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SYNTHESIS, CHARACTERIZATION AND IN VIVO
ANTIHYPERLIPIDEMIC EFFECTS OF GREEN SILVER
NANOPARTICLES OF ALLIUM SATIVUM
A Thesis submitted
In the partial fulfillment of the requirement for the degree of
MASTER OF PHILOSOPHY
(Eastern Medicine)
By
ABDUL SAMI
(BEMS)
University College of Conventional Medicine
Faculty of Medicine & Allied Health Sciences
The Islamia University of Bahawalpur
Pakistan
Registration Number 132/ IU. M.Phil./2019 Session 2019-2021
Table of Contents
1. Declaration………………………………………………..iii
2. Certificate…………………………………………………v
3. Dedication………………………………………………...vi
4. Acknowledgement………………………………………..vii
5. Content…………………………………………………...ix
6. List of Figure…………………………………………….xxi
7. List of Tables…………………………………………….xxvi
8. List of Abbreviations…………………………………….xxix
9. Abstract………………………………………………….xxxi
1.INTRODUCTION……………………………………………………1
1.1. Background………………………………………………………………….……2
1.2. Medicinal Plants……………………………………………………………..……6
1.3. Medicinal Plants in Pakistan…………………………………………..…………9
1.4. Aim and Objectives………………………………………………………………10
1.4.1. Aim…………………………………………………………………….10
1.4.2. Objectives………………………………………………………………11
1.4.3. Methodology to be adopted………………………...………………….11
1.4.4. Significance of the study………………………………………………11
2.LITERATURE REVIEW……………………………………………13
2.1. Drug delivery system………………………………………….…………………14
2.1.1. Classification of Drug Delivery System……………………………….14
2.1.1.1. Conventional drug delivery system………………………....15
2.1.1.2. NDDS (Novel drug delivery system)………………………..16
2.1.1.3. Use of NDDS………………………………………….…….16
2.1.1.4. Controlled release drug delivery system (CRDDS)………..17
2.1.1.4.1. Approaches for Controlled release drug delivery
system………………………………………………..………..17
2.1.1.4.2. LDDS (Localized drug delivery system)…………..18
2.1.1.4.3. SRDDS (Sustained release drug delivery system)...18
2.1.1.4.4. MDDS (Modulated drug delivery system)…….…..18
2.1.1.4.5. Feedback-controlled drug delivery system…….….18
2.1.1.4.6. Targeted drug delivery system………………..……19
2.1.1.5. Novel drug delivery system composition …………………...19
2.1.1.6. Nano-carrier…………………………………………………20
2.2. Nanotechnology………………………………………………………………….20
2.2.1. Types of nanoparticles………………………………………...………21
2.2.2. Nanoparticles properties………………………………………………21
2.2.3. Nanomaterial Classification…………………………………………..22
2.2.4. Synthesis methods of nanoparticles…………………………….……..24
2.2.5. Benefits of green synthesis of nanoparticles………………………….25
2.2.5.1. Green synthesis method……………………………………..25
2.2.6. Nano Silver…………………………………………………….………28
2.2.6.1. Properties of silver…………………………………….…….29
2.2.6.2. Importance of Silver nanoparticles…………………..……..30
2.2.6.3. Mode of action of Nano silver………………………..……..31
2.2.6.3.1. Anti-Bacterial Properties…………………..……..32
2.2.6.3.2. Anti-Fungal Properties………………….…..……32
2.2.6.3.3. Antiviral Properties………………………..……..32
2.2.6.3.4. Anti-inflammatory Properties……………….......32
2.2.6.4. Synthetic Routes Adapted for the Synthesis of
Ag-NPs………………………………………………………..33
2.2.6.5. Biological Synthesis of Silver nanoparticles………..33
2.3. Hyperlipidemia……………………………………………………………….….37
2.3.1. Classification of hyperlipidemia………………………………………39
2.3.2. Etiology………………………………………………………………...39
2.3.3. Epidemiology………………………………………………………......40
2.3.4. Pathophysiology……………………………………………………….40
2.3.5. Histopathology………………………………………………………...43
2.3.6. History and Physical Examination…………………………………...43
2.3.7. Evaluation……………………………………………………….…….43
2.3.8. Treatment / Management…………………………………….…….....45
2.3.9. Non-pharmacological therapy……………………………….….….…45
2.3.10. Pharmacological therapy……………………………………..……..46
2.3.11. Differential Diagnosis………………………………………..……...47
2.3.12. Prognosis…………………………………………………….….…...48
2.3.13. Complications……………………………………………….….……48
2.3.14. Postoperative and Rehabilitation Care……………………..….……49
2.3.15. Herbal treatment of Hyperlipidemia………………………..………49
2.4. Diabetes Mellitus …………………………………………………………..…..51
2.4.1. Prevalence…………………………………………………….……...52
2.4.2. Classification of Diabetes Mellitus…………………………….…....54
2.4.2.1. Diabetes Mellitus Type 1……………………………..……54
2.4.2.2. Diabetes Mellitus Type 2…………………………………….54
2.4.2.3. Gestational diabetes (Type 3………………………………...55
2.4.2.4. Other specific Types of Diabetes……………………….…....55
2.4.2.4.1. Diseases of the exocrine pancreas………………...55
2.4.2.4.2. Genetic defects in insulin action………………….55
2.4.2.4.3. Genetic defects of beta cell function………...…...56
2.4.2.4.4. Endocrinopathies…………………………….……56
2.4.2.4.5. Drug- or chemical-induced……………….………56
2.4.2.4.6. Infections…………………..………………………57
2.4.2.4.7. Uncommon forms of immune-mediated diabetes..57
2.4.2.4.8. Other genetic syndromes sometimes associated
with diabetes………………………………………….………57
2.4.3. Pathophysiology of Diabetes Mellitus………………………….…......57
2.4.3.1. Insulin resistance………………………………………….………...59
2.4.3.2. Insulin deficiency…………………………………………..………..60
2.4.4. Symptoms ……………………………………………………...………62
2.4.5. Role of lipid profile in Diabetes Mellitus ……………………………..63
2.4.6. Complications of Diabetes Mellitus …………………………………..63
2.4.7. Diagnosis of Diabetes………………………………………………….64
2.4.8. Criteria for Diagnostic of Diabetes Mellitus……………….…………65
2.4.9. Differential diagnostic criteria for type 1 and type 2 diabetes after
