 Tert-Butylbicyclophosphorothionat C8H15O3PS 222.0475 219.033 5.773 (Fresh juice) X √ X X
29 Soyasaponin III C42H68O14 796.4597 819.4493 5.81 (Fresh juice) X √ X X 9S-hydroxy-12R,13S-epoxy- 30 C18H30O4 310.2142 333.2034 7.066 (Fresh juice) X √ X X 10E,15Z-octadecadienoic acid University
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84 Elemental Mass m/z Fresh No Compound name RT(min.) Maceration Sonication Soxhlet composition (g/mol) Juice 7.273 (Fresh juice); 31 Eplerenone C24H30O6 414.2044 437.1937 X √ X √ 7.377 (Soxhlet) 279.257 32 Linoleamide C18H33NO 302.2463 0.599 (Sonication) X X √ X
24-Nor-5β-cholane-3α,12α,22,23- 33 C23H40O4 380.2912 381.2985 3.24 (Sonication) X X √ X tetrol 4.565 (Sonication); 34 Palmitic amide C16H33NO 255.2566 278.2459 X X √ √ 3.368 (Soxhlet) 7.326 (Sonication); 36 Exserohilone C20H22N2O6S2 450.094 451.1012 X X √ √ 7.534 (Soxhlet) 14,14,14-Trifluoro-11Z-tetradecenyl 37 C16H27F3O2 308.1968 309.2042 0.3 (Soxhlet) X X X √ acetate
38 gamma-Pentachlorocyclohexene C6H5Cl5 251.8844 274.8734 4.406 (Soxhlet) X X X √
39 Cycloleucine C6H11NO2 129.0794 130.0867 4.527 (Soxhlet) X X X √
40 5-Aminopentanoic acid C5H11NO2 117.079 118.0863 4.761 (Soxhlet)Malaya X X X √
41 Derrone C20H16O5 336.1009 337.1081 4.792 (Soxhlet) X X X √
42 Sophoracoumestan A C20H14O5 334.0856 335.0923 4.798 (Soxhlet) X X X √ 43 Erythrinin A C20H16O4 320.106 321.1132of 4.831 (Soxhlet) X X X √ 44 Neuraminic acid C9H17NO8 267.0953 268.1025 4.833 (Soxhlet) X X X √
45 L-Galactose C6H12O6 180.0636 203.0529 4.887 (Soxhlet) X X X √
46 Benserazide C10H15N3O5 257.1016 258.1087 4.892 (Soxhlet) X X X √
47 Corynebactin C39H42N6O18 882.2566 905.246 4.919 (Soxhlet) X X X √
48 Quebrachitol C7H14O6 194.0793 217.0685 4.95 (Soxhlet) X X X √
49 Xestoaminol C C14H31NO 229.2408 230.2481 4.954 (Soxhlet) X X X √
50 Linamarin C10H17NO6 247.1063 270.0955 4.958 (Soxhlet) X X X √ (2S,3S)-2,3-Dihydro-2,3- 51 C7H8O4 156.0418 157.0491 5.052 (Soxhlet) X X X √ dihydroxybenzoate
52 p-Acetaminobenzoic acid C9H9NO3 179.0594 180.0661 5.078 (Soxhlet) X X X √
53 (+)-Eudesmin C22H26O6 386.1734 409.1627 6.398 (Soxhlet) X X X √ Note: x = Not detected, √ = DetectedUniversity
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Table 4.4: List of identified phytoconstituents in the leaf aqueous extracts of S. polyanthum by LC-MS
Elemental Mass Fresh No Compound name m/z RT(min.) Maceration Sonication Soxhlet composition (g/mol) Juice
1 Stearamide C18H37NO 283.2875 306.2768 3.387 (Maceration) √ X X X 3.675 (Maceration); 2 Palmitic amide C16H33NO 255.256 278.2452 √ √ X X 3.597 (Fresh Juice)
4.183 (Maceration); 3 2-Amino-3-methyl-1-butanol C5H13NO 103.0996 104.1069 4.411 (Sonication); √ X √ √ 4.188 (Soxhlet) 4.626 (Maceration); 4 5-Aminopentanoic acid C5H11NO2 117.0788 140.068 √ X √ X 4.732 (Sonication) 4.8 (Maceration); 5 Theobromine C7H8N4O2 180.0643 203.0535 4.837 (Sonication); √ X √ √ 4.807 (Soxhlet) 4.842 (Maceration);Malaya 4.828 (Fresh Juice); 6 Sphinganine C16H35NO2 273.2667 274.274 √ √ √ X 4.864 (Sonication); 4.834 (Soxhlet) of5.001 (Maceration); 7 1-Deoxy-D-xylulose C5H10O4 134.058 157.0473 5.003 (Fresh Juice); √ √ √ X 5.087 (Sonication) 5.002 (Maceration); 8 Karanjin C18H12O4 292.0736 293.0809 √ √ X X 5.008 (Fresh Juice) 5.107 (Maceration); 9 3-propylmalic acid C7H12O5 176.0687 199.058 √ √ X X 5.108 (Fresh Juice) 5.312 (Maceration); a-Methyl-3,4- 10 C10H12O4 196.0738 219.063 5.321 (Fresh Juice); √ √ √ X dihydroxyphenylpropionic acid 5.408 (Sonication) 6.257 (Maceration); 11 (+)-Eudesmin C22H26O6 386.1734 409.1628 √ X X √ 6.269 (Soxhlet) 6.257 (Maceration); 12 Adifoline C22H20N2O7 424.1284 425.1362 √ √ X X 6.272 (Fresh Juice) 6.43 (Maceration); 6.442 (Fresh Juice); 13 Apiole UniversityC12H14O4 222.0896 245.0789 √ √ √ √ 6.52 (Sonication); 6.445 (Soxhlet) 86 86
Elemental Mass Fresh No Compound name m/z RT(min.) Maceration Sonication Soxhlet composition (g/mol) Juice 7.29 (Maceration); Exserohilone C20H22N2O6S2 450.094 451.1011 7.299 (Fresh Juice); √ √ X √ 7.305 (Soxhlet)
15 Elaidamide C18H35NO 281.2719 304.2619 7.751 (Maceration) √ X X X 12.343 (Maceration); 16 Proanthocyanidin A2 C30H24O12 576.1282 599.1174 12.372 (Fresh Juice); √ √ √ X 13.603 (Sonication)
17 Allo-Inositol C6H12O6 180.0641 203.0534 4.807 (Fresh Juice) X √ X X
18 Tiaprofenic acid C14H12O3S 260.0514 261.0586 4.89 (Fresh Juice) X √ X X
19 Anthraquinone C14H8O2 208.0526 209.0607 5.075 (Fresh Juice) X √ X X 6.272 (Maceration) 20 Burseran C22H26O6 386.1722 425.1362 X √ √ X 6.368 (Sonication)
21 2,8-Dihydroxyadenine C5H5N5O2 167.043 168.0509 3.993 (Sonication) X X √ X
22 Linamarin C10H17NO6 247.1048 248.1122 4.202 (Sonication) X X √ X 23 Perindoprilat C17H28N2O5 340.2008 341.2086 4.324(Sonication)Malaya X X √ X 24 D-Glucoheptose C7H14O7 210.0739 329.3161 4.843 (Sonication) X X √ X
25 Phytosphingosine C18H39NO3 317.2927 318.3001 4.862 (Sonication) X X √ X 26 Phenazine-1,6-dicarboxylic acid C14H8N2O4 268.0482 269.0556 of5.119 (Sonication) X X √ X 28 δ-Valerolactam C5H9NO 99.0685 100.0758 5.275 (Sonication) X X √ X
30 DiallylTrisulfide C6H10S3 177.9946 219.0332 5.813 (Sonication) X X √ X Catechin 3-O-(1-hydroxy-6- 31 oxo-2-cyclohexene-1- C22H20O9 428.1112 451.1006 7.506 (Sonication) X X √ X carboxylate) Apigenin 7-(3''acetyl-6''-E-p- 32 C32H28O13 620.1517 643.1419 11.373 (Sonication) X X √ X coumaroylglucoside)
33 3,4-Dihydroxybenxylamine C7H9NO2 139.0637 140.071 4.643 (Soxhlet) X X X √
34 GlnCys Asp C12H20N4O7S 364.1061 365.1134 4.689 (Soxhlet) X X X √
36 Disialyllactose C34H56N2O27 924.3043 947.2937 5.025 (Soxhlet) X X X √
37 Aminofurantoin C8H8N4O3 208.0587 109.066 5.08 (Soxhlet) X X X √
38 Syringic acid C9H10O5 198.0532 199.0602 5.115 (Soxhlet) X X X √
39 Salvianolic acid C26H22O10 494.1188 495.1263 7.102 (Soxhlet) X X X √ Note: x = Not detected, √ = DetectedUniversity
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4.4 Antioxidant activities of the extracts
4.4.1 DPPH radical scavenging activity (RSA) of the extracts
4.4.1.1 DPPH-RSA for M. charantia single extracts
The DPPH-RSA test was performed for the M. charantia extracts as shown in
Table 3.1, and as per the procedure described in section 3.2.4. The decrease in absorbance of DPPH solution at 517 nm implies the reduction of DPPH to yellow coloured DPPH which is initiated by antioxidants. Quercetin was used as a standard for this assay as it has highest DPPH scavenging potential among flavonoids in view of its phenolic structure. The DPPH scavenging potential for the extracts of M. charantia, differed based on the method used for extraction and the % inhibition of DPPH free radical by each extract ranged between 8.53 to 19.76 %. The order of activity of the extracts was soxhlet (19.76 %) > fresh juice (14.19Malaya %) > maceration (9.16 %) > sonication (8.53 %). The positive control (quercetin) had 69.21 % DPPH radical inhibition. The data was analysed by one ofway ANOVA test followed by LSD Test for multiple comparisons. The results suggest a significant difference (p < 0.001) in the
DPPH radical scavenging activity of the four extracts indicating the influence of extraction method on the inhibition. It is evident that extract obtained by soxhlet method had highest DPPH radical scavenging potential than its counterparts. Thus, soxhlet extract was significantly different from fresh juice (p < 0.01), maceration (p < 0.001) and sonication (p < 0.001) Therefore, sonication method was ineffective in extracting hydrophilicUniversity antioxidants from the plant material when compared to the other extraction methods tested as far as M. charantia is concerned.
