Antiglycation and Antioxidant Potential of Selected Plant Materials
By Asia Atta M.Phil. (UAF)
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
in
BIOCHEMISTRY
DEPARTMENT OF BIOCHEMISTRY
FACULTY OF SCIENCES UNIVERSITY OF AGRICULTURE, FAISALABAD PAKISTAN 2015
DECLARATION I hereby declare that the contents of the thesis, “Antiglycation and antioxidant potential of selected plant materials” are product of my own research and no part has been copied from any published source (except the references, standard mathematical or genetic models/equations/formulae/protocols etc). I further declare that this work has not been submitted for award of any diploma/degree. The University may take action if the information provided is found inaccurate at any stage.
Asia Atta 2002-ag-743
The Controller of Examinations, University of Agriculture, Faisalabad.
“We, the Supervisory Committee, certify that the contents and form of the thesis
submitted by Ms. Asia Atta, Regd. No. 2002-ag-743, have been found satisfactory and recommend that it be processed for evaluation, by the External Examiner(s) for the award of
Ph.D. degree”.
Supervisory Committee
1. Chairman ______Prof. Dr. Munir Ahmad Sheikh
2. Member ______Dr. Muhammad Shahid
3. Member ______Prof. Dr. Tanweer Khaliq
DEDICATION
To My loving Parents, Who mean the world to me
I dedicate this thesis to the best gift given to me from almighty Allah, My Parents and my entire family Who always provide me compassion throughout my life and my success is really the fruit of their prayers
ACKNOWLEDGMENT In the name of Allah, the merciful, the beneficent I have the pearls of my eyes to admire countless blessings of Allah Almighty because the words are bound, knowledge is limited and time of life is too short to express his dignity. Then the trembling lips and wet eyes praise the greatest man of Universe, the last messenger of Allah, HAZRAT MUHAMMAD (PBUH), whom Allah has sent as mercy and the illuminating torch for the world. I deem it my utmost pleasure to avail an opportunity to express my gratitude to my honorable supervisor, Dr. Munir Ahmad Sheikh, Department of Biochemistry , University of Agriculture, Faisalabad for his kind behavior, moral support and constructive criticism during my study period. I am passionately thankful to Dr. Muhammad Shahid, (Associate professor, Department of Biochemistry) for his sincere and encouraging attitude, guidance and moral support throughout my PhD studies. I am extremely obliged to Prof. Dr. Tanweer khaliq (Chairman, Department of physiology and pharmacology, UAF) for his special attention with regard to my research work and exam. I would like to express my gratitude and profound admiration to my International Research Support Initiative Program (IRSIP) supervisor Dr. Hang Xiao (Associate profess) and his team for their immense support toward my research work in the Department of Food Science, University of Massachusetts, Amherst (MA), USA. I also ought to financial support (Indigenous scholarship and IRSIP) provided by Higher Education Commission (HEC), Islamabad, Government of Pakistan without this financial support it could not possible for me to complete my doctoral studies. At the same time I express my special thanks to my research groups of Medicinal Biochemistry Lab, Department of Biochemistry, UAF and Department of Food Science, University of Massachusetts, Amherst (MA), USA for their unreserved love, benevolent help and encouragement during the whole studies and research. I need to thank all my near and dear friends and fellows, Dr. Sumaira Sharif, Dr. Mazhar Abbas, Dr. Ghulam Mustafa, Dr. Sammar Abbas, Dr. Ali Raza, Dr. Abid Ali, Dr. Nazia kanwal, Dr. Sumia Akram, Dr. Mushtaq, Salman bhai, Ali bhai and Sir Shamshad, Runa khan, Saira and Andleeb for their love providing amenities and sincere friendship. I pay my special thanks to Nadeem bhai for his unreserved affection, care and sincere prayers for my success. May ALLAH bless them with success and happiness, Ameen! Many thanks are due to my nephews M. Zaighum, Zaryab, Aalyan, M. faateh and to my nieces Meerab, Wardah and Irha for their innocient concern which give me heartiest pleasure. I express special appreciation to Uncle Iqbal Hussain Qureshi, for his unreserved affection guidance, encouragement and everlasting prayers. I owe immense feelings of love and thanks to my affectionate parents as their prayers are always behind my each and every success and for my entire family for being there in times of need, their great patience, continuous encouragements and untiring efforts while I did this work. A special thanks to my siblings for bearing me throughout my degree. All of my prayers, good habits and sacred emotions are for them. May Allah bless them all here and hereafter (Amin).