diagnosis…………………………………………………………….…….….66
2.4.10. Treatment of diabetes mellitus…………………………….…..……..68
2.4.10.1. Insulin and oral hypoglycemic drugs……………………...68
2.4.10.2. Herbal treatment of diabetes ………………………………68
2.5. Allium sativum…………………………………………………………………...70
2.5.1. Botanical Description…………………………………………............70
2.5.2. Bulb…………………………………………………………………….71
2.5.3. Leaves………………………………………………………………….72
2.5.4. Flowers…………………………………………………………...........72
2.5.5. Fruits………………………………………………………….………..73
2.5.6. Seeds……………………………………………………………...........73
2.6. Classification of Allium sativum………………………………………...74
2.7. Chemistry and Pharmacology…………………………………………...74
2.8. Pharmacological activities………………………………………………75
2.8.1. Antibacterial activity…………………………………………..76
2.8.2. Antiviral activity………………………………………............76
2.8.3. Antifungal activity……………………………………………..77
2.8.4. Antiprotozoal activity………………………………………….77
2.8.5. Anti-parasitic activity………………………………….………78
2.8.6. Wound Healing activity……………………………….………78
2.8.7. Anti- Diabetic activity……………………………….………...79
2.8.8. Antihypertensive activity…………………………….………..79
2.8.9. Anti-tumor Effects………………………………….………...79
2.8.10. Liver Protective/Detoxification Effects…………….……….79
2.8.11. Antioxidative and Radioprotective Effects…………………80
2.8.12. Diuretic and Digestive activity……………………………...80
2.8.13. Anti-cancer Activity……………………………….………….80
2.8.14. Cardio protective activity…………………………….……….81
2.8.15. Alzheimer’ Disease Protective activity……………………….81
3. MATERIALS AND METHOD……………………………………83
3.1. Materials…………………………………………………………………………84
3.1.1. Instruments and Equipments………………………………………….84
3.1.2. Chemicals……………………………………………………………...87
3.2. Method……………………………………………………………………….…..89
3.2.1. Plant Collection and Identification……………………………….…..89
3.2.2. Synthesis of Allium sativum Linn. Bulb extract………………..…….90
3.2.3. Percentage yield of Allium sativum extract……………………..……90
3.2.4. Phytochemical Analysis………………………………………..……...90
3.2.4.1. Tests for Alkaloids…………………………………………..90
3.2.4.1.1. Mayer’s reagent test……………………………….91
3.2.4.1.2. Wagner’s test……………………………………...91
3.2.4.1.3. Hager’s test………………………………………..91
3.2.4.2. Tests for Carbohydrates…………………………………….91
3.2.4.2.1. Molish test………………………………………...91
3.2.4.2.2. Barfoed’s test……………………………………..91
3.2.4.3. Tests for Reducing Sugars………………………………....91
3.2.4.3.1. Fehling’s test…………………………….……….91
3.2.4.3.2. Benedict’s test……………………………….…...91
3.2.4.4. Tests for Flavonoids………………………………….…....91
3.2.4.4.1. Alkaline reagent test……………………………….91
3.2.4.4.2. Lead Acetate Test………………………………….92
3.2.4.4.3. Ammonia solution test……………………….…….92
3.2.4.5. Tests for Glycosides………………………………….……....92
3.2.4.5.1. Borntrager’s test…………………………….……..92
3.2.4.5.2. Legal’s test…………………………………….…..92
3.2.4.5.3. 10% NaOH test…………………………………....92
3.2.4.6. Test for cardiac glycosides………………………………….92
3.2.4.6.1. Keller-Killani test………………………………....92
3.2.4.7. Tests for Tannin and Phenolic compounds……………….92
3.2.4.7.1. Ferric chloride test 5% ………………………….92
3.2.4.7.2. Lead Acetate Test………………………………..93
3.2.4.7.3. Dilute iodine solution test…………………….....93
3.2.4.7.4. Ferric chloride test 10% or ferric chloride test…93
3.2.4.7.5. Hydrolysable tannin………………………….....93
3.2.4.8. Test for Saponins……………………………………..……93
3.2.4.8.1. Froth test……………………………………..…..93
3.2.4.9. Tests for Protein and Amino acids…………………..…….93
3.2.4.9.1. Ninhydrin test………………………………..…..93
3.2.4.9.2. Biuret test…………………………………..…….93
3.2.4.10. Tests for Triterpenoids and Steroids………………..…...94
3.2.4.10.1. Salkowski’s test…………………………..…….94
3.2.5. Biosynthesis of silver nanoparticles ………………………….……94
3.2.6. Percentage yield of Nanoparticles…………………………………….96
3.2.7. Characterization of Silver Nanoparticles …………………………….96
3.2.7.1. Ultraviolet visible spectroscopy (UV- vis) ……………….….96
3.2.7.2. Fourier Transform Infrared Spectroscopy (FTIR) …….….97
3.2.7.3. X-ray Diffraction (XRD) ……………….……………….….97
3.2.7.4. Transmission Electron Microscopy (TEM) and Energy
Dispersive X-Ray (EDX). ………………………………….….……..98
3.2.7.5. Scanning Electron Microscopy (SEM)………………..….…98
3.2.8. Preparation of Buffer…………………………………………..….…..99
3.2.8.1. HCl Buffer 1.2 pH………………………………….…..……99
3.2.8.2. Phosphate Buffer (p H 6.8)……………………….….……..99
3.2.9. Allium sativum λ max measuring……………………………..……...99
3.2.9.1. Stock solution……………………………………………....100
3.2.9.2. Determination of λ max…………………………………....100
3.2.9.3. Calibration of standard curve……………………….……..100
3.2.10. Determination of Entrapment efficiency…………..……….101
3.2.11. Release of Drug Studies (In-vitro)……………………..…...102
3.2.12. Kinetics of Drug Release………………………………...….102
3.2.12.1. Model Zero-Order……………………………..….103
3.2.12.2. First-Order Release Kinetic Model……………….103
3.2.13. Anti-oxidant activity…………………………………..….....104
3.2.13.1. DPPH assay……………………………………….104
3.2.14. In vitro anti-diabetic activity of Allium sativum silver
nanoparticles………………………………………………….…….106
3.2.14.1. a-Amylase and a-Glucosidase inhibition activity of
AS.AgNPs…………………………………………………...106
3.2.15. In vitro anti-diabetic activity of Allium sativum Extract......106
3.2.15.1. α-Amylase inhibition assay……………………….106
3.2.15.2. α-Glucosidase inhibition assay…………………...107
3.2.16. Experimental Animals………………………………………108
3.2.17. Induction of Hyperlipidemia Along with Treatment Plan....108