4.4.1.2 DPPH-RSA for S. polyanthum single extracts
All the extracts of S. polyanthum exhibited remarkable DPPH radical scavenging activity and superior antioxidant power than M. charantia. The DPPH free radical
88 scavenging, inhibitory activities of M. charantia extracts were relatively less when compared to S. polyanthum extracts. The DPPH assay conducted on S. polyanthum extracts. The antioxidant inhibitory activities of the extracts were between 58.03 to
64.93 %. Current study supported that S. polyanthum leaves have abundant hydrophilic antioxidant molecules which were extracted successfully that might have reacted intensively with that of DPPH radicals to scavenge them in a significant manner. The order of activity of the extracts was sonication (64.93 %) > maceration (64.18 %) > soxhlet (63.15 %) > fresh juice (58.03 %). Among the four extracts tested maceration, soxhlet and sonication were statistically similar and the difference between them was insignificant (p > 0.05). However, fresh juice extract was significantly different from the other three extracts (p < 0.05). This has suggested that irrespective of method of extraction applied to S. polyanthum the extracts possessedMalaya similar concentrations of hydrophilic antioxidants except fresh juice, that might have shown scavenging potential against DPPH free radicals. However, the ofscavenging efficiency was not comparable to that of standard quercetin as the difference was significant (p < 0.01).
4.4.1.3 DPPH-RSA for M. charantia and S. polyanthum combination extracts
As many as 28 combinations of 8 extracts (Four from M. charantia and 4 from
S. polyanthum) as shown in Table no. 3.1 were tested for DPPH free radical scavenging activity to estimate the synergistic effects of the extracts. Initially combination within theUniversity plant extracts was carried out and later between the two different plant extracts was performed.
Six within combinations of M. charantia extracts were used and the overall
DPPH radical inhibitory ability of them was ranged between 5.49 to 18.37 %. The order of activity of the extracts was Fresh Juice MC-Soxhlet MC (18.37 %) > Sonication MC-
Soxhlet MC (14.17 %) > Fresh Juice MC-Sonication MC (11.3 %) > Maceration MC-
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Fresh juice MC (11.16 %) > Maceration MC-Soxhlet MC (10.88 %) > Maceration MC-
Sonication MC (5.49 %). Despite the combination of extracts there was no significant increase in the DPPH scavenging activities rather decreased in comparison to single extracts tested. This suggests that hydrophilic antioxidants were reduced to a greater extent upon pooling the extracts. Like M. charantia six within combinations of S. polyanthum were also tested and as expected the results were similar to the individual extracts tested with an inhibition ranging from 61.7 to 64.67 %. The order of activity of the extracts was Sonication SP-Soxhlet SP (64.67 %) > Fresh Juice SP-Sonication SP
(64.57 %) > Fresh Juice SP-Soxhlet SP (64.35 %) > Maceration SP-Soxhlet SP (63.79
%) > Maceration SP-Fresh juice SP (62.25 %) > Maceration SP-Sonication SP (61.7
%). This difference between highest and lowest is marginal, nevertheless these combinations of S. polyanthum were far superior to MalayaM. charantia in their DPPH radical scavenging efficiency. However, “Maceration SP-Sonication SP” and “Maceration SP- Fresh juice SP” scavenging were statisticallyof dissimilar (p < 0.05) with the rest of the combinations.
Sixteen between combinations of M. charantia and S. polyanthum were tested to evaluate the presence and absence of synergism in their DPPH radical scavenging effects. In this combination study 10 µg/mL of S. polyanthum and 10 µg/mL of M. charantia extracts were combined to match with individual extract concentration of 20
µg/mL, to evaluate a meaningful comparison during the analysis of the results. The valuesUniversity ranged from 58.4 to 65.35 %, the highest being 65.35 % demonstrated by “Maceration MC-Sonication SP” and the lowest expressed by “Maceration MC-
Maceration SP”. Interestingly, despite only half the concentration (10 µg/mL) of S. polyanthum in the tested samples the scavenging potential was not varied much but had negligible differences to that of single extracts tested which actually had double the concentration (20 µg/mL). This brings us to the conclusion that presence of half the
90 concentration (10 µg/mL) of M. charantia in combinations has potentiated the effect of
S. polyanthum extracts despite lower in concentration. Hence, it can be assumed that different plant extract combinations exhibit synergistic DPPH radical scavenging power. As shown in Figure 4.7 the 65.35 % inhibition of “Maceration MC-Sonication
SP” was statistically different from that of standard quercetin (69.21 %) and other combinations (p < 0.05). The combinations exhibiting similar activity were depicted with similar alphabetical letter, the values ranged between 5.49 to 65.35 %. The results are presented in Figure 4.7.
Malaya
of
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g 80 f f f 69.21 70 64.18 e 64.93 63.15 58.03 A 60 50 40 Momordica charantia d c 30 19.76 Syzygium polyanthum % Inhibition b b 20 14.19 9.16 8.53 Quercetin 10 0 Maceration Fresh Juice Sonication Soxhlet Quercetin Treatments of extraction (20 µg/mL)
Quercetin 69.21 h Soxhlet MC-Soxhlet SP 62.3 e Soxhlet MC-Sonication SP 64.12 f Soxhlet MC-Fresh Juice SP 61.81 e Soxhlet MC-Maceration SP 62.13 e
Sonication MC-Soxhlet SP 63.26 f
SP Sonication MC-Sonication SP - 65.13 f Sonication MC-Fresh Juice SP 62.88 f
MC B Sonication MC-Maceration SP 63.67 f Fresh Juice MC-Soxhlet SP 61.74 e Fresh Juice MC-Sonication SP Between Combination 63.69 f Fresh Juice MC-Fresh Juice SP Malaya61.07 e Fresh Juice MC-Maceration SP 61.72 e
Maceration MC-Soxhlet SP 63.84 f Maceration MC-Sonication SP 65.35 g Maceration MC-Fresh Juice SP of 61.49 e Maceration MC-Maceration SP 58.4 e
(20 µg/mL) Sonication SP - Soxhlet SP 64.67 f
Fresh Juice SP - Soxhlet SP 64.35 f Fresh Juice SP - Sonication SP 64.57 f Maceration SP - Soxhlet SP 63.79 f Maceration SP - Sonication SP 61.7 e Within Within Combination SP Maceration SP - Fresh Juice SP 62.25 e Sonication MC - Soxhlet MC 14.17 c Fresh Juice MC – Soxhlet MC 18.37 d Fresh Juice MC - Sonication MC 11.3 c Maceration MC - Soxhlet MC 10.88 c Maceration MC - Sonication MC 5.49 b Within Within Maceration MC – Fresh Juice MC 11.16 c Blank 0 Combination MC University-10 0 10 20 30 40 50 60 70 80 % Inhibition
Figure 4.7: DPPH radical scavenging activity of different aqueous extracts of M. charantia and S. polyanthum, A. Single extracts; B. Combination extracts, data was presented as percent DPPH inhibition, mean ± SD (n=3) data was analysed according to one way ANOVA and LSD multiple comparison test, same alphabetical letters denote non-significant difference at p < 0.05.
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4.4.2 FRAP activity of the extracts
4.4.2.1 FRAP activity of M. charantia single extracts
The FRAP test was performed in a microplate for the test samples given in
Table 3.1 and as per the procedure described in section 3.2.4. Quercetin was used as a positive standard for this assay. The FRAP activity for single extracts, The FRAP inhibitory values of M. charantia were in between 3.12 to 4.19 %. The order of activity of the extracts was soxhlet (4.19 %) > maceration (3.40 %) > sonication (3.30 %) > fresh juice (3.12 %). ANOVA test between all extracts were significantly lower (p <
0.05). The statistical analysis of the data revealed that the difference in activities of extracts obtained from different methods was insignificant (p > 0.05). This suggests that
M. charantia extracts, irrespective of the method of extraction were identical in showing FRAP activity and was negligible as it was significantlyMalaya similar to blank tested.
4.4.2.2 FRAP activity of S. polyanthum singleof extracts The ferric reducing antioxidant power (FRAP) for extracts of S. polyanthum were in the order of fresh juice (69.05 %) > sonication (29.24 %) > soxhlet (28.21 %) > maceration (17.56 %). Interestingly the % inhibition of fresh juice of S. polyanthum was better than the positive control, quercetin (63.27 %) and it was statistically significant (p
< 0.05). The method of extraction had significant difference (p < 0.001) in extracting the electron donating phytoconstituents that are capable of eliciting FRAP activity. In contraryUniversity to the fresh juice (69.05 %) maceration method of extraction had significantly less (p < 0.001) ferric reducing antioxidant power and it was the least among the extracts tested. However, extracts obtained by sonication and soxhlet were statistically similar (p > 0.05). Our study has supported the antioxidant potential of S. polyanthum extracts over M. charantia extracts. But, contrary to the DPPH activity results, FRAP activity results of S. polyanthum had influence of extraction method. Similar results
93 were also noticed with M. charantia extracts in DPPH assay though not as intense as S. polyanthum samples. This can be ascribed to the method chosen for extraction that might have influenced the concentrations of proton donating and electron donating antioxidant compounds extracted differently in aqueous plant extracts. Hence, fresh juice of S. polyanthum demonstrated highest FRAP activity, in contrary to the highest
DPPH scavenging activity by sonication extract. Therefore, determine of the best extraction method is prerequisite before herbal formulations to reduce the disparity in the therapeutic efficacy.
4.4.2.3 FRAP activity for M. charantia and S. polyanthum combination extracts
Similar 28 combinations of 8 extracts (4 from M. charantia and 4 from S. polyanthum) as shown in Table 3.1 were tested Malaya for FRAP antioxidant activity to estimate the synergistic effects of the extracts like in DPPH assay. To understand the pattern of combination effect on FRAP, firstof within the plants extracts were tested and later tests were carried out between the two different plant extract combinations.