Asia Atta
TABLE OF CONTENTS
Sr. No. TITLE Page No. 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 8 2.1 Concepts of functional and nutraceutical foods 8
2.2 Oxidative stress and safety concerns 10 2.3 Phytoconstituents as antioxidant 11 2.3.1 Tocopherols composition 12
2.3.2 Phenolic content 12 2.3.3 Flavonoids 13
2.3.4 Carotenoids 14
2.3.5 Alkaloids 14 2.3.6 Saponins 15 2.4 Antioxidant capacities of plant materials 15 2.4.1 Antioxidant activities of vegetables 15 2.4.2 Antioxidant activities of dry fruits 16 2.5 Anticancer potential of selected plant materials 17 2.5.1 Anticancer potential of vegetables 17 2.5.2 Anticancer potential of dry fruits 19 Glycation and formation of advanced glycation end 2.6 19 products 2.6.1 Advanced glycation end products 20 2.6.2 Intracellular AGEs and their harmful effects 21 2.6.3 Mechanisms of actions of AGEs 22
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2.6.3.1 Cross linking extracellular matrix proteins 22
2.6.3.2 Crosslinking intracellular proteins 22
2.6.3.3 RAGE dependent effects of AGEs 22 Protein modification to form Amadori in diabetes & its 2.6.4 24 complications 2.7 Carbohydrate hydrolyzing enzymes inhibition 25 Biochemistry of carbohydrate hydrolyzing enzymes and their 2.7.1 26 inhibitors 2.7.2 Inhibitors of carbohydrate hydrolyzing Enzymes from plants 27 Phytoconstituents with carbohydrate hydrolyzing enzymes 2.7.3 27 inhibitory activity 3 MATERIAL AND METHODS 29 3.1 List of chemicals 29 3.2 Instruments used 29 3.3 General procedure 30 3.3.1 Collection and identification of plant materials 30
3.3.2 Grinding and sample size/extraction 31 3.3.2.1 Extraction of free phenolic compounds 31 3.3.2.2 Extraction of bound phenolic compounds 32 3.3.3 Proximate analysis of plants materials 32 3.3.3.1 Estimation of crude protein 32 3.3.3.2 Estimation of crude fats 33 3.3.3.3 Estimation of crude fibers 33 3.3.3.4 Determination of ash contents 34 3.3.3.5 Determination of total carbohydrates 34 3.3.3.6 Total energy 34 3.3.4 Mineral analysis 34
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3.3.5 Phytochemistry of plants 35
3.3.5.1 Qualitative analysis 35
3.3.5.2 Quantitative analysis 35 3.3.6 Antioxidant activity of plant extracts 36 3.3.6.1 Total phenolic contents (TPC) of selected plant extracts 36 3.3.6.2 Total flavonoids content (TFC) of selected plant extracts 37 3.3.6.3 DPPH radical scavenging assay of selected plant extracts 37 3.3.6.4 Determination of reducing power of selected plant extracts 37 3.3.7 Brine shrimp lethality assay 37
3.3.7.1 Lethality concentration determination 38
3.3.8 Thrombolytic assay 38 Measurement of inhibition of cell proliferation by plants 3.3.9 39 extracts 3.3.9.1 Cell lines and culture 39 3.3.9.2 In vitro anti-proliferative assay 39 3.3.10 Antiglycation activity 40 3.3.10.1 Non-enzymatic protein glycation 40 Spectrophotometric analyses of nitro-blue tetrazolium (NBT) 3.3.10.2 40 reductive assay 3.3.10.3 Spectrophotometric analyses of Girard-T assay 40 3.3.10.4 AGEs analysis 40 3.3.11 Enzyme assay 41 3.3.11.1 α-Glucosidase assay 41 3.3.11.2 α-Amylase inhibition assay 41 3.3.12 Statistical analysis 42 4 RESULTS AND DISCUSSION 43 Extraction and % age yield of extracts from selected 4.1 43 plant materials
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4.2 Proximate analysis of selected plant materials 45 4.2.1 Proximate analysis of selected vegetables 46 4.2.2 Proximate analysis of selected dry fruits 47 4.3 Mineral Analysis of the selected plant materials 48 4.3.1 Mineral analysis of selected vegetables 49 4.3.2 Mineral analysis of selected dry fruits 50 4.4 Preliminary phytochemical analysis 53 4.5 Quantitative analysis of phytochemicals 54 4.5.1 Quantitative analysis of phytochemicals of vegetables 54 4.5.2 Quantitative analysis of phytochemicals of selected dry fruits 55 4.6 Antioxidant potential of plant materials 57 4.6.1 Antioxidant concentration of selected plant materials 57 4.6.1.1 