3.2.18. Diabetes Induction Along with Treatment Plan…………..110
3.2.19. Blood Sampling…………………………………………….114
3.2.20. Measurement of Body Weight, Relative Liver, and Heart
Weight of Rats ……………………………………………………...115
3.2.21. Histological assessment of liver, kidney and pancreas sample
by Heamatoxylin Eosin (H/E) staining ……………………………116
3.2.22. Biochemical analysis……………………………………….117
3.2.22.1. Serum glucose……………………………………117
3.2.22.2. Lipid profile………………..………………….….117
3.2.22.1.1. Triglycerides (mg / dl)…………………117
3.2.22.1.1.1. Principle………………………….…..117
3.2.22.1.1.2. Reagent and Standard…………….…118
3.2.22.1.1.3. Procedure……………………………118
3.2.22.1.1.4. Calculation…………………………..118
3.2.22.1.2. Total cholesterol (mg/dl)……………………...118
3.2.22.1.2.1. Principle ………………………….…118
3.2.22.1.2.2. Reagent preparation…………….…..119
3.2.22.1.2.3. Procedure …………………………....119
3.2.22.1.2.4. Calculation ……………………….....119
3.2.22.1.3. High Density lipoprotein (HDL) …………..…119
3.2.22.1.3.1. Principle Reagent and Standard.…...119
3.2.22.1.3.2. Procedure……………………………120
3.2.22.1.3.3. Calculations ……………………......120
3.2.22.1.4. LDL-Cholesterol (mg/dl) …………………….120
3.2.22.1.5. VLDL-Cholesterol (mg/dl) ……………….….121
4. RESULTS AND DISCUSSION……………………………………122
4.1. Percentage yield of Allium sativum extract………………………………123
4.2. Allium sativum extract’s phytochemical analysis………………….……..123
4.3. Percentage Yield of Silver Nanoparticles………………………….……..125
4.4. Characterization of AgNPs ……………………………………….……...125
4.4.1. UV-Visible Spectroscopy ………………………………….…...126
4.4.2. FTIR Analysis ……………………………………………..…...127
4.4.3. SEM Analysis ……………………………………………..…....129
4.4.4. EDS Analysis…………………………………………….…..….130
4.4.5. XRD analysis………………………………………………...…131
4.5. Determination of λ max of Allium sativum Extract………………….....132
4.6. Allium sativum Standard Curve Phosphate Buffer (pH 6.8)……….….133
4.7. Entrapment efficiency……………………………………………….......134
4.8. In-vitro nanoparticles drug release study…………………………….…134
4.9. Antioxidant effect of Allium sativum extract by DPPH scavenging
Activity………………………………………………………………….……...……137
4.10. Invitro Antidiabetic Potential of Allium sativum and Allium sativum
nanoparticles…………………………………………………………..………...….139
4.11. Effects of Extract and Silver nanoparticles of Allium sativum on High Fat Diet
Induced Hyperlipidemic Rats…………………………………………………...…..142
4.11.1. Effect of Allium sativum on Body Weight………………………….142
4.11.2. Effects of Extract and Silver nanoparticles of Allium sativum on
Relative Liver and Heart Weight ……………………………………….….144
4.12. Effects of Extract and Silver nanoparticles of Allium sativum on Diabetes
Induced Rats………………………………………………………………….….….146
4.12.1. Effect of Allium sativum on Body Weight ……………………..…..146
4.12.2. Effects of Extract and Silver nanoparticles of Allium sativum on
Relative Liver, Pancreas and Kidney Weight ………………………..…….148
4.13. Anti-Diabetic activity of Diabetic Rats Group…………………………...…...150
4.13.1. Plasma Glucose Test …………………………………………...…..150
4.14. Lipid Profile of Hyperlipidemic Rats …………………………………….…..152
4.14.1. Total-Cholesterol level ……………………………………………..152
4.14.2. Triglycerides level…………………………………………….……..153
4.14.3. HDL Cholesterol level………………………………………..……..154
4.14.4. VLDL Cholesterol level………………………………………..…....155
4.14.5. LDL Cholesterol level ……………………………….…………...…156
4.15. Lipid Profile of Diabetic Rats………………………………..…………….....158
4.15.1. Total-Cholesterol level……………………………..…………….....158
4.15.2. Triglycerides level………………………………...……………...….159
4.15.3. HDL Cholesterol level……………………………………...……….160
4.15.4. VLDL Cholesterol level ………………………...…………………..161
4.15.5. LDL Cholesterol level……………………………………………….162
4.16. Histopathological examination…………………………………………….…163
4.16.1. Histopathological examination of liver in Hyperlipidemic rats…...163
4.16.2. Histopathological examination of Pancreas, Liver and Kidney in
Diabetic rats…………………………………………………………………165
DISCUSSION……………………………………………………….. 170
5. CONCLUSION……………………………………………………176
6. REFERENCES……………………………………………………178
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ABSTRACT
Background
Hyperlipidemia has been identified as one of the most important risk factors for the development
and progression of coronary heart disease. The leading causes of mortality are coronary heart
disease, stroke, atherosclerosis, and hyperlipidemia. Diabetes mellitus, also known as diabetes, is
a group of metabolic disorders that may be described as hyperglycemia, and is caused by a lack
of insulin secretion, insulin resistance, or both for a long time. Diabetes mellitus (DM) has
achieved epidemic proportions in the last few decades of the twentieth century and is recognized
as a global public health concern because of its multifactorial surfaces influencing basic
biochemical processes in the body. Antimicrobial, anticancer, antioxidant, antidiabetic,
antiemetic, antihypertensive, hypoglycemic, hypolipidemic and immunomodulatory are several
of the biological functions attributed to Allium sativum. Phytochemical analysis of this species
have shown that it is abundant in alkaloids, tannins, carotenoids, saponins, phenols and
flavonoids all of which have been shown to have high antioxidant activity and may be used to
reduce silver to silver nanoparticles.