Six within combinations of M. charantia extracts showed insignificant FRAP activities ranged between 2.97 to 5.36 %. The order of activity of the extracts was Fresh
Juice MC-Soxhlet MC (5.36 %) > Sonication MC-Soxhlet MC (4.95 %) > Maceration
MC-Soxhlet MC (4.44 %) > Maceration MC-Fresh juice MC (4.15 %) > Maceration
MC-Sonication MC (3.55 %) > Fresh juice MC-Sonication MC (2.97 %). It was observedUniversity that FRAP activity of combined M. charantia extracts were akin to individual extracts and the FRAP values were not worth considering. The FRAP values of S. polyanthum within combinations were as presented in Figure 4.8. The extracts of S. polyanthum exhibited the highest FRAP values and interestingly fresh juice containing combination extracts were superior in their activity. The order of activity of the extracts was Fresh Juice SP-Sonication SP (68.12 %) > Maceration SP-Fresh Juice SP (66.6 %)
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> Fresh Juice SP-Soxhlet SP (63.69 %) > Sonication SP-Soxhlet SP (36.07 %) >
Maceration SP-Soxhlet SP (27.56%) > Maceration SP-Sonication SP (23.81 %). Upon analysing the data by LSD multiple comparison test, it was found that fresh juice containing juices among the above combinations were statistically similar (p > 0.05) and they were with statistically higher (p < 0.05) ferric reducing power than positive control, quercetin (63.27 %).
Sixteen between combinations of M. charantia and S. polyanthum were tested to know the synergism in FRAP activity. The values ranged from 14.54 to 59.16 %, the highest was 59.16 % expressed by “Soxhlet MC-Fresh Juice SP” and the lowest was observed in “Maceration MC-Maceration SP”. In between combination results
“Maceration MC-Maceration SP” was found to be least antioxidant in both DPPH and FRAP assays indicating its poor radical scavengingMalaya and ferric reducing effects. Out of sixteen combinations only four combinations were closely comparable to positive control, statistically all the four were similarof and significantly lower than standard (p < 0.05), only fresh juice of S. polyanthum containing extracts were consistently produced superior activities, in contrary fresh juice extracts of M. charantia were ineffective in showing reducing capacity. The top four extracts among the tested combinations were
Soxhlet MC-Fresh juice SP (59.16 %) > Fresh juice MC-Fresh juice SP (57.96 %) >
Sonication MC-Fresh juice SP (54.45 %) > Maceration MC-Fresh juice SP (53.71 %).
The results were presented in Figure 4.8. University
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90 e d 80 69.05 A 63.27 70 60 50 c c Momordica charantia 40 29.24 28.21 b Syzygium polyanthum
% Inhibition 30 17.56 20 a a a Quercetin 10 3.40 3.12 3.30 4.19 0 Maceration Fresh Juice Sonication Soxhlet Quercetin Treatments of extraction ( 10 µg/mL)
Quercetin 63.27 i Soxhlet MC-Soxhlet SP 15.49 b Soxhlet MC-Sonication SP 22.77 b Soxhlet MC-Fresh Juice SP 59.16 h Soxhlet MC-Maceration SP 19.09 b Sonication MC-Soxhlet SP 25.57 e Sonication MC-Sonication SP 24.15 e SP - Sonication MC-Fresh Juice SP 54.45 h
MC Sonication MC-Maceration SP 15.92 b B Fresh Juice MC-Soxhlet SP 25.93Malaya e Fresh Juice MC-Sonication SP 15.77 b Between Combination Fresh Juice MC-Fresh Juice SP 57.96 h
Fresh Juice MC-Maceration SP 14.93 b Maceration MC-Soxhlet SP of23.16 c
Maceration MC-Sonication SP 22.4 b Maceration MC-Fresh Juice SP 53.71 h Maceration MC-Maceration SP 14.54 b Sonication SP - Soxhlet SP 36.07 g Fresh Juice SP - Soxhlet SP 63.69 i
( 10 µg/mL) ( 10 µg/mL) Fresh Juice SP - Sonication SP 68.12 k Maceration SP - Soxhlet SP 27.56 f
Within Combination SP Combination Within Maceration SP - Sonication SP 23.81 d Maceration SP - Fresh Juice SP 66.6 j Sonication MC - Soxhlet MC
4.95 a Fresh Juice MC – Soxhlet MC 5.36 a
Fresh Juice MC - Sonication MC 2.97 a Maceration MC - Soxhlet MC 4.44 a MC
Within Within Maceration MC - Sonication MC 3.55 a Maceration MC – Fresh Juice MC 4.15 a Combination UniversityBlank 0 -10 0 10 20 30 40 50 60 70 80 % Inhibition
Figure 4.8: FRAP radical scavenging activity of different aqueous extracts of M. charantia and S. polyanthum, A. Single extracts; B. Combination extracts, data was presented as percent FRAP inhibition, mean ± SD (n=3) data was analysed according to one way ANOVA and LSD multiple comparison test, same alphabetical letters denote non-significant difference at p < 0.05.
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4.5 Antidiabetic enzyme inhibitory activity of the extracts
4.5.1 In vitro α-amylase inhibitory activity of the extracts
4.5.1.1 In vitro α-amylase inhibitory activity of M. charantia single extracts
First and foremost, four individual extracts of M. charantia were obtained by different extraction methods were tested for α-amylase inhibitory activity. The data was analysed by one way ANOVA, a significant difference (p < 0.05) was observed in α- amylase inhibitions between the four different extracts. Thus, the method of extraction applied had significant impact in extracting constituents responsible for enzyme inhibitory activity. The fresh juice of M. charantia exhibited highest α-amylase inhibition of 61.24 % followed by sonication (57.06 %), maceration (51.27 %) and soxhlet (43.2 %) compared with the standard, acarbose (88.51 %). Unlike DPPH, FRAP results of M. charantia, significant α-amylase enzymeMalaya inhibitory activity was noticed though not to the level of standard, acarbose. 4.5.1.2 In vitro α-amylase inhibitory activityof of S. polyanthum single extracts S. polyanthum individual extracts obtained by maceration, sonication, soxhlet and fresh juice were investigated for α-amylase inhibitory activity. The order of their activities were fresh juice (92.21 %) > sonication (80.49 %) > soxhlet (48.46 %) > maceration (34.49 %). A remarkable difference was noticed between the extracts, and corresponding α-amylase inhibitory activities, they were significantly different (p <
0.001). Surprisingly, once again fresh juice of S. polyanthum showed an activity superiorUniversity (92.21 %) to the standard, acarbose (88.51 %) and the next best was the extract derived from sonication (80.49 %). Soxhlet and maceration extracts were significantly
(p < 0.05) lower in demonstrating α-amylase inhibitory activity than others (Figure 4.9).
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4.5.1.3 In vitro α-amylase inhibitory activity of M. charantia and S. polyanthum
combination extracts
A combination of 14 extracts of M. charantia and S. polyanthum were tested to know the synergism. Unlike antioxidant assays, in antidiabetic assays within the plant extract combinations were not considered as they were inferior and hence not tested.
The α-amylase inhibitory values of the combined extracts ranged from 15.18 to 90.86
%, the highest was expressed by “Soxhlet MC-Fresh Juice SP” and the lowest was observed in “Sonication MC-Sonication SP”. Out of the fourteen combinations only three combinations were closely comparable to positive control (88.51 %), statistically they were similar to standard (p > 0.05), Soxhlet MC-Fresh Juice SP (90.86 %),
Sonication MC-Fresh juice SP (90.3 %) and Maceration MC-Fresh juice SP (87.64 %). Fresh juice of S. polyanthum consistently producedMalaya superior activities yet again this time on α-amylase. The data suggests that there is an existence of synergism among these three combinations this is becauseof, despite the half the concentrations of S. polyanthum in combinations the inhibitory values were improved significantly. The rest of the combinations were ineffective and no synergism was observed and the activities were nearly half to the inhibition of standard, acarbose. The results were presented in
Figure 4.9.
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100 e e 92.21 88.51 A 90 d 80.49 80 b c b c 48.46 70 61.24 51.27 57.06 60 b b 50 34.49 43.2 Momordica charantia 40 Syzygium polyanthum % Inhibition 30 Acarbose 20 10 0 Maceration Fresh Juice Sonication Soxhlet Acarbose Treatments of extraction (20 µg/mL)
Acarbose 88.51 h B Soxhlet MC-Sonication SP 36.95 d A Sonication MC-Soxhlet SP 35.5 c Soxhlet MC-Fresh Juice SP Malaya 90.86 h
µg/mL) Maceration MC-Soxhlet SP 23.21 b Sonication MC-Fresh Juice SP of 90.3 h Fresh Juice MC-Soxhlet SP 59.92 g Maceration MC-Sonication SP 24.59 b Fresh Juice MC-Sonication SP 41.46 e Soxhlet MC-Soxhlet SP 33.41 c Fresh Juice MC-Maceration SP 47.15 f Sonication MC-Sonication SP 15.18 b Sonication MC-Maceration SP 26.53 b Soxhlet MC-Maceration SP 28.28 c Treatmentsof extract combination(20 Maceration MC-Fresh Juice SP 87.64 h Blank 0 University-20 0 20 40 60 80 100 120 % Inhibition
Figure 4.9: α-amylase inhibitory effects of different aqueous extracts of M. charantia and S. polyanthum, A. Single extracts; B. Combination extracts, data was presented as percent α-amylase inhibition, mean ± SD (n=3) data was analysed according to one way ANOVA and LSD multiple comparison test, same alphabetical letters denote non- significant difference at p < 0.05.
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4.5.2 In vitro α-glucosidase inhibitory activity of extracts
4.5.2.1 In vitro α-glucosidase inhibitory activity of M. charantia single extracts
The α-glucosidase inhibitory values of the extracts ranged between 16.47 to
21.77 % and the standard, acarbose had 32.22 %. All four different extracts of M. charantia possessed statistically similar α–glucosidase inhibitory activity (p > 0.05).
The extract produced by maceration showed highest 21.77 % inhibition in comparison to the sonication extract that had the least 16.47 %. Therefore, maceration extract has the highest α-glucosidase enzyme inhibitory activity among the extracts. However, all four of them were significantly lower in showing the inhibition in comparison to standard, acarbose (p < 0.05).