Total phenolic contents 57 4.6.1.2 Total flavonoid content (TFC) 59 4.6.2 Antioxidant activities of plant materials 60 4.6.2.1 Reducing power assay 60 4.6.2.2 DPPH radical scavenging activity 61 Toxicological screening of plant materials against brine 4.7 64 shrimp nauplii 4.7.1 Brine shrimp lethality bioassay of vegetables 64 4.7.2 Brine shrimp lethality bioassay of dry fruits 65 4.8 Thrombolytic activity of plant materials 66 Thrombolytic activity of methanolic extracts of selected 4.8.1 67 vegetables Thrombolytic activity of methanolic extracts of selected dry 4.8.2 67 fruits 4.9 Antiproliferative activity of selected plant materials 68 4.9.1 Antiproliferative activity of selected vegetables 69 4.9.2 Antiproliferative activity of selected dry fruits 70 4.10 Antiglycation activities of selected plant materials 72 iv
4.10.1 Effect of selected plant materials on the reduction on NBT 74 4.10.1.1 Effect of selected vegetable materials on the reduction of NBT 74 4.10.1.2 Effect of selected dry fruit materials on the reduction of NBT 75 Effect of selected plant materials on the formation of α- 4.10.2 76 dicarbonyl 4.10.2.1 Effect of selected vegetables on the formation of α-dicarbonyl 77 4.10.2.2 Effect of selected dry fruits on the formation of α-dicarbonyl 78 Effect of methanolic extracts of selected plant materials on 4.10.3 82 the formation of AGEs Effect of methanolic extracts of selected vegetables on the formation 4.10.3.1 82 of AGEs Effect of methanolic extracts of selected dry fruits on the formation 4.10.3.2 83 of AGEs Carbohydrate hydrolyzing enzymes inhibition by plant 4.11 88 materials Carbohydrate hydrolyzing enzymes inhibition through 4.12 88 selected vegetables 4.12.1 α-amylase inhibitory activity 88 4.12.2 α-glucosidase inhibitory activity 88 Carbohydrate hydrolyzing enzymes inhibition through 4.13 90 selected dry fruits 4.13.1 α-amylase inhibitory activity 90 4.13.2 α-glucosidase inhibitory activity 90 5 SUMMARY 92 LITERATURE CITED 95
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LIST OF TABLES
Table Title Page No. No. 2.1 Amadori products associated with diabetic complications 24 3.1 Scientific and vernacular name of selected vegetables 31 4.1 Proximate composition of selected vegetables (%) 46 4.2 Proximate composition of selected dry fruits (%) 48 4.3 Mineral composition of selected vegetables (mg/100g) 52 4.4 Mineral composition of selected dry fruits (mg/100g) 52 4.5 Qualitative analysis of phytoconstituents of selected plant materials 53 Quantitative analysis of phytoconstituents (mg/g) of selected 4.6 55 vegetable Quantitative analysis of phytoconstituents (mg/g) of selected dry 4.7 56 fruits 4.8 Antioxidant potential of selected vegetables 63 4.9 Antioxidant potential of selected dry fruits 63 Cytotoxic activity of methanolic extracts of vegetables against brine 4.10 65 shrimp nauplii at different concentrations Cytotoxic activity of methanolic extracts of dry fruits against brine 4.11 66 shrimp nauplii at different concentrations 4.12 Thrombolytic activity of methanolic extracts of selected vegetables 67 4.13 Thrombolytic activity of methanolic extracts of selected dry fruits 68 Effect of methanolic extracts of different vegetables on the 4.14 75 reduction of NBT induced by glycated BSA Effect of methanolic extracts of different dry fruits on the reduction 4.15 76 of NBT induced by glycated BSA Effect of methanolic extracts of different vegetables on the 4.16 78 formation of α-dicarbonyl compounds in glucose/BSA system Effect of methanolic extracts of different dry fruits on the formation 4.17 80 of α-dicarbonyl compounds in glucose/BSA system Effect of methanolic extracts of different vegetables on the 4.18 84 formation of AGEs in glucose/BSA system Effect of methanolic extracts of different dry fruits on the formation 4.19 86 of AGEs in glucose/BSA system
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LIST OF FIGURES
Fig. No. Title Page No.