Aim
The aim of this research study is the green synthesis and characterization of silver nanoparticles
by using extract of Allium sativum and to evaluate antidiabetic and antihyperlipidemic potential
of silver nanoparticles.
Method
The bulbs of Allium sativum Linn. were stripped away. The bulbs were sterilized with
doubledistilled water after being washed thoroughly 2–3 times under running tap water. The bulb
sample was dried at room temperature (25 degrees Celsius). Approximately, 6g of Allium
sativum Linn. bulb was cut (not crushed) into 1/4 bits, then applied to 50ml of hydro-ethanolic
30/70 V/V Solution and allowed to sit for 3 days at room temperature. The soaked substance of
Allium sativum Linn. was filtered with Muslin cloth after 72 hours of soaking. Parts that were
solid were withdrawn. The filtrate was re-filtered on Whatman filter paper no. 1 to produce a
clean filtrate. To concentrate crude extract, the solvent (Ethanol) was evaporated on the rotary
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evaporator at 35°C. The semi-solid mass of crude drug was obtained by air drying the condensed
crude sample. The coarse extract obtained was collected in a jar. This blunt extract was kept at
4°C in the refrigerator for later use. The phytochemical analysis of different compounds in crude
extract was performed. 0.01mM AgNO3 and different concentrations of plant extract were used
for the green synthesis of silver nanoparticles. The synthesis of silver nanoparticles was
confirmed by color change and UV visible spectroscopy while presence of crude extract in silver
nanoparticles was confirmed by FTIR spectroscopy. The particles size surface morphology of
silver nanoparticles was observed with SEM. The antioxidant activity of silver nanoparticles was
performed by 96 well plate methods. Invitro antidiabetic activity was performed by using α-
amylase and α-glucosidase. Invivo antihyperlipidemic and antidiabetic activity was performed on
male Sprague Dawley rats.
Results
The color of silver nanoparticles formulation was changed light yellowish to dark brown which
indicated the formation of silver nanoparticles. It was further confirmed with UV visible
spectroscopy, in which maximum absorbance band of silver nanoparticles was observed at 426
nm. The FTIR spectra of optimized formulation of silver nanoparticle confirm the reduction of
silver and presence of plant extract in silver nanoparticles. The particle size of optimized
formulation was 103.7 nm. The SEM analysis showed that silver nanoparticles of Allium sativum
are circular and tube like shape, nanocapsules. The Invitro and Invivo activity showed that silver
nanoparticles have potent antidiabetic activity. So, it suggested that silver nanoparticles have
potent antihyperlipidemic and antidiabetic activity against hyperlipidemia and diabetes mellitus.
Conclusion
It had been concluded that the green synthesis of silver nanoparticles by using Allium sativum
plant extract is possible and these silver nanoparticles have potent antihyperlipidemic,
antidiabetic, antioxidant activity. So, these nanoparticles can be used against different heart
diseases related to hyperlipidemia and diabetes.
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Keywords
Allium sativum, nanotechnology, Silver nanoparticles, antihyperlipidemic, antidiabetic,
phytochemical analysis, antioxidant, histopathology
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CHAPTER 1
INTRODUCTION
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1. INTRODUCTION
1.1. Background
Nanotechnology is the study of the preparation, characterization, fabrication, modification of
arrangements, tools and materials with at least one dimension (or elements with at least in one
dimension) of 1–100 nanometers. Materials with particle sizes below this threshold have
physical and chemical properties that are substantially changed from macro scale materials made
of the same material (Duncan, 2011). Due to a rising need to improve environmentally
sustainable materials synthesis technologies, biosynthesis of nanoparticles has gotten a lot of
attention in the last years. Only in recent years has the biosynthetic approach using plant extracts
gained some attention as an easy and feasible alternate to chemical and physical procedures for
synthesising metal nanoparticles. Nanotechnology is becoming more relevant in the food and
healthcare industries. Bioactive nano encapsulation, biosensors to identify and measure
pathogens, and innovative tools for assessment and advancement of fresher, cleaner, and reliable
drug preparations have also yielded promising findings and implementations in the fields of
nutrient and drug distribution systems.
Plants, plant wastes, microbes, and fungi are widely used to synthesize nanoparticles, which has
recently increased the practice of biological particles as targets for "green nanotechnology."
(Duncan, 2011)(Singhal et al., 2011) Plants are better candidates for nanoparticle synthesis,
according to (Krithiga, Rajalakshmi and Jayachitra, 2015), since they are largely nontoxic, have
natural capping agents, and reduce the expense of microorganism separation and culture media.