4.5.2.2 In vitro α-glucosidase inhibitory activity of S. polyanthum single extracts In contrary to M. charantia, the results of αMalaya-glucosidase inhibitory effects of S. polyanthum were highly significant (p < 0.001). A threefold higher activity was observed with fresh juice (96.06 %) thanof acarbose (32.22 %), the fresh juice of S. polyanthum exhibited excellent α-glucosidase inhibitory activity. Extract obtained from sonication also had two fold α-glucosidase inhibitory activity than acarbose. The % inhibitory activities were in between 16.57 to 96.06 % and the decreasing order of their activities were, fresh juice (96.06 %) > sonication (64.74 %) > soxhlet (18.16 %) > maceration (16.57 %). The fresh juice of S. polyanthum was more effective than others, activities of the extracts found to be significantly different from one another (p < 0.001), henceUniversity it is established that method of extraction has significant impact on antidiabetic activities.
4.5.2.3 In vitro α-glucosidase inhibitory activity of M. charantia and S. polyanthum
combination extracts
Like in α-amylase inhibitory study a combination of 14 extracts of M. charantia and S. polyanthum were tested to know the synergism against α-glucosidase. The α-
100 glucosidase inhibitory values of the combined extracts ranged from 11.49 to 95.52 %, the highest was expressed by “soxhlet MC-fresh juice SP” and the lowest was observed in “sonication MC-soxhlet SP”. Out of the fourteen combinations remarkably five combinations were significantly (p < 0.05) higher than positive control (32.22 %), three among them exhibited nearly threefold higher activity than acarbose, soxhlet MC-fresh juice SP (p < 0.001; 95.52 %), sonication MC-fresh juice SP (p < 0.001; 93.99 %) and maceration MC-fresh juice SP (p < 0.001; 87.29 %). The data suggests that there is an existence of synergism among these three combinations. The same above three combinations also possessed strong α-amylase inhibitory effects, all had one thing common in them that is having 50 % of extract derived from fresh juice. Fresh juice of
S. polyanthum consistently produced superior enzyme inhibitory antidiabetic activities both individually and in combination. However, sonicationMalaya extract of S. polyanthum in combinations had reduced inhibitory values and this reduction is in the following order, soxhlet MC-sonication SP (51.46 %) > ofsonication MC-sonication SP (45.23 %) > maceration MC-sonication SP (18.03 %) > fresh juice MC-sonication SP (16.11 %).
The rest of the combinations were ineffective and no synergism was observed and the activities were marginal. The results were presented in Figure 4.10.
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120 e A 100 96.06
80 d 64.74 60 Momordica charantia c Syzygium polyanthum % Inhibition 40 b 32.22 b b b b b 21.77 Acarbose 16.57 18.63 16.47 19.78 18.16 20
0 Maceration Fresh Juice Sonication Soxhlet Acarbose Teatments of extraction (40 µg/mL)
Acarbose 32.22 d B
Soxhlet MC-Sonication SP 51.46 e
Sonication MC-Soxhlet SP 11.49 b
Soxhlet MC-Fresh Juice SP 95.52 f µg/mL) Maceration MC-Soxhlet SP 25.04 c Malaya Sonication MC-Fresh Juice SP 93.99 f Fresh Juice MC-Soxhlet SP 14.87 b of Maceration MC-Sonication SP 18.03 b
Fresh Juice MC-Sonication SP 16.11 b
Soxhlet MC-Soxhlet SP 31.36 d
Fresh Juice MC-Maceration SP 16.19 b
Sonication MC-Sonication SP 45.23 e Treatmentsof extract combination(40 Sonication MC-Maceration SP 14.28 b
Soxhlet MC-Maceration SP 29.64 d
Maceration MC-Fresh Juice SP 87.29 f
Blank 0 University-20 0 20 40 60 80 100 120 % Inhibition
Figure 4.10: α-glucosidase inhibitory effects of different aqueous extracts of M. charantia and S. polyanthum, A. Single extracts; B. Combination extracts, data was presented as percent α-glucosidase inhibition, mean ± SD (n=3) data was analysed according to one way ANOVA and LSD multiple comparison test, same alphabetical letters denote non-significant difference at p < 0.05.
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4.6 Evaluation of herbal tablet formulation
In the current study the best extracts of the two selected plants were formulated as solid herbal formulations as described in section 3.2.6 and as per the formula given in
Table 3.2 and Figure 3.1.
The quality control tests for granules such as angle of repose, Carr’s index and
Hausner’s ratio were carried out as per the procedures described in section 3.2.6.1 and the results were presented in Table 4.6 and Figure 4.11. The results have indicated that the prepared granules had angle of repose (29.89), Carr’s index (10) and Hausner’s ratio
(1.11) which are considered as ‘Excellent’ according to USP-34 (2009). The prepared herbal tablet formulations were also evaluated for compendial and non-compendial quality tests, such as weight variation, friability, disintegration, hardness, thickness and diameter. The weight variation was within the USP Malayalimits as none of the tablet deviated from the acceptable range (± 5 % allowance for > 324 mg tablets). The hardness of the tablets was within the range of 51-62 N andof it was acceptable. For the above batch of herbal tablets the disintegration time was within the range of 10.19 to 13.15 min. and all the six tablets tested in disintegration apparatus (Electrolab ED-2 SAPO) disintegrated from the basket rack assembly within 15 min. Therefore, the disintegration test for the prepared formulations was considered passed. The herbal formulations prepared were tested for friability test (Erweka TAR 10 friabilator) to assess their ability to withstand shock during transportation and packaging and it was 0.72 %, the acceptable range as perUniversity USP-34 is 0.5-1 %, therefore the test was passed. Prepared batch of herbal tablet formulations were consistent in thickness and diameter as observed by low SD values.
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Table 4.5: Evaluation results of herbal tablet formulations
Observed value Parameters (Mean ± SD)
Angle of repose (Ɵ) 29.89 ± 0.16
Carr’s Index (%) 10 ± 0.57
Hausner’s ratio 1.11 ± 0.005
Weight variation (mg) 554.5 ± 1.45
Thickness (mm) 3.57 ± 0.17
Diameter (mm) 13.8 ± 0.04
Hardness (N) 56.8 ± 4.3
Friability (%) 0.72 ± 0.05
Disintegration time 10.19 to 13.15 (minutes) (RangeMalaya for six tablets) of
Figure 4.11: Prepared herbal tablet formulations of the best extracts of M. charantia and S. polyanthum. University
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CHAPTER 5: DISCUSSION
There is a rapid growth in the number of cases of type 2 diabetes globally over the last few decades. Multiple factors such as obesity, sedentary life style, genetic factors and dietary habits are a few among known that are responsible for its rise.
Hyperglycaemia, a symptom of diabetes, or a high blood glucose level, has been shown to increase reactive oxygen species and end products of oxidative damage in humans.
Most studies show that oxidative damage is increased in people with diabetes.
Endogenous antioxidants are capable of preventing oxidative damage and associated health complications, like inflammation, cardiac diseases, diabetes and cancer. Natural products in recent times were extensively studied as antioxidants, most of the natural antioxidants come from spices and herbs andMalaya are known to contain many phytoconstituents that can scavenge free radicals generated during the metabolism. These include vitamin C, vitamin E, carotenoidsof such as β-carotene and lycopene, and other phytonutrients, or substances found in fruits, vegetables such as bay leaf (S. polyanthum) having phytoconstituent eugenol (Ismail, Mohamed, Sulaiman, & Ahmad,
2013), bitter gourd (M. charantia) having phytoconstituents charantin, momordicosides and flavonoids. Hence, a detailed investigation on M. charantia and S. polyanthum was explored in this study, because in addition to ready availability, efficacy, they believed to have milder side effects than synthetic drugs (Richard & Matthew, 2009). University 5.1 Identification of selected plants for the study
The term herbal medicine is quite frequent in usage and it refers to herbs, herbal materials, herbal preparations and finished herbal products, which contain active plant parts, either as powders or as extracts, or as their combination (WHO, 2014). One of the common problems associated with natural derived products is inconsistency and
105 heterogeneity in the quality. Therefore, standardization of medicinal plants and herbal products is of paramount importance. The quality of herbal raw materials fluctuates greatly due to geographical location, soil environment and mode of collection, in addition the influence of diverse climatic conditions, their habit and habitats can alter the quality. Standardization of herbal products has been strongly recommended to overcome disparity among them in recent times. Important attributes of standardization procedure include proper authenticity and purity of the samples selected for the herbal products. It is often observed that improper identification will lead to several complications associated with them. Authenticity of the sample can be done either by macroscopy or microscopy or by chemical evaluation or by genotypic analysis.
Macroscopy can be applied to distinguish the desired herb from its common adulterant by observing the herbaceous, woody or succulent Malaya nature of the plant. While the leaf (shape, size, margin etc.), flower (simple or compound, inflorescence, arrangement of carpels and stamens, sex of the flowers etc.)of and fruit (type, dehiscence or indehiscence etc.) morphologies will be utilised to discriminate from adulterants. On the contrary microscopy is helpful to analyse the microscopical characters employing microscopes to visualize type of trichomes, epidermis, oil glands, vascular tissues, cells and cell inclusions. Therefore, detailed morphological and microscopical characters of the two selected plant species were carried out to confirm the authenticity and identity
(Lachumy & Sasidharan, 2012). The macro and microscopical characters of the two selectedUniversity species were in correlation to the existing available reference and the identity of the plants was established by their microscopical characters shown in Figure 4.1 and
Figure 4.2.