2.1 Structure of the advanced glycation end products 20 AGEs formation pathways incorporating the polyol pathway and 2.2 by the α-oxoaldehydes, methyl glyoxal (MGO) and 3- 21 deoxyglucosone (3-DG). 2.3 Receptor for advanced glycation end product 23 Competitive inhibition of enzymatic hydrolysis of carbohydrate 2.4 26 substrates by α-glucosidases in the small intestine 3.1 Flow chart of phytochemical extraction of selected plant materials 32 Percentage yield of extracts from selected plant materials using 4.1 44 methanol and ethanol as solvents 4.2 Energy values of the selected plant species 48 Percent inhibition of proliferation by methanolic extracts of 4.3 70 selected vegetables Percent inhibition of proliferation by methanolic extracts of 4.4 72 selected dry fruits Fluorescence intensity of albumin in various glycation systems of 4.5 79 selected vegetables at different concentrations Fluorescence intensity of albumin in various glycation systems of 4.6 82 selected dry fruits at different concentrations Inhibitory effects on α-amylase and α-glucosidase of selected 4.7 89 vegetables extracts Inhibitory effects on α-amylase and α-glucosidase of selected dry 4.8 91 fruits extracts
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LIST OF ABBREVIATIONS:
AGEs Advanced glycation end products WHO World health organization AR Aldose reductase NADH Nicotinamide adenine dinucleotide FOSHU Foods for special health use ROS Reactive oxygen species RNS Reactive nitrogen species RSS Reactive sulfer species PUFAs Polyunsaturated fatty acids GAE Gallic acid equivalents AOA Antioxidant activities DPPH 2, 2 diphenyl-1-picrylhydrazyl TPC Total phenolic content TFC Total flavonoid content
H2O2 Hydrogen per oxide SPGE Sweet potato greens extract ALI Arginine-lysine imidazole AFGP Alkyl formyl glycosyl pyrrole CML N-carboxymethyllysine GOLD Glyoxal lysine dimer MOLD Methylglyoxal lysine dimer MGO Methylglyoxal RAGE Receptor advanced glycation end products DMSO Dimethyl sulfoxide MTT 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide NBT Nitro-blue tetrazolium BSA Bovine serum albumin PNPG p-nitrophenyl-a-d-glucopyranoside BSLB Brine shrimp lethality bioassay
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Antiglycation and antioxidant potential of selected plant materials Abstract Considering, the nutraceutical worth of phytoconstituents, current research was designed to focus on the antioxidant and antiglycation properties as well as the biochemical analysis of different edible species locally available and readily consumed. The species dealt in current study include vegetables Ipomoea batatas, Trigonella foenum-graecum, Daucus carota, Solanum Melongena, Brassica rapa rapa and dry fruits i.e. Phoenix dactylifera, Juglans, Prunus dulcis, Pistacia vera, Arachis hypogaea, Vitis vinifera L., Cocos nucifera, Prunus armeniaca. In this context, optimization of solvent extraction (methanolic/ethanolic) for the isolation of bioactive molecules was carried out. Maximum yield was recorded for methanol used for further biological potential. Nutritional analysis revealed that all the edibles were rich sources of crude protein, carbohydrate, fat and dietary fiber. Micro and macro minerals analysis also exhibited the significant presence of Na, K, Ca, Mg and P. Preliminary phytochemical screening unveiled the extraordinary incidence of alkaloids, saponins, tannins, flavonoids that was also proved by quantitative analysis. Maximum total phenolic and flavonoid content was responsible for their significant antioxidant capicities and biologicl activities. Toxicological secreening exhibited slightly higher lethality toward dry edibles whereas no cytotoxicity was observed for the tested vegetables against the brine shrimps nauplii. In order to thrombolytic activity, all the investigated samples exhibited moderate clotlytic percentage except Juglans and Trigonella foenum-graecum. Colon and lung tumor derived cell lines (Ht-29 and H1299) revealed different sensitivity for the tested extracts. Early and intermediary glycation products through NBT reduction assay and Girard T test revealed considerable inhibitory effects at different incubation periods. A strong inhibition for glucose-induced advanced glycation end-product (AGE) in a dose and time dependent manner was recorded. In contrast to α-amylase, a significantly higher inhibition towards α- glucosidase was shown by the selected samples; an impending remedial approach connected to postprandial hyperglycemia. In this scenario, plant derived foods enriched with bioactive moieties are effective to tailor specific healthy diet for the target population. ix
CHAPTER # 1 INTRODUCTION
Throughout the ages, nature has fulfilled all the basic needs of humans including food stuffs, clothing, and means of transportation, shelters, flavours and medicine which form the basis of locally used medicine systems and provide remedies to mankind since ancient times (Heinrich, 2010). Although all the properties attributed to therapeutic nature of plants are not true, medicinal therapy of plants is the result of findings of many centuries (Ahmad et al., 2013). Moreover, the use of herbal remedies has risen in the developed countries in the last few decades because of their lesser side effects. Among the natural biodiversity of flora and fauna approximately 70,000 green species are well-known in folk and modern medicinal systems all over the world (Schippmann et al., 2006). Many medicinal plants may also have direct nutritional benefits of which we know little.