Silver nanoparticles (AgNPs) are gaining popularity due to their simple formation process and
electrical, special optical, and thermal properties, that improve near-infrared absorption,
electrical conductivity and charge separation (Khan et al., 2011).
Plant extracts are favoured over additional biological foundations due to their wide range of
decreasing metabolites and abundant supply. Secondary plant derivatives hold a wealth of
promise as nutraceuticals, medicine, and additive of food. Polyphenols of plants are one of the
most abundant classes of antioxidant secondary metabolites found in nature (Cieśla et al., 2013).
The skill of plant extracts to synthesize nanoparticles with improved properties has been
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attributed to the reduction properties of these antioxidant metabolites (Kharissova et al., 2013).
Plant-based biosynthesis of AgNPs has many advantages over conventional synthesis processes,
including not expensive, environmental friendliness and the removal of high strain, electricity,
temperature, and toxic chemicals (Geetha et al., 2013). AgNPs have gained a lot of interest
among the numerous inorganic metal nanoparticles as stabilizers, anticancer agents, effective
antimicrobial, detectors and biomedical sensors with low toxicity for in vivo and in vitro
applications (Cieśla et al., 2013)(Geetha et al., 2013). AgNPs have been used for larvicidal,
anticoagulant, thrombolytic,(Raja, Ramesh and Thivaharan, 2015)(Lateef et al., 2016) and anti-
inflammatory (Chouhan, 2018) purposes by other studies.
Medicinal plants with well-established medicinal properties and no reported side effects have
risen to the top of the pharmacopoeia. However, permeability, poor solubility, low
bioavailability, and heterogeneity the biological setting make the distribution of plant/herbal
therapeutic molecules as drugs difficult. These drawbacks of herbal medicines may be solved by
encapsulating or adding them to appropriate nanomaterials, which can dramatically increase
pharmacokinetics and efficiency (Okafor et al., 2013). Medicinal plants are of particular interest
because they have capping layers that regulate the size and shape of nanoparticles (Ansari, Islam
and Sameem, 2012). AgNPs have been developed using a variety of medicinal plants, including
species (Geetha et al., 2013)(Rauwel et al., 2015)(Huang et al., 2007)(Lal and Nayak, 2012).
Garlic (Allium sativum) is a natural species with medicinal properties used in most cuisines
around the world. Individually, this species is thought to be a general cure for a variety of
ailments. Hypolipidemic, Antimicrobial, antidiabetic, anticancer, antihypertensive, antioxidant,
antiemetic, immunomodulatory and hypoglycemic are several of the biological functions
attributed to these spices (Muthukumaran et al., 2015)(Ahuja et al., 2006)(Petrovska and
Cekovska, 2010)(Kubra and Rao, 2012)(Haniadka et al., 2012)(Li et al., 2012). Phytochemical
analysis of this species have shown that it is abundant in alkaloids, flavonoids, tannins,
saponins, carotenoids and phenols, and all of which have been shown have great antioxidant
activity (Mikaili et al., 2013)(Otunola et al., 2010) and may be used to reduce silver to silver
nanoparticles.
Hyperlipidemia is a condition described by high serum total cholesterol, triglycerides, very low
density, low-density lipoprotein cholesterol and low levels of high-density lipoprotein [30].
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Hyperlipidemia was identified as one of most important risk factors for development,
progression of coronary heart disease (Grundy, 1986). The leading causes of mortality are
coronary heart disease, hyperlipidemia, atherosclerosis and stroke (Smith, Song and Sheldon,
1993). Liver produces two-thirds of all cholesterol produced in the body. 3-hydroxy-3-
methylglutaryl (HMG)-Co A reductase is rate-limiting enzyme that offers feedback regulation by
regulating concentrations of cholesterol in cells. Dietary management, use of lipid-lowering
diets, exercise and medications are all used to treat hyperlipidemia (Stone, 1996).
Hydroxymethylglutarate coenzyme A (HMG-CoA) reductase inhibitors, also known as statins,
are the most widely used medicines to treat hyperlipidemia. Bile acid sequestrants (anion-
exchange resins) like cholestyramine and colestipol; fibrates like clofibbrate, fenofibrate,
gemfirozil, ciprofibrate, and cholesterol absorption inhibitors like ezetimibe; bezafibrate; niacin;
and omega-3 fatty acids are some of the other medicines used to treat hyperlipidemia (Lin et al.,
2010).
Despite the stock of variety of medicine to treat hyperlipidemia, antihyperlipidemic therapy lacks
efficacy, protection, and, most importantly, "price." Statins, for example, which are especially
well suited for lowering LDL, carry the possibility of severe muscle injury (Kobayashi et al.,
2008). Niacin, a common medication for lowering triglycerides, has been linked to
hyperglycemia and liver damage (Guyton and Bays, 2007). Since adding bile acid sequestrants
and niacin to continuing statin treatment in patients with hypercholesterolemia, xanthomas of the
Achilles tendon have been identified (Lakey, Greyshock and Guyton, 2013). Fibrate-related side
effects also include the skeletal muscle, liver and kidneys. Rhabdomyolysis caused by
fenofibrate was complicated by acute renal failure (Kiskac et al., 2013). As a result, more
effective antihyperlipidemic agents are also required. Plant-based chemicals are also thought to
be less toxic and free of side effects than synthetic products. Allium sativum (Yeh and Liu,
2001), Commiphora mukul (Singh, Niaz and Ghosh, 1994), soy or Glycine max (Wong et al.,
1998), Nigella sativa (Lateef et al., 2016), and Plantago ovata (Sabzghabaee et al., 2012) are
some of the plants that have been shown to have antihyperlipidemic efficacy in clinical trials.