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5.2 Extraction of plant materials using different extraction methods
Extraction of plant materials is critical in isolation and purification of phytoconstituents. There are several extraction techniques, solvents and duration of extractions available in the literature with diverse yields. In the current study water was selected as a solvent of choice primarily due its association to life and dependence of humans in daily routine. Four different extraction methods were selected, maceration, soxhlet extraction, sonication and fresh juice, and plant materials were extracted as described in section 3.2.2. Among the M. charantia extracts sonication extraction produced more yield (65.93 g; 26.37 % w/w) probably by a mechanism described earlier by Chemat et al. (2017), according to this study cavitation phenomena creates surface disruption during sonication that can result in surface peeling, erosion and particle breakdown offering increased yield. The effectMalaya of sonication on the media and effects of micro mixing, macro-turbulence due to cavitation bubbles created during the process could be the key factors for the higherof yield. Similar mechanisms might have played in the extraction of M. charantia and the temperature during sonication was less than 30 oC and based on the above observations this temperature is ideal for extraction of M. charantia. Soxhlet extraction also exhibited good yield (60.63 g; 24.25 % w/w) probably by enhanced diffusiveness of the solvent into the material due to decreased viscosity and solubility induced desorption of the compounds to contribute to the effect
(Chemat et al., 2017). Low maceration and fresh juice yields also might have followed theUniversity similar mechanisms though to lesser extent. In contrary to the M. charantia extraction S. polyanthum showed varied yields ranging from 7.44 % (18.6 g) to 10.07 % (100.73 g), fresh juice being the highest
(100.73 g; 10.07 % w/w). Large 1 kg fresh sample of S. polyanthum selected for fresh juice could have been contributed greatly to its high yield. The main reason for selection of higher plant material for fresh juice was based on the assumption that 1 kg of fresh
107 sample is equivalent to dry 250 g of plant material that was used in other extraction methods. Since fresh samples were subjected to shear forces in a blender that might have enhanced solvent penetration and extraction. Among the S. polyanthum extracts maceration produced least (18.6 g; 7.44 %) in comparison to heat treated soxhlet (21.79 g) and ultrasound treated sonication (20.54 g). In maceration the material was soaked in solvent for 3 days without agitation or turbulence at 5-8 °C, due to poor solvent penetration and lack of mixing in the extraction procedure the yield obtained was very low. Though fresh juice gave higher yield during extraction interestingly the yield values of S. polyanthum and M. charantia fresh juices were significantly different (p <
0.05). It could be due to relative differences in the hardness, porosity of the plant materials during blending, particle size and also might be due to differences in surface fatty substances, polar soluble components. Therefore,Malaya it is revealed that method of extraction had an effect on the yield and subsequently different pharmacological activities. The yield values of S. polyanthumof and M. charantia obtained by different extraction methods are shown in Figure 4.3.
5.3 GC-MS and LC-MS profiling of the M. charantia and S. polyanthum extracts
In GC-MS it was noticed that heat inducing extraction methods such as soxhlet and sonication have shown less peaks indicating either destruction or evaporation of volatile components. Comparison of the mass spectra of individual components with NIST08University library data identified about 21 phytocostituents in both M. charantia and S. polyanthum (Table 4.2 & Table 4.3). Though individual extract had less number of peaks each plant possessed more than 21 compounds from four different extractions.
This suggests that individual extracts may have different volatile constituents corresponding to the extraction procedure adopted. Nevertheless, two volatile chemical compounds, phenol, 2, 4-bis (1, 1-dimethyl) and octadecanoic acid, methyl ester among
108 them were with the highest peak area percentage in both the plants suggesting their high concentrations. Content of these compounds in various extraction methods were shown in Figure 4.4 & Figure 4.5. Among them the former is reported in the literature for antifungal (Rangel, Castro, & Garcia, 2014) and the later for its antioxidant and antihyperglycaemic activity (Rahman, Ahmad, Mohamed, & Rahman, 2014).
In LC-MS several phytoconstituents were identified by their mass fragmentation patterns in comparison to the standard literature data. The identified compounds were classified as phenolic compounds (catechin 3-O-(1-hydroxy-6-oxo-2-cyclohexene-1- carboxylate, apigenin 7-(3”-acetyl-6”-E-p-coumaroylglucoside, proanthocyanidin A2, apiole), lignans (burseran), isoflavones (derrone, sophoracoumestan A), sphingolipids
(spinganine, phytosphingosine), saponins (soyasaponin), steroids (eplerenone), glycosides (linamarin), quinones (anthraquinone),Malaya aminoacids (arginine, tyrosine, cysteine), fatty acids and phenolic acids (11-amino-undecanoic acid, 9S-hydroxy- 12R,13S-epoxy-10E,15Z-octadecadienoic of acid, p-aminobenzoic acid), sugars (allo- inositol) and hormones ((+)-eudesmin). In comparison to M. charantia, the S. polyanthum extracts had lesser, 39 phytoconstituents than its counterpart which showed
53. Though many of the identified compounds are similar to M. charantia extract,
Phenolic compounds (catechin 3-O-(1-hydroxy-6-oxo-2-cyclohexene-1-carboxylate, apigenin 7-(3”-acetyl-6”-E-p-coumaroylglucoside, proanthocyanidin A2, apiole), lignans (burseran), sphingolipids (spinganine, phytosphingosine), glycosides (linamarin),University quinones (anthraquinone) and hormones ((+)-eudesmin) were common in both plants. However, few characteristic phytoconstituents were noticed in S. polyanthum, an alkaloid (adifoline), a flavonol (karanjin), a lactam (valerolactam), a phytotoxin (exserohilone), polyhydroxy sugars (1-deoxy-d-xylulose, d-glucoheptose, disialyllactose), abundant fatty acid derivatives (theobromine, stearamide, palmitic amide, salvianolic acid) and phenolic acids (5-aminopentanoic acid, 3-propylmalic acid,
109 a-methyl-3,4-dihydroxyphenylpropionic acid, tiaprofenic acid, phenazine-1,6- dicarboxylic acid, syringic acid). There was a significant difference in the peak area of the detected constituents, varied with the method of extraction in both the plant species as shown in Table 4.3 and Table 4.4 corresponding to the applied method of extraction.
5.4 Antioxidant activities of the extracts
Free radicals including ROS and reactive nitrogen species are generated in our body by various endogenous systems, exposure to different physiochemical conditions, or pathological states, and have been implicated in the pathogenesis of many diseases
(Cheng, Lin, Yu, Yang, & Lin, 2003; Slater, 1984). ROS such as, superoxide anion, hydroxyl radical, hydrogen peroxide, singlet oxygen can cause DNA damage (Halliwell & Gutteridge, 1981), protein damage (Bartold, Wiebkin,Malaya & Thonard, 1984), cellular damage by oxidation of polyunsaturated fatty acids of cell membranes (Halliwell, 1977; Comporti, 1985; Wu & Ng, 2008), classificationof of oxidative damage can be done as mild oxidative stress (MOS), temperate oxidative stress (TOS), and severe strong oxidative stress (SOS) based on its severity (Lushchak, 2014). As a defence against oxidative damage, the body normally maintains a variety of mechanisms to prevent such damage while allowing the use of oxygen for normal functions. Such “antioxidant protection” derives from sources both inside the body (endogenous) and outside the body (exogenous). Endogenous antioxidants include molecules and enzymes. ExogenousUniversity antioxidants are derived usually from food, food derived antioxidants and phytoconstituents of plants. The 1, 1-diphenyl-2-picrylhydrazine (DPPH) free radical scavenging assay is one of the most preferred antioxidant assays. The method is based on the principle of decolourization of DPPH solution by the sample and intensity of measurement of the absorbance at 517 nm. DPPH free radical reacts with compounds capable of donating hydrogen and a significant reduction in the absorbance of reaction
110 mixture is considered as significant free radical (DPPH) scavenging effect of the sample
(Krishnaiah, Sarbatly, & Nithyanandam, 2011). Earlier reports on fruit constituents of
M. charantia and M. cochinchinensis have identified flavonoids, coumarins, anthraquinones, anthocyanins and phenolic acids responsible for the antioxidant activity
(Daniel, Supe, & Roymon, 2014; Nagarani, Abirami, & Siddhuraju, 2014). Wide varieties of phenolic acids were reported in the fruits of both species, such as gallic acid, protocatechuic acid, vanillic acid, chlorogenic acid, tannic acid, caffeic acid, p-coumaric acid, p-hydroxybenzoic acid, gentisic acid, syringic acid and ferulic acids. The report further detected higher proportion of gallic acid in ripe fruits than in unripe fruits and other parts of the plant (Nagarani, Abirami, & Siddhuraju, 2014). It has been determined that antioxidant activity of M. charantia fruits is due to polyphenolic contents. The plants cultivated at different climaticMalaya conditions, soil, agricultural methods, time of harvest, post harvesting condition, storage conditions, processing parameters during extraction and type of cultivatedof sub-species often show differences in contents of phytoconstituents (Nagarani, Abirami, & Siddhuraju, 2014; Horax,
Hettiarachchy, & Chen, 2010). Probably due to the above reasons not all the constituents reported in the literature were identified in the LC-MS chromatograms of the current study. Phytoconstituents such as flavonoids, anthocyanins, anthaquinones were also identified in the current study which were reported earlier. However, flavonoids such as epicatechin, quercetin, myricetin, luteolin and kaempferol were not identifiedUniversity from the list of documented flavonoids of M. charantia. Only catechin, apigenin, proanthocyanidin and few isoflavones were detected in the current samples.
Thermal treatment was found to have important effect on M. charantia fruits, their phenolic contents and antioxidant activity of heat treated extracts than their untreated ones (Nagarani, Abirami, & Siddhuraju, 2014). DPPH scavenging ability was very low for M. charantia individual extracts it was ranged between 8.53-19.76 % (Figure 4.7).