Ethnomedicinal study is considered as an important tool for the discovery of plant based medication. Right from the commencement of ethnobotany, the documentation of folk medicinal acquaintance of plants, has discovered a variety of contemporary medications (Gilani and Rahman, 2005). Currently, one quarter of drugs included in the modern pharmacopeia are plant-derived and many others are synthetic analogs built on proto-type compounds isolated from plants (WHO, 2006). For example, aspirin a popular analgesic, was derivative of species of Salix and Spiraea originally and some valuable anti-cancer agents like paclitaxel and vinblastine are solely derived from plant sources (Ahmad et al., 2013).
Dietary guidelines emphasize the dynamic aspects of phytonutrients as they exert beneficial effects on human health particularly against cardiovascular complications, diabetes, neurodegenerative diseases and oncogenic events. The veracity has encouraged the supplementation of plant-derived nutraceuticals to modulate the onset of chronic ailments. There are significant evidences supporting the presence of bioactive moieties in fruits and vegetables, helpful to attenuate various ailments (Alipoor and Rad, 2012). The proven facts also provide an insight regarding balanced nutrition and disease prevention. Thereby, functional and nutraceutical foods are imperative due to their assuaging nature, nutritional worth, sustainability and safe status. Phytonutrients are plant-derived materials performing key role in maintaining human health, especially in disease prevention and cure. In recent
1 Chapter # 1 Introduction
era, phytochemicals-based nutraceuticals particularly those from fruits and vegetables are becoming popular due to consumer’s awareness of their health-enhancing potential. Epidemiological studies have correlated the consumption of these constituents with declining incidences of several physiological threats. These bioactive ingredients like phenolics and carotenoids are diversified in nature which shows considerable antioxidative activity (Shahidi, 2009). Understanding the complex role of diet in chronic diseases is challenging since a typical diet provides more than 25,000 bioactive food constituents (Wang et al., 2012), many of which may modify a multitude of processes that are related to these diseases. Phenolic compounds are a much diversified group of phytochemicals that are endemic in human nutrition from ancient times by virtue of their chemical structures that signify numerous health enhancing properties (Su, 2012). These are secondary metabolites over 8000 structural variants such as phenolic acids, stilbenes, coumarins, lignins and flavonoids (Wang et al., 2012). Total dietary intake of these identified subtances ranged up to 1g/day, which is 10-100 folds higher than other antioxidants like vitamin C and E (Manach et al., 2004). Currently, epidemiological studies suggest that the consumption of natural polyphenol-rich food such as fresh fruits, vegetables or teas reduce oxidative stress and degenerative ailments owing to their intrinsic antioxidant potential (Serrano et al., 2007; Patel et al., 2012). Besides, they contribute indirect protection by activating the endogeneous defense systems by modulating the cellular signaling processes (Pal et al., 2012). Flavonoid is also an enormous group of polyphenol phytonutrients that are water-soluble pigments and also a predominant component of Eastern folk medicines for thousands of years (Tapas et al., 2008). It is important for human health because of their high pharmacological activities. Flavonoids are potent free radical scavengers by donating their alcoholic hydrogen atoms to free radicals to prevent coronary heart disease and exert anticancer activities. Flavonoids, especially quercetin, are potent antidabetic agents because they exert multiple actions that are both hypoglycemic and antihyperglycemic (Pereira et al., 2011). They brings about the regeneration of pancreatic islets to stimulate insulin release and enhance Ca2+ uptake from isolated islets cell which suggest a place for flavonoids in noninsulin-dependent diabetics (Tapas et al., 2008). Currently, life style matters in a variety of ways; ease in life, diet with less hectic preparation and to relish palatability have adversely affected dietary pattern thereby altered the metabolic pathways along with production of free radicals (Shahidi, 2009). Among various dietary
2 Chapter # 1 Introduction