Diabetes mellitus, also known as diabetes, is a collection of metabolic abnormalities that may be
described as hyperglycemia, and is caused by a lack of insulin secretion, insulin action efficacy,
or both for a long time (Atkinson and Maclaren, 1994)(Teixeira et al., 2013). It is thought that
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many pathogenic pathways could be involved in the progression of diabetes. They're thought to
have a spectrum that includes autoimmune obliteration of β-cells in the pancreas. The deficiency
of insulin action by target tissues is the basis of various irregularities in protein , lipid and
particularly carbohydrate breakdown during diabetes (Ozougwu et al., 2013). Diabetes mellitus
(DM) has achieved epidemic proportions in the last few decades of the twentieth century
(Raveendran et al., 2018) and is recognized as global public health apprehension because of its
multifactorial surfaces influencing basic biochemical processes in the body (Abraham et al.,
2017).
Diabetic classification is generally founded on the etiology and clinical demonstration. In
particular, type-I, type-II, gestational diabetes, and a few other distinct forms of diabetes mellitus
are classified as type-I, type-II, and gestational diabetes, respectively (Sicree, Shaw and Zimmet,
2006). In general, there are two main forms of diabetes, the first of which is defined as
inadequate insulin development and the second of which is defined as non-responsiveness of
target cells to insulin (Suchy et al., 2008). Typically, type-I diabetes is thought to be produced by
the loss of pancreatic Beta cells, which are responsible for insulin synthesis and are unique to the
autoimmune method. Auto antigens on beta cells, B lymphocytes, T lymphocytes, macrophages
and dendritic cells have all been shown to performance a role in pathogenesis of autoimmune
diabetes (Bei, Yoon and Abur, 2005).
Type-1 Diabetes is characterized by an inability to release insulin, which results in lower glucose
absorption by muscles and adipose tissue (Lehninger et al., 2015). Type-I diabetes, often
recognized as insulin-based diabetes mellitus (IDDM), commonly handled with exogenous
insulin, while type-II diabetes, also famous as non-insulin dependent diabetes (NIDDM), is
commonly managed by oral hypoglycemic agents such as biguanides, sulphonyl ureas, among
other miscellaneous uses (Felig, Brusilow and Boyer, 1995)(Rosak, 2002). Diarrhea, lactic
acidosis, liver complications, and other adverse side effects have been identified in recent years
as a result of available diabetes treatment (Mittal et al., 2010). This disease has been estimated to
affect about 424.9 million people globally (8.8% of the global population), with a projected
growth of around 628.6 million people by the year 2045 (IDF., 2017).
Medicinal plants are thought to be rich reservoirs of natural health promoting combinations like
phytoalexins and phytochemicals, which contain polyphenols, vitamins E, C, and A, carotenoids,
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flavonoids, and a variety of other constituents. In contrast to allopathic medications and
narcotics, the use of plants as medicines for treatment of wounds, their regeneration is not only
cost effective and inexpensive, but it has also been shown to be healthy with no side effects
(Cotropia, Quillen Jr and Webster, 2013). Nearly 800 plant species have been identified as
having anti-diabetic behaviour and properties.
Several plant species have been used as a diabetes preventive or treatment tool by Chinese, Asian
Indians, South Americans, and Native Americans since ancient times (Jafri, Rajalakshmi and
Ramaprabhu, 2010). Several plants were shown to be beneficial for management of diabetes
mellitus. It is self-evident that medicines sold on the market are derived from plants, either
directly or indirectly, as primary sources. Since ancient times, plans have been used as medicine
in numerous parts of the world for both preventative and curative purposes. Another explanation
for widespread use of medicinal herbs for management of diabetes around the world may be their
low cost and widespread availability (Deepashree and Prakash, 2007).
Herbal compounds were used as traditional medicine for the management of variety of diseases
for a long time. Traditional pharmaceutical manufacturers have an advantage that they do not
have to prove their arguments of curing illnesses are true; but, if they work with a substance that
is a vaccine, they would have multiple proofs. Any of the compounds used in herbal products
may be healthy, but others may not. These findings are based on the presumption that the
products were tainted or came into contact with chemicals, heavy metals, or narcotics, or that the
products did not contain the claimed /specified ingredients. Any traditionally used herbal
remedies may interfere with medications, causing severe side effects, or they may be deemed
dangerous for a large number of patients with particular conditions (Hannibal and Bishop, 2015).
1.2. Medicinal Plants
Plants have been used for medicinal purposes since the dawn of time. Plants have been used in
therapeutics since about 400 – 500 B.C., according to literature. The Chinese were the ones who
pioneered the use of natural herb preparations in medicine. Traditional medication offered by the
primary health care system is now the most reasonable and accessible healthcare option in
disadvantaged neighborhoods. And it's been estimated that indigenous peoples have been using
medicinal plants as medicine for a long time (Prakash and Gupta, 2005). For alternative health
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care facilities, the majority of people in emerging countries such as India, China, and Pakistan
depend on traditional medicine to heal a variety of diseases due to its healthy use and efficacy for
hundreds of years. Therapeutic plants are used in local or home procedures in numerous parts of
Pakistan for the management of a wide range of ailments (Muhammad, Saeed and Khan, 2012).
Medicines that are directly obtained from common sources are also commonly used. Medicinal
plants contain chemicals that have potential therapeutic effects. Various species of herbal plants
are widely used as a medical mediator for the treatment of a host of communicable diseases
around the world. We may estimate that about a quarter of currently accepted medicines are
derived from botanical sources (Sahreen et al., 2014)(Vane and Botting, 1995). Herbal drugs are
commonly used to promote primary health in developed countries. Seventy to eighty percent of
the world's population accepts these drugs because they are culturally acceptable. Herbal drugs
are less likely to cause adverse effects. The use of herbal drugs to treat different diseases has
been a novel trend of drug research (Eldahshan and Abdel-Daim, 2015). While the
pharmaceutical industry has produced a vast range of clinical drugs, native medicinal methods of
treatment are still used in rural areas. The WHO has recognized the value of traditional
homegrown medicines, which are used as the first line of protection in health care (Goleniowski
et al., 2006). Plants are responsible for around 85% of the drugs used in primary health care
around the world (Farnsworth, 1988).