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DPPH reacts with hydrogen radical or an electron to become stable molecule (El-Maati,
Mahgoub, Labib, Al, & Ramadan, 2016). The results of S. polyanthum extracts indicated that they had high ability to donate these hydrogen radicals and electrons to
DPPH. The entire individual extracts of S. polyanthum showed consistent DPPH scavenging effect with inhibition ranging from 58.03-64.93 % (Figure 4.7). As far as combination of extracts is concerned the results were interesting to note in both within plant and between plants extracts. M. charantia and S. polyanthum within combinations had no significant differences from their individual extracts, they retained DPPH scavenging values, except “maceration MC-sonication MC” with a significant reduction to 5.49 % than their individual values (Figure 4.7). Despite half the concentration of extract (10 µg/mL) in combinations than individual extracts (20 µg/mL) the results of between combinations retained DPPH radical scavengingMalaya values with maximum observed in “maceration MC-sonication SP”, 65.35 %. According to Kiokias et al. (2008) phenolic compounds have the abilityof to donate hydrogen radicals to DPPH and thereby scavenge these radicals. Similarly the number of hydroxyl groups of phenolic compounds will decide the antioxidant potential of the test sample (Cao, Sofic, & Prior,
1996; Sang, Laplsey, Jeong, Lachance, Ho, & Rosen, 2002). In order to donate its hydrogen atom by a phenolic compound its reduction potential should be lower than the reduction potential of the free radical (Shahidi & Ambigaipalan, 2015). Though, many of the identified phytoconstituents were similar in M. charantia and in S. polyanthum extractsUniversity there were considerable differences in DPPH scavenging. Both had phenolic compounds (catechin 3-O-(1-hydroxy-6-oxo-2-cyclohexene-1-carboxylate, apigenin 7-
(3”-acetyl-6”-E-p-coumaroylglucoside, proanthocyanidin A2, apiole), lignans
(burseran), sphingolipids (spinganine, phytosphingosine), glycoside (linamarin), quinone (anthraquinone) and hormone ((+)-eudesmin). With the above findings, we assume that the phenolic compounds of S. polyanthum had higher reduction potential to
112 donate hydrogen than M. charantia. In addition, S. polyanthum has specific phenolic acids like 5-aminopentanoic acid, 3-propylmalic acid, a-Methyl-3, 4- dihydroxyphenylpropionic acid, tiaprofenic acid, phenazine-1,6-dicarboxylic acid and syringic acid which were not identified in M. charantia. Earlier researchers like
Kshirsagar & Upadhyay. (2009), reported potent DPPH free radical scavenging effects for S. cumini methanolic leaf extracts of the same genus, Syzygium support the current results.
There is no single standardized method to determine the antioxidant properties of food products and beverages, therefore it is always recommended to use more than one antioxidant method to evaluate antioxidant capacity of the samples. Antioxidant methods can be classified on account of their mechanism. DPPH and ferric reducing antioxidant power (FRAP) antioxidant assays are twoMalaya frequently used methods. FRAP assay gives the reducing power of the sample by transferring an electron to the targeted molecule, in contrary DPPH measures the offree radical scavenging ability of the samples to transfer hydrogen atom. Though, some researchers consider that DPPH has both the capacities (Foti, Daquino, & Geraci, 2004; Prior, Wu, & Schaich, 2005). FRAP is the ability of a sample to donate electrons. The phytoconstituents with high level of electron donating potential usually exhibit high FRAP activity. The principle of FRAP assay is by reaction of antioxidant sample with ferric (Fe3+) tripyridyltriazine complex and subsequent colour change from green to blue due to formation of ferrous (Fe2+) tripyridyltriazineUniversity (Kubola & Siriamornpun, 2008; Benzie & Strain, 1996). It has been noticed that FRAP activity of plant extracts was due to their phenolic content (El-Maati,
Mahgoub, Labib, Al, & Ramadan, 2016). Common phenolic compounds in M. charantia are catechin, gallic acid, gentisic acid, chlorogenic acid and epicatechin
(Horax, Hettiarachchy, & Chen, 2010; Prior, Wu, & Schaich, 2005). M. charantia extracts showed negligible FRAP activity with 3.12 - 4.19 % inhibition (Figure 4.8).
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Earlier studies in literature about the effect of boiling, microwaving and pressure cooking on phenolic contents and antioxidant activities revealed that boiling improves antioxidant activity of vegetables. The study was carried out on four vegetables, Vigna unguiculata, Momordica charantia, Ipomoea aquatica and Brassica olearancea. Upon subjecting for boiling, microwaving and pressure cooking there was significant variations in the antioxidant activities among the vegetables. The ferric reducing antioxidant power of boiled vegetables has shown significant increase in antioxidant activity ascribing to increased phenolic contents. Pressure cooking and microwaving did not had significant decline (Ng, Chua, & Kuppusamy, 2014). In contrary in the current study, the selected solvent and extraction methods affected phenolic content in M. charantia that resulted in its insignificant effect. However, significant differences between the DPPH and FRAP activities of M. charantiaMalaya were noticed. The fresh juice of S. polyanthum was considered very significant in showing FRAP activity among the four individual extractsof with 69.05 % inhibition (Figure 4.8). While in combined extracts (Figure 4.8), within combinations of S. polyanthum extracts, surprisingly “fresh juice SP-sonication SP)” combination had significant (p < 0.05)
FRAP value (68.12 %) than standard quercetin (63.27 %) due to synergism. Between combination extracts of the two plants has marginally reduced the FRAP values unlike within combination with maximum inhibition of 59.16 % by “soxhlet MC-fresh juice
SP” (Figure 4.8) (Yong, Zaiqi, Shuping, Xiaoli, Xuegang, & Kai, 2014). The LC-MS profilingUniversity of the S. polyanthum extract has identified electron rich amides, theobromine, stearamide, palmitic amide and antioxidant phenolic acids (5-aminopentanoic acid, 3- propylmalic acid, a-methyl-3,4-dihydroxyphenylpropionic acid, tiaprofenic acid, phenazine-1,6-dicarboxylic acid, syringic acid). S. polyanthum commonly referred as
Indonesian bay-leaf has long history of usage in traditional jamu preparations of locals to control diabetes (Elfahmi, Herman, & Oliver, 2014). Other species of the genus
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Syzygium, Syzygium aromaticum (Clove) (El-Maati, Mahgoub, Labib, Al, & Ramadan,
2016), Syzygium cumini (Syn: Eugenia jambolana Lam. or Syzygium jambolana Dc or
Eugenia cuminii Druce.) (Prince, Kamalakkannan, & Menon, 2004; Prince,
Kamalakkannan, & Menon, 2003), Syzygium guineense, (Nguyen, Rusten, Bugge,
Malterud, Diallo, & Paulsen, 2016) Syzygium paniculatum (Vuong, Hirun, & Chuen,
2014) and several others of the family Myrtaceae have been reported for rich polyphenolic contents. The above characteristics support the antioxidant potential of S. polyanthum, its taxonomical association with phenolic constituent rich genus further strengthens our view (Arumugam, Manaharan, Heng, Kuppusamy, & Palanisamy, 2014;
Khrishnasamy & Muthusamy, 2015; Chandran, Primelazhgan, Shanmugam, &
Thankarajan, 2016). These phytoconstituents might have contributed to a great extent for the FRAP activity of fresh juice. The maceratedMalaya extract of S. polyanthum had the least FRAP activity with 17.56 % inhibition. This can be attributed to prolonged duration of extraction (3 days) that of might have caused the hydrolysis of phytoconstituents in the solvent reducing its antioxidant power. In addition to the presence of polyphenolics in the extracts, presence of water soluble theobromine, a methylxanthine alkaloid, a compound rich in Theobroma cacao beans might have influenced the FRAP activity of S. polyanthum. Reports suggest that the theobromine content in the beans reduces upon storage due to fermentation (Benzie & Strain, 1996;
Niemanak, Rohsius, Elwers, Ndoumou, & Lieberei, 2006; Maleyki & Ismail, 2010). Chocolates,University especially dark chocolates are documented to be rich in this theobromine along with caffeine, influence the cognitive performance, changes in mood and behaviour (Smit, Gaffan, & Rogers, 2004).
Free radicals are generated either due to metabolism within the biological systems or externally acquired from environment, severely affect human health leading to several diseases, such as aging, cardiovascular, neurogenerative diseases, diabetes
115 and cancer. In recent times there are an increased number of cases of diabetes and cancer worldwide (Dasgupta & De, 2007). Free radicals and oxidative damage was identified as one of the major causes of diabetes (Yokozawa, Kim, Kim, Okubo, Chu, &
Juneja, 2007). Nutraceuticals, vitamins, vegetables, fruits and beverages can avoid or limit oxidative damage (Wu & Ng, 2008). Polyphenols are rich in food and green vegetables, hence can protect oxidative damage by scavenging free radicals.
Tocopherols, vitamin C are natural antioxidants in plants capable of scavenging lipid peroxyl radicals and quenching of oxygen free radicals (Munne &Alegre, 2002). M. charantia has been well advocated for its antidiabetic effects for various reasons; some researchers found that its fresh fruit juice can restore key antioxidant enzymes, superoxide dismutase, xanthin oxidase and catalase, in diabetic patients (Tayyab & Lal, 2013; Lin, Liua, Yang, & Fu, 2012). Apart fromMalaya that several mechanisms were proposed to explain the antidiabetic qualities of M. charantia extracts, having insulin- like peptide mimicking its action (Khannaof, Jain, Panagariya, & Dixit, 1981; Baldwa, Bhandari, Pangaria, & Goyal, 1977; Ng, Wong, Li, & Yeung, 1986), increased glucose uptake and glycogen synthesis (Shibib, Khan, & Rahman, 1993; Sarkar, Pranava, &
Marita, 1996; Welihinda & Karunanayake, 1986; Miura, Itoh, Iwamoto, Kato, Kawai,
& Park, 2001; Rathi, Grover, Vats, 2002; Ahmed, Adeghate, Cummings, Sharma, &
Singh, 2004; Cummings, Hundal, Wackerhage, Hope, Belle, & Adeghate, 2004;
Yibchok, Adisakwattana, Yao, Sangvanich, Roengsumran, & Hsu, 2006), compounds thatUniversity enhance the insulin secretion (Ahmed , Adeghate, Sharma, Pallot, & Singh, 1998; Kameswararao, Kesavulu, & Apparao, 2003; Fernandes, Lagishetty, Panda, & Naik,
2007) and inhibition of intestinal glucose absorption (Meir & Yaniv, 1985;
Mahomoodally, Fakim, & Subratty, 2004; Mahomoodally, Fakim, & Subratty, 2007).
Similarly, as mentioned earlier S. polyanthum leaf extracts have been traditionally used by local folk for diabetes in the name of “Jamu” preparation. Therefore, current study
116 was carried out to evaluate α-amylase and α-glucosidase inhibitory and subsequent glucose absorption inhibition by M. charantia fruit and S. polyanthum leaf extracts.