interventions to cope with life-threatening ailments, such as polyphenols, carotenoids like β- carotene and lutein are the promising components such constituents are effective against an array of non-communicable disorders like dyslipidemia, diabetes mellitus and oxidative stress thus exerting hypoglycemic, hypocholesterolemic and some other beneficial effects. Consumption of diet rich in polyphenolis increases the antioxidant concentration in blood and body tissues thereby protects against oxidative stress (Pennathur et al., 2010). In modern era, diet therapy with special reference to polyphenols has been invigorated worldwide and people are using natural food materials as an intervention against various maladies. Among different regimen tools, polyphenolic enriched functional and nutraceutical foods engrossed attention due to their acceptability, easy access, low cost and long administration safety (Thielecke and Boschmann, 2009). Functional and nutraceutical foods resemble with traditional ones, however, provide some additional health benefits. Thereby provision of such bioactive moieties is one of the prime benefits of fruits and vegetables consumption (Shahidi, 2009). Scientists are covering a predominant focus on the reduction of various metabolic ailments by introducing suitable alterations in the dietary pattern. An inverse relationship is found between some fruits and vegetable consumption and morbidity as well as mortality of degenerative diseases that may be partly due to their antioxidants (Wootton-Beard and Ryan, 2014; Zia-ul-Haq et al., 2014; Zhang and Liu, 2015). Because of potential effects of antioxidants from plants on reactive oxygen species, they are thought to be health promoting. In addition antioxidants prolong the shelf life of commercial food. However it is acclaimed that some synthetically originated antioxidants like butylhydroxytoluene and butylhydrxyanisole, due to their toxicity and potential health risks are needed to be replaced with natural ones (Li et al., 2013). In 21st century, diabetes mellitus is considered the most severe metabolic syndrome as a “third killer” of mankind along with the cancer and coronary complications (Jain and Saraf, 2010). Currently, Pakistan is holding 6th position in terms of diabetic patients (WHO, 2006). The global prevalence was estimated at 2.8% in 2000 (171 million people) and it is projected that 4.8% (366 million people) will be affected in 2030 if no action is taken. Chronological evidences proved diabetes mellitus a renowned and divergent disorder which can be distinguished into two types one was genetic and the other was the result from dietary imprudence (Wild et al., 2004).
3 Chapter # 1 Introduction
Development of diabetes involves several pathogenic processes ranging from autoimmune pancreatic β-cell destruction resulting insulin deficiency and cause abnormalities leading to resistant insulin action. Basically, metabolic abnormalities of fat, carbohydrate and protein are due to deficient insulin action on target tissues. Deficiency of insulin action is due to inadequacy of insulin secretion and/ or less tissue response to insulin at certain points during complex pathway of its action. Impaired insulin secretion parallels with defective insulin action and coexists in the patient frequently to such an extent that it is difficult to determine the primary cause of hyperglycemia (American Diabetes Association, 2013). Complications lead to mortality in diabetes mellitus. These complications arise due to loss of control of blood glucose. For many individuals achievement of good control is not easy because the hormonal control which maintains glucose balance is destructed by the disease (Kazeem et al., 2013). As diabetes is a potential hazard for quality of life due to its chronic problems, people are turning towards other therapies to combat this disease (Yao, et al., 2013). Due to their health-promoting properties of phytoconstituents more than 800 plants have been introduced as herbal medicines for the treatment of diabetes (Mentreddy, 2007). A number of plant derivatives are representatives of chemical compounds and show variety of mechanisms of action are likely to be involved in lowering blood glucose levels; with their role in the treatment of diabetes mellitus (Ali et al., 2012). These include glycosides, alkaloids, polysaccharides, galactomannan gum, hypoglycans, peptidoglycans, steroids, amino acids, glycopeptides, carbohydrate, terpenoids, guanidine, and inorganic ions. High levels of glucose activate many important biochemical mechanisms in diabetes. The elevation of advanced glycation end-products (AGEs) formation is one of the forfeits of such hyperglycemic condition (Yan et al., 2008). The Maillard reaction is a process which links chronic hyperglycemia to a series of physiopathological alterations considered important in the development of chronic complications of diabetes and different diseases (Goldin et al., 2006). Initially, the formation of Schiff bases between the free amino groups of the proteins (predominantly the ε-amino group of lysine and the guanidine group of arginine) and carbonyl groups of reducing sugars or other carbonyl compounds which is followed by various Amadori rearrangements, generating a relatively stable aminoketose derivative. These derivatives undergo further degradative changes, giving rise to highly reactive dicarbonyls, such as glucosone, 3-