Medicinal plants are widely consumed by natives in the form of unprocessed spices, and they are
often useful in the pharmaceutical industry for manufacturing a variety of loaded medicines. The
majority of people use herbal remedies as a moderate and rational source of health and well-
being in order to live a comfortable and healthy life. Therapeutic plants contain a wide range of
compounds that have limited therapeutic applications (Shinwari, Jamil and Zahra,
2014)(Shinwari et al., 2015). Plants have long been used to relieve pain, and in this day and age,
the key goal of research is to discover their role and capacity in the treatment and control of a
range of ailments. The majority of studies indicate that a variety of herbs and medicinal plants
have beneficial effects on miscarriage, hormone deficiencies, liver disorders, anemia, kidney
diseases, and neurologic and psychiatric disorders (Kooti and Daraei, 2017). Medicinal plants are
a key component of most native medicinal systems around the world, as we can see. Ethno
botany is a valuable resource for the production and study of innate medicines (Farnsworth,
1990). Furthermore, ―traditional‖ herbal medicine use implies a broad range of historical uses,
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which is unquestionably true for a large number of products marketed as ―traditional herbal
medicines.‖ In most developing nations, a large proportion of the population is reliant on
traditional physicians and their arsenal of curative plants to fulfill their well-being needs. Despite
the fact that modern medicine and traditional practice coexist, herbal remedies have sustained
their popularity due to historical and academic factors (Vishwakarma et al., 2013). Furthermore,
natural yield has long played a major role in the management and prevention of human ailments
all over the world. Sea animals, aquatic microorganisms, terrestrial plants, terrestrial vertebrates
and invertebrates, and terrestrial vertebrates and invertebrates are among the sources of material
for making natural medicines (Newman, Cragg and Snader, 2000), and their importance in
current medicine has been addressed in numerous studies and reviews (Jones, Chin and
Kinghorn, 2006). In current years, there have been increased waves of interest in natural goods
chemistry in the field of science. This level of awareness may be explained to a quantity of
factors, containing unmet therapeutic needs, remarkable variety similarly substance structure and
organic properties of naturally occurring optional metabolites, the suitability of novel bioactive
common mixes as biochemical tests, the development of novel and sensitive systems to see
organically dynamic regular products, and improved procedures to detach, clean, and essentially
portray these dynamic constituents, and advances in revealing the interest for resource of
complex characteristic substances (Clark, 1996).
WHO (World Health Organization) has also recognized importance about conventional
medicines and has established methods, guiding principles, and values for botanical medicines.
The application of verified agro-industrial technologies is involved in the formation and
dispensation of herbs, as well as the production of plant-based medicines (Organization, 1993).
The majority of current medicines are derived from medical plants, which are the primary origins
of new drugs. There are now expected to be over 250,000 flowering plant species. The toxicity
of plants can be determined by studying medicinal plants, which aids in the protection of animals
and humans from natural toxins. Herbs and simple remedies have long been used in traditional
civilizations around the world, and they are becoming increasingly common in modern culture as
a natural alternative to synthetic chemicals. For everyday health care, the vast majority of people
on the planet also dependent on conventional material medicine. ―A medicinal plant is one in
which compounds contained in one or more of its organs have therapeutic properties or are
useful in the manufacture of effective drugs‖ (Fazal and Singla, 2012). Curative plants play a
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significant part in well-being of vulnerable publics all over world. The majority of medicinal
plants are flowering plants, as has been observed. Furthermore, the overall estimate predicts that
by 2050, the global medicinal plant industry will be worth $5 trillion (US). Still now, people are
turning to herbal ingredients to find a cure to their misery from the adverse effects of other drugs
(Shinwari, 2010).
1.3. Medicinal Plants in Pakistan
Almost all of Pakistan's plant varieties have fulfilled the requirements of 84 percent of the
population. Medicinal plants predate the evolution of humans. Common medicines claim to have
their roots in household medications, and this knowledge is passed on over the years. Plants have
medicinal properties, and conventional or Native herbal medicine is based on them. Hippocrates'
famous quote, "Let medicine be your food and food be your medicine", emphasizes the
importance of herbal remedies (Wariss et al., 2014) Pakistan is unique in its climate, with the
Hindu-Kush Himalayas and Karakorum ranging in altitude from 0 to 8611 meters, and as a
result, it has a wide range of climatic zones and a diverse floral array. There are over 6,000
varieties of high plants in Pakistan. A minimum of 12% of the flora is used for medicinal
purposes, and many plants are transported. In Pakistan, wild plant species are the primary source
of a massive crude drug [Pansar] market chain. Herbal medicines are used to treat both human
and animal illnesses. Some plant varieties are considered specific for a specific disease in most
cases, but they seldom have many uses (Shinwari and Qaiser, 2011). Since the dawn of time,
plants were used as a supply of medicine in nearly all cultures. Standard flora is a crucial
preserve for enhancing wellbeing, and it is the main source of remedies for a variety of ailments
throughout most human societies. Most countries in South America, Asia, and Africa have a
diverse plant population that is used to treat a wide range of ailments. That is why, according to
WHO, conventional medicines are primary health care system for about 60% world's population.
However, a wide variety of plant species are still unfamiliar which have with potential However,
there is still a considerable number of plant species with unexplained biological behaviors
(Mustafa et al., 2016). Due to its diverse climate and a multitude of other factors, the most
significant of which are soil conditions and various ecological areas, Pakistan has a peculiar
geographical distribution. As a result, it has a diverse variety of pungent and medicinal plants, as
well as an array of botanical assets. Plants are used by the majority of Cholistan natives to cure a
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range of illnesses due to their extraordinary medicinal properties. Medicinal plants are used as a
natural way to treat extensive range of health issues. Capsaicin, a pain killer originating from
peppers, does not compete with other body sensations (Akhter and Arshad, 2006). The demand
for medicinal plants is increasing on a daily basis, not just in the local market but also on the
international market, necessitating the emergence of new strategies to meet this demand. Since
plants bind to 70% of synthetic drugs, a substantial number of medicines are now derived
directly from medicinal plants (Pattanaik, 2006).
The current study is directed at the formation, characterization, assessment of antihyperlipidemic
potentials of silver nanoparticles made from aqueous ethanolic extract of Allium sativum as part
of our study on the therapeutic, nutraceuticals, and economic uses of these species.
1.4. Aim and Objectives
1.4.1. Aim
Conventional method for preparation of metal nanoparticle have many limitation like slower rate
of reaction, higher cost and use of chemical reducing agent such as sodium borohydride,
dimethyl foramide, trisodium citrate, which can increase burden on environment. The important
consideration in green synthesis are utilization of non-toxic chemicals, use of ecofriendly solvent
and use of renewable materials and in this consequences in recent years, green synthesized
biocompatible metal nanoparticle are gaining considerable attraction in the field of biomedicine,
due to the use of natural resource, rapid rate of synthesis, ecofriendly and safer method. Other
advantage is greener synthesis gives well defined and controlled size of nanoparticle and also it
is devoid of contamination and scale up is very easy. Silver nanoparticle has many application
such as antibacterial, antifungal, antimalarial, larvicidal, anti-acne, anti-dandruff, anti-
plasmodial, anticancer and anti-wounds activity. In the view of the above, it was felt worthwhile
to proceed with Synthesis, Characterization and in vivo antihyperlipidemic effects of green
nanoparticles of Allium sativum which has wide spectrum of biological effects.
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1.4.2. Objectives
The objectives of present study are
1. To synthesize extract of Allium sativum
2. Phytochemical analysis of Allium sativum extract
3. Biosynthesis of silver nanoparticles of Allium sativum
4. To evaluate the characterization of silver nanoparticles of Allium Sativum
5. Evaluation of in-vitro antidiabetic potential of silver nanoparticles of Allium sativum
6. Evaluation of in-vivo anti hyperlipidemic effect of silver nanoparticles of Allium sativum
7. Evaluation of in-vivo antidiabetic effects of silver nanoparticles of Allium sativum
8. Histopathological examination of organs such as liver, kidney and pancreas
1.4.3. Methodology to be adopted
1. Selection of plant and procurement of chemicals
2. Collection of plant
3. Authentication of plant
4. Preparation of plant extract by using solvent
5. Green Synthesis of silver metal nanoparticle by using plant extract
6. Characterization of nanoparticle
7. Evaluate antihyperlipidemic potential
8. Evaluate antidiabetic potential
9. Histopathological examination
1.4.4. Significance of the study
The use of nanotechnology to different plant extracts (crude drugs) has shown various
advantages for herbal medicines which are sustained release, increased bioavailability, solubility
and enhanced pharmacological activity. It also provides less toxicity and protects the crude drug
from physical and chemical degradation. Among the metal nanoparticles, noble metal
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nanoparticles have demonstrated potential biomedical applications. Due to the small size,
nanoparticles can easily interact with biomolecules both at surface and inside cells, yielding
better signals and target specificity for diagnostics and therapeutics. Silver nanoparticles have
played a main role in the field of nanotechnology and nano medicine. Development of
nanoparticles (NPs) as a part of new medicine has given rise to a new field of research. In
comparison to traditional anti-hyperlipidemic drugs, NPs provide a targeted approach which
prevents undesirable effects. In this communication, we have reviewed the role of silver NPs
(AgNPs) in antihyperlipidemic nano medicine. Natural plant extracts are relatively safer than
conventional synthetic drugs. So, developing nanoparticles formulation of these natural crude
extracts will be safer and effective for the treatment of hyperlipidemia.
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6. Conclusion
In this study, the phytochemical analysis of Allium sativum plant was performed and results
showed that this plant is rich with many phytochemicals like alkaloids, glycosides and
antioxidant agents. Conclusively, our results confirm that size-controlled AgNPs were
successfully synthesized by using the ethanolic extract of Allium sativum with the reduction of
Ag+ into Ag° at room temperature identified by UV-Vis, XRD and SEM analysis. FTIR analysis
showed that Allium sativum aqueous ethanolic extract has such compounds which helped in the
reduction of silver into its NPs along with the capability of stabilizing these particles. The
synthesis of AgNPs through the green route is cost-effective and eco-friendly methods. The
results showed that synthesized AgNPs were circular and tube in shapes with size ranging from
103.7-106.6 nm through SEM analysis. In this study the DPPH scavenging activity of optimized
formulation of silver nanoparticles was performed which showed that these silver nanoparticles
have potent antioxidant activity. Invivo antidiabetic activity of silver nano particles that these
silver nanoparticles have potent antidiabetic activity. Silver nanoparticles and Allium sativum
aqueous ethanolic extract may be explored for its potential in the prevention and treatment of
clinical hyperlipidemia and diabetes mellitus.
Future prospects
Green synthesis of nano particles using plant extracts is an easy one step coast effective
bio synthesis process, and it could have great potential in the development of nano
medicine alternative to the conventional therapies against hyperlipidemia and diabetes
mellitus.
Furthermore, we recommend In vivo studies for the evaluation toxicological study of
these nanoparticles using hyperlipidemic rat‘s models.
Furthermore, we recommend clinical study for the evaluation of these nanoparticles for
the treatment of hyperlipidemia and diabetes in human models.
We recommend the evaluation of these nanoparticles for the treatment of other heart
diseases.