5.5 Antidiabetic enzyme inhibitory activity of the extracts
Diabetes mellitus is a chronic disease associated with high blood glucose levels also known as hyperglycaemia, it is prevalent throughout the world and especially in developing countries. Annually the number of diabetic cases is growing substantially and it will be among the leading causes of mortality and morbidity in the decades to come. Tissue damage in diabetes is the major cause of concern leading to retinopathy, nephropathy, inflammation, cancer, and high risk of cardiovascular diseases. The tissue damage is mediated by free radicals generated as a result of diabetes leading to lipid peroxidation of unsaturated fatty acids associated withMalaya cell membranes. Herbs, herbal medicine received lot of attention in the treatment of diabetes mellitus over the years. The herbal medicine are serving as a saferof alternative treatment approaches in the management of hyperglycaemia associated with diabetes. Herbs and herbal derived products are capable of decreasing the high blood glucose levels by interfering with the carbohydrate metabolism and glucose absorption in the intestine. α-amylase is one of the key hydrolysing enzymes in the mucosal lining gastrointestinal tract involved with carbohydrate digestion and assimilation, down regulation of this enzyme can interfere with conversion of complex carbohydrates to simple monosaccharides like glucose, thus leadingUniversity to reduced glucose absorption. Multicomponent plant extracts act as inhibitors of α-amylase, thereby block the release of glucose from dietary carbohydrates and lead to reduced glucose levels in plasma, decreased postprandial hyperglycaemia (Sailaja &
Khrisna, 2016).
Several plant derived products have shown significant results in preclinical studies against α-amylase and α-glucosidase enzymes to prove antidiabetic status. M.
117 charantia and S. polyanthum were two such plants extensively reported in literature for antioxidant and antidiabetic properties. M. charantia commonly named as bitter melon is amongst the widely explored plant and widely advocated plant for its antidiabetic and hypoglycaemic effects, especially in indigenous population in Asia, Africa and South
America. Several preclinical studies on animal models have suggested its effect on controlling diabetes, improved protection from insulin resistance and obesity.
Nevertheless, active principles responsible neither isolated nor their mechanisms were properly established (Andreas, Christine, Hans, Caludia, Staab, & Edmund, 2012). S. polyanthum commonly referred as Indonesian bay leaf, is considered to have anti- inflammatory, antipyretic, antioxidant, antihyperglycaemic and detoxificant properties by indigenous populations of Asia. The plant is rich in terpenes like citral and eugenol, polyphenolics like tannins and flavonoids, believedMalaya to be responsible for most of its activities. Previous investigations of antidiabetic effect of the plant have attributed to its polyphenolic components. However, preciseof mechanism of the effect were unknown and therefore, in the current research an in-depth study was conducted on both the aqueous extracts of the plants to further investigate their individual effects and their synergistic combined effects (Ratna, Ferawati, Wahyu, Lucia, Iwan, & Elisabeth, 2015).
As presented in Figure 4.9, fresh juice of M. charantia showed significant α- amylase inhibitory activity with 61.24 % inhibition. Constituents of M. charantia such as flavonoids, charantin, glycosides and steroids have been reported for the hypoglycaemicUniversity activity (Kumar, Balaji, Um, & Sehgal, 2009). However, some of these compounds like charantin were not identified in the LC-MS analysis. Charantin is insoluble either in highly polar solvent (water) or highly non-polar solvent (benzene) instead has high solubility in chloroform and dichloromethane. These solvents possess asymmetric molecular arrangement of chlorine, hydrogen atom surrounding carbon atom, such arrangement provides enhanced solubility for compounds like charantin.
118
Another probable reason may be due to low temperature of extraction, literature has suggested 120-150 °C is favourable for charantin extraction. None of the current extraction methods exceeded 100 °C. For the above reasons charantin might have not extracted in the current study. Nevertheless, the hypoglycaemic effects of the extracts may not be ruled out as there are multiple mechanisms for M. charantia extracts to elicit such action by multiple constituents (Pitipanapong, Chitprasert, Goto, Jiratchariyakul,
Sasaki, & Shotipruk, 2007). The next best extraction to show α-amylase inhibitory activity was sonication (57.06 %) which had statistically similar activity like fresh juice
(p > 0.05). Soxhlet had least 43.2 % inhibition indicating that heating causes loss of α- amylase inhibitory constituents in M. charantia. Maceration with its 51.27 % inhibition was statistically similar to soxhlet (p > 0.05). The results reiterated the α-amylase inhibitory effect of M. charantia. Malaya Fresh juice of S. polyanthum showed highly significant (p < 0.001) α-amylase inhibitory activity with 92.21 % inhibition.of Fresh juice, of S. polyanthum consistently showed better results in DPPH, FRAP, and also in α-amylase inhibitory studies.
Phytochemical investigation by Widyawati et al. (2015) on the S. polyanthum has flavonoids, glycosides, alkaloids and tannins from the leaves that might have showed the effect. Few earlier studies have demonstrated the hypoglycaemic activity of S. polyanthum, in vitro antidiabetic actions were investigated on methanolic extracts, an in vivo study on isolated abdominal muscle study of rats revealed glucose inhibitory actionsUniversity by leaf methanolic extract, effect similar to acarbose (Gray & Flatt, 1998). Similar antidiabetic results were reported upon administration of alcoholic extracts in alloxan induced mice for 7 days (Ratna et al., 2015). The order of α-amylase inhibition between the extracts was fresh juice (92.21 %) > sonication (80.49 %) > soxhlet (48.46
%) > maceration (34.49 %). The least effect in soxhlet and maceration indicates hydrolysis of the responsible constituents upon storage as in case of maceration. When
119 the combination of two aqueous extracts of the plants are tested as given in Figure 4.9, remarkable activity was noticed with fresh juice of S. polyanthum containing samples, all three fresh juice combinations of S. polyanthum were comparable to standard, acarbose. They showed 90.86 %, 90.03 % and 87.64 % despite half the concentration than their individual extracts demonstrating synergism.
The α-glucosidase inhibitory effect of M. charantia was poor for all the extracts with values ranging from 16.47 to 21.77 % as shown in Figure 4.10. Despite poor α- glucosidase inhibitory effect of M. charantia its antidiabetic effects were well demonstrated in literature, the activity was ascribed most of the time to the constituents such as charantin, glycosides, steroids, cucurbitanes, vicine, peptides and alkaloids
(Chuang, Hsu, Chao, Wein, Kuo, & Huang, 2006; Kumar, Balaji, Um, & Sehgal, 2009). M. charantia is one of the most widely studied plantsMalaya for antidiabetic effects. Some of the earlier studies have discovered the mechanisms of its actions, few of them were, by enhancing the glucose uptake by liver, of reduced gluconeogenesis by a process of inhibiting essential enzymes of gluconeogenesis (glucose-6-phosphatase and fructose-
1,6-biphosphatase), increased glucose oxidation, enhanced glucose uptake by cells, promoting the number of insulin producing β-cells and potentiating insulin release
(Alternative medicine review, 2007). A polypeptide from the M. charantia fruits has demonstrated insulin like effect when administered subcutaneously. In recent times several such peptides have been isolated involved in insulin signalling pathway. Oral administrationUniversity of its aqueous extracts at a dose of 400 mg/day for 15 days in rats fed on fructose rich diet demonstrated significant (p < 0.001) reduction in hyperglycaemia
(Grover & Yadav, 2004). Even though the α-glucosidase inhibitory effects of M. charantia were not reproduced as documented in the literature its antidiabetic effect cannot be ruled out as it is believed to act by multiple mechanisms. Therefore, few researchers have considered M. charantia as herbal remedy for type 2 diabetes;
120 evidence has shown glucose tolerance and suppression of postprandial glucose in rats by M. charantia (Nagarani, Abirami, & Siddhuraju, 2014). The α-glucosidase inhibitory effects of S. polyanthum were 16.57 to 96.06 % as given in Figure 4.10. The fresh juice of S. polyanthum exhibited excellent 96.06 % α-glucosidase inhibition than acarbose standard (32.22 %). S. polyanthum and its phylogenetically close family members were found to have abundant phytoconstituents with pharmacological significance (Elfahmi,
Herman, & Oliver, 2014; Prince, Kamalakkannan, & Menon, 2004; Prince,
Kamalakkannan, & Menon, 2003; Nguyen, Rusten, Bugge, Malterud, Diallo, &
Paulsen, 2016; Vuong, Hirun, & Chuen, 2014; Arumugam, Manaharan, Heng,
Kuppusamy, & Palanisamy, 2014; Krishnasamy & Muthusamy, 2015; Chandran,
Primelazhgan, Shanmugam, & Thankarajan, 2016). S. cumini has shown moderate hypoglycaemic effect upon administration of 12 g ofMalaya seed powder in three divided doses for three months, the effect was similar to chlorpropamide (Kohli & Singh, 1993). When combination of S. polyanthum and M.of charantia aqueous extracts were tested like in α-amylase inhibitory study only fresh juice S. polyanthum containing samples had superior activity and it was 3 fold higher (p < 0.001) than the standard, acarbose. This suggests that fresh juice of S. polyanthum due to its ability to inhibit α-amylase and α- glucosidase can significantly lower post-prandial hyperglycaemia. LC-MS analysis have presented a list of phenolic compounds in S. polyanthum, including few flavonoids, studies has shown the inhibitory effect of flavonoids on α-glucosidase leading to antidiabeticUniversity effect (Widyawati, Nor, Mohd, & Mariam, 2015). Therefore, current study has given full insight into the antioxidant and antidiabetic activities of M. charantia and
S. polyanthum. The above findings justify the folklore use of these herbs for diabetes.
Many natural product discoveries were retrieved from the folklore evidences. For instance, Crataegus is a spiny shrub, native to Europe and North America whose leaves, flowers and berries were used for congestive heart failure by the local folk. It contains
121 flavonoids and oligomeric procyanthins, responsible for the activity. It was reported to have antioxidant, inotropic, vasodilator and anti-hyperlipidaemic actions, as well as decreased capillary permeability (Valli & Giardina, 2002). In Japan and China a green tea (Camellia sinensis) is popular as folklore medicine, the chemo preventive effects of green tea are probably attributed in particular to the catechin polyphenolic components.
Green tea is normally consumed as a brewed tea, though most chemo preventive applications have used a concentrated extract. Green tea catechins may act on multiple pathways to prevent cancer, including oxidative stress, elimination of carcinogens, and inhibition of enzymes (Shahidi & Ambigaipalan, 2015).
5.6 Evaluation of prepared herbal tablet formulations
Herbs were proven to be beneficial in controlling hyperglycaemia and can effectively contain diabetic complications upon long-termMalaya use. Due to increasing use of herbal medicine their standardization has been emphasised on several platforms. Currently, identifying the marker compoundsof in an herbal products and its quantitative measurement is employed to ascertain its quality and efficacy. However in recent times this approach of identifying one or two marker compounds has raised serious doubts as they proved ineffective in estimating the therapeutic activities. As it is evident herbal preparations contain multiple components that can target multiple targets leading a unique pharmacological response which is not comparable to one or two constituents in it. Hence, detection of effective compound combinations has been recommended to assessUniversity the quality of herbal products (Long et al., 2016). The main objective of the study was to initially extract the two plant materials using different extraction techniques and check whether method of extraction can exhibit significant differences in antioxidant and antidiabetic effects on the aqueous extracts, finally to prepare a herbal tablet dosage form employing the best extracts. Several polyherbal formulations were prepared and evaluated for antidiabetic management and for other alternative
122 treatments. The purpose of developing herbal formulations of the above extracts is to provide significantly efficacious, safe and economical treatment alternatives to the diabetic patients (Margret & Jayakar, 2010). In this study we combined the two best plant extracts in equal proportions (300 mg/ tablet) equivalent to one or two serving of these vegetables in our daily food intake. The details of the excipients used in the formulation were as given in Table 3.1. Finely powdered ingredients were manually mixed using geometrical dilution technique to ensure homogeneity of the mix as described in section 3.2.6. Conventional granulation technique using HPMC (1 %) as granulating agent was employed and the resulting granules were evaluated for flow properties. The prepared granules had excellent angle of repose, Carr’s index and
Hausner’s Ratios as shown in Table 4.6. The prepared herbal tablets were evaluated for tabletting characters to estimate their quality parameters.Malaya The evaluation results of prepared herbal formulations were presented in Table 4.6. The uniformity of weight, diameterof and thickness of the herbal formulations were given in Table 4.6. The general requirement of USP for mass uniformity is that no more than two tablets should deviate from the average weight by more than ± 5 %. The deviation in weight of the tablets manufactured from formula was within these limits of the Pharmacopoeia. The average weight of the 20 tablets was 554.5 ± 1.4 mg and none of the tablet deviated from the limit. The average diameter and thickness of the tablets were 13.8 ± 0.04 and 3.57 ± 0.17 mm respectively, and the variation diameter and in thicknessUniversity were within the 5 % deviation limit. To evaluate the hardness of the prepared formulations 10 tablets were randomly selected and hardness was found to be 56.8 ± 4.3
N, which may be considered to be ideal (Binega, Dawit, & Bayew, 2013). Low standard deviation values of above tablet evaluation parameter have suggested that the prepared tablets were uniform and acceptable pharmacopoeial standards of USP.
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Friability test was conducted to ascertain the ability of the tablets to withstand transportation, handling and packing stress without chipping, cracking and breaking.
The general specification is that loss in weight of less than 1 % is required to pass the friability test. The formulations exhibited a loss of 0.72 % after the test is completed on
20 randomly selected tablets. The results indicated the disintegration of the tablets as shown in Table 4.6. The tablets disintegrated within 10.19 to 13.15 min. All the six tablets disappeared from the basket rack assembly of the instruments within 15 min. as required by Pharmacopoeia for an uncoated tablet. The herbal tablets thus showed acceptable disintegration characteristics. The tablets appeared to disintegrate by dissolving in the medium, rather than through a process of breaking up and releasing the particles of the tablets into the disintegration medium. The current study has extracted two selectedMalaya plant materials by four different extraction methods, the extracts were subjected for chemical profiling and were evaluated successfully for antioxidant andof antidiabetic activities, an herbal tablet formulation was developed from the best extracts.
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CHAPTER 6: SUMMARY AND CONCLUSION
Diabetes mellitus, a chronic metabolic disorder characterised by hyperglycaemia, can lead to macro and micro vascular complications which eventually can damage vital organs such as eyes, kidneys, heart and brain. Despite advancements in the medical field it is still a major cause of morbidity and mortality in the world.
Changing life style and food habits were found to aggravate the diabetic complications.
According to world health organisation (WHO) reports 70-80 % of the world population rely on traditional systems of medicine and natural derived products for their ailments.
Fruits and vegetables in diet have proved to be beneficial in reversing the incidence of diabetic complications. There is substantial evidence to support their role in preventing negative health outcomes related to cardiac diseases,Malaya diabetes, aging and cancer. Increased consumption of recommended levels of fruits and vegetables can minimize overall healthcare spending and can achieveof socio-economical goals. Since natural products are very good source of antioxidants invariably due to rich polyphenolics, terpenoids and vitamins the current study was aimed at selecting two food grade plants and evaluating their antioxidant, enzyme inhibitory potential upon exposing them to different procedures of extraction. The rationale behind selecting M. charantia and S. polyanthum was because both have well documented evidences to support their antioxidant and antidiabetic activities. In addition, ample folklore data is available on theseUniversity two plants and therefore public acceptance, awareness on them as a food supplement would be high.
The two selected plants, M. charantia and S. polyanthum were successfully extracted by four different extraction methods. Our study revealed that, method of extraction has a significant effect on extractive values and on the number of constituents in an extract, significant differences in antioxidant and antidiabetic enzyme inhibitory
125 activities were also observed depending on the method employed for extraction. The extractive values in M. charantia were in the order of sonication (26.37 %) > soxhlet
(24.25 %) > maceration (12.98 %) > fresh Juice (3.06 % w/w). The extractive values in
S. polyanthum were fresh juice (10.07 %) > soxhlet (8.70 %) > sonication (8.22 %) > maceration (7.44 % w/w). GC-MS and LC-MS profiling of the extracts was carried out to correlate their activities to that of marker compounds detected in them. Each plant material was extracted by four different extraction procedures, a total of 8, 10, 10 and
11 peaks were observed in GC-MS chromatograms of S. polyanthum soxhlet, sonication, fresh juice and maceration extracts respectively. Similarly, a total of 9, 10,
12 and 15 peaks were observed in GC-MS chromatograms of M. charantia sonication, soxhlet, fresh juice and maceration extracts respectively. The two volatile chemical compounds, phenol, 2, 4-bis (1, 1-dimethyl) andMalaya octadecanoic acid, methyl ester amongst them which are in highest proportion in both the plants, their presence matched to earlier literature. LC-MS profiling has revealedof the presence of polypenolics such as apigenin, catechin, proanthocyaninin, lignans and also steroids, quinones, sphingolypids in both the plant extracts. Despite similar constituents detected in LC-MS chromatograms of both plants their activities differed to a great extent this can be attributed to some phytoconstituents, karanjin, adifoline, phenol acids, theobromine, stearamide, palmitic amide, and others in the extracts. Nevertheless, further studies are needed to label them as “effective compounds combination” to consider as multiple markersUniversity responsible for activities. The study has reiterated the necessity for standardization of herbal products and traditional medicine. DPPH scavenging ability was very low for M. charantia individual extracts it was ranged between 8.53-19.76 %.
S. polyanthum showed consistent DPPH scavenging effect ranging from 58.03-64.93 %.
M. charantia extracts showed negligible FRAP activity with 3.12-4.19 % inhibition, whereas S. polyanthum had 17.56-69.05 % and it was significantly higher than standard,
126 quercetin, α-amylase and α-glucosidase inhibitory activities of S. polyanthum extracts were remarkable especially by fresh juice. Though moderate α-amylase inhibition was noticed by M. charantia extracts their α-glucosidase results were poor. Combined extracts of the two plants exhibited mixed results and only few combinations, those with fresh juice of S. polyanthum consistently produced better antioxidant and antidiabetic outcomes.
Fresh juices of both plants had shown remarkable antioxidant and antidiabetic effects especially fresh juice of S. polyanthum. The fresh juices of M. charantia and S. polyanthum due to their rich antioxidant and antidiabetic enzyme inhibitory characters are suitable for inclusion as a health supplement. In conclusion the study favours natural dietary supplements rich in antioxidants, in amounts sufficient to prevent complications of oxidative stress and act as health promoters. ChoosingMalaya the right food with controlled sugar levels and the antioxidant content becomes the perfect combination for diabetes to be in check. Thus, choice of diet and dietaryof products is critical in human health; foods and vegetables rich in antioxidant phytoconstituents such as polyphenolic flavonoids, phenolic acids, tannins and vitamins are beneficial and reduce the risk associated with oxidative stress and degenerative diseases. Both plants are considered to have rich nutritional values with lot of polyphenols, vitamins, antioxidants and minerals.
Therefore, M. charantia and S. polyanthum could be an effective, readily available, inexpensive, acceptable and renewable plant based health promoting entities available as University food supplements, can reduce postprandial glucose levels by interfering with carbohydrate metabolism and by reducing oxidative stress. The study has successfully developed herbal formulations of the above plants with concentrations of the best plant extracts equivalent to one or two servings of the fruit and leaves that possesses free radical scavenging and antidiabetic profiles. However, the formulations were neither tested for in vitro drug release nor for in vivo performance. Hence, further studies on
127 these formulations should be needed to correlate the activities to that of marker compounds, formulation factors responsible for the activity.
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APPENDIX
LIST OF PUBLICATIONS AND CONFERENCE PRESENTATIONS
Publication
1. Muhammad Jihad Sandikapura, Shaik Nyamathulla & Ibrahim Mohamed
Noordin. (2018). Comparative antioxidant and antidiabetic effects of Syzygium
polyanthum leaf and Momordica charantia fruit extracts. Pakistan Journal of
Pharmaceutical Sciences. 31(2) (Suppl), 623-625.
Conference presentation
1. Muhammad Jihad Sandikapura, Shaik Nyamathulla & Ibrahim Mohamed
Noordin. (2016). In vitro antidiabetic activities of Momordica charantia fruit
and Syzygium polyanthum leaf extracts obtained from different extraction
methods. Multidisciplinary International Conference Resilience and Empowered
Communities for Sustainable Development. Jakarta, Indonesia: November 16,
2016. (Poster Presentation) University of Malaya
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