4 Chapter # 1 Introduction
deoxyglucosone, and glyoxal, all capable of acting as protein cross linking agents and forming additional Schiff bases (Berrou et al., 2009). The ultimate effect of protien glycation is generation of high molecular weight protein aggregates and other fluorescent entities, referred to as advanced glycation end products (Lapolla et al., 2005). During chronic hyperglycemia, excessive glucose uptake in tissues also affects the key enzyme aldose reductase (AR) in the polyol pathway. This leads to the reduction of various sugars to sugar alcohols, such as glucose to sorbitol, followed by NADH-dependent sorbitol dehydrogenase-catalyzed fructose production. Increased fructose formation in turns leads to the production of reactive carbonyl species which are key factors in AGEs formation. Furthermore, sorbitol and its metabolites accumulate in the nerves, retina, kidney, and lens due to poor penetration across membranes and inefficient metabolism, resulting in the development of diabetic complications (Cooper, 2008; Yan et al., 2008). Millard reaction is a spontaneous reaction depending in vivo on the degree and duration of hyperglycemia (Berrou et al., 2009) thus the excessive constant supply of glucose increases
the formation of AGEs in diabetic patients. HbA1c, a very good biomolecule, used to assess glycaemic control, as blood glucose levels is in proportion to the degree of glycation.
HbA1c allows estimating glucose levels in blood over a relatively long time period i.e.at least 2 months. In hyperglycaemic conditions, autooxidation of glucose takes place generating OH radicals (Noire and Tsukahara, 2005; Goldin et al., 2006). Epidemiological evidences revealed that the formation and accumulation of AGEs can be the consequence in aging, while extreme acceleration was found in the progression of diabetes along with the pathogenesis of many other diseases such as neurodegenerative diseases and diabetic vascular complications (Lee et al., 2007). Under these consequences, inhibition of AGE formation is specifically a promising target for the assessment of therapeutic potential in AGE-related disorders (Zhang et al., 2011). Hyperglycemia leads to oxidative stress in diabetes, causing increased free radical formation via glucose auto-oxidation and interrupting the electron transport chain. It also happens during AGE formation (Ahmed, 2009). A high oxidants level mainly affects cell components by lipid peroxidation, and then triggering the signaling pathways specifically as in protein kinase C and nuclear factor-κB releasing proinflammatory cytokines. Thus ineffective scavenging and increased production of reactive oxygen species play an important role in diabetes mellitus. Impairment in the metabolism of glutathione, alteration
5 Chapter # 1 Introduction
in levels of antioxidant enzymes and decrease in levels of ascorbic acid cause the disturbance in the defense system of antioxidants leading to diabetes (Patel et al., 2012). Advanced glycation end-products and oxidative stress are associated with the pathogenesis of diabetes and its complications. Interaction of both antioxidants and antiglycation activities is well known. Therefore, it is found that natural extracts or compounds that have both activities as antioxidants and antiglycatin agents may have great therapeutic potential for treatment of diabetes and complications (Chen et al., 2011). Numerous studies have also authenticated that the anti-glycation potential significantly correlates with the plant based phenolics (Ho et al., 2010; Ignat et al., 2011), a most abundant dietary antioxidants (Ignat et al., 2011). Therefore, the process of AGEs formation can also be retarded through antioxidants by preventing further oxidation of intermediary compounds of glycation and metal-catalyzed glucose oxidation. Presently, the upshots regarding antiglycation potential of vegetables and fruits has gained a considerable attention among scientific community and numerous plant families Anarcardiaceae, Curcubitaceae, Moraceae, Annonaceae, Sapotaceae, Phyllanthus, Myrtaceae, Rutaceae etc., have been documented for their noteworthy role as antioxidant and antiglycation agents (Ahmad et al., 2007; Peng et al., 2008). Several natural compounds like curcumin, rutin and garcinol have been reported to possess antioxidant property as well their role in the prevention of formation of AGEs in vitro and in vivo (Elya et al., 2012). Although some studies highlighted the anti-glycating potential of natural sources still there need comprehensive study to relate antioxidant properties of extracts to the anti-glycating potentials. Recent interests in the study of phytonutrients, polyphenols have also focused on their potential to inhibit carbohydrate-digesting enzymes due to their redox properties along with their other health promoting benefits (Wang et al., 2012). Prospective studies showed that carbohydrate-hydrolyzing enzymes such α-amylase and α-glucosidase linked to postprandial hyperglycemia in the digestive tract (Hays et al., 2008; Kwon et al., 2008; Ranilla et al., 2010. Epidemiological studies connect postprandial hyperglycemia and oxidative stress as an earliest metabolic aberration in the progression and prevention of metabolic ailments (Kim et al., 2005; James and David, 2007). In this regard, inhibitors can retard the uptake of dietary carbohydrates, suppress postprandial hyperglycemia combined with antioxidative properties
6 Chapter # 1 Introduction
could be therapeutic for metabolic ailments (Kwon et al., 2008; Shibano et al., 2008; Kim et al., 2010). Natural resources especially, fruits and vegetables provide a huge and highly diversified chemical bank from which we can explore for potential therapeutic agents by bioactivity- targeted screenings (Lam et al., 2008) including aproteic, e.g. antibiotics, alkaloids, terpenes, cyanogenic glucosides and digestive enzymes inhibitor. Even though the antidiabetic activity has been reported in the plant species, no work on antiglycation has been explored in the edible plants species. Opportunity exploration of therapeutic potential of fruits and vegetables is still very wide open in line with the development of herbal industry, herbal medicine, and phytopharmaca (Elya et al., 2012). To more fully characterize the antioxidant profile and possible associated health benefits of different fruits and vegetables, it is worth quantifying these bioactive compounds. Considering the eminence of plant bioactive constituents as one of the tools in diet-based therapy, functional components of fruits and vegetables have potential to address various metabolic syndromes among the vulnerable segments. Keeping in view, the nutraceutical worth of phytonutrient including polyphenols, current research project was designed to focus on the antioxidant and antiglycation properties as well as the biochemical analysis of different edible species those are locally available and readily consumed. The species dealt with in the research project include vegetables Ipomoea batatas (Sweet potato), Trigonella foenum-graecum (Fenugreek), Daucus carota (Carrot), Solanum melongena (Eggplant), Brassica rapa rapa (Turnip) and dry fruits Phoenix dactylifera (Dates), Juglans (Walnut), Prunus dulcis (Almonds), Pistacia vera (Pistachio), Arachis hypogaea (Peanuts), Vitis vinifera L (Raisins)., Cocos nucifera (Coconut), Prunus armeniaca (Apricot). In this context, Optimization of extraction conditions for the isolation of bioactive molecules was carried out. The specific objectives set to be attained are herein:
Comprehensive exploration of biochemical and biological activities of selected plant materials. Antiglycation & antioxidant activities of the selected plant materials to investigate their inherent potential as natural inhibitors of AGEs and oxidative stress. Potential of the selected green materials against the cancer using human cancer cell lines.
7 CHAPTER # 2 REVIEW OF LITERATURE
Fundamental nexus between nutrition and health diverting the consumers focus towards plant based products as a remedy against various metabolic syndromes. In diet-based regimen, functional/nutraceutical foods are gaining the core attention of researchers to be a therapeutic device against the maladies (Sohaimy, 2012). The escalating consumption of these phytonutrients provides a viable approach towards nutrient optimization and food synergy. No doubt, medicines are inevitable for curing various physiological disorders nevertheless, high treatments cost predominantly in the developing countries along with associated side effects demanding some other rational approaches to meet the peril. In this scenario, plant- derived foods enriched with bioactive moieties are effective to tailor specific healthy diet for the target population. There are established facts that fruits and vegetables are concentrated source of natural constituents that alone or in combination beneficent for the human health (Hwang et al., 2012). Consequently, fruit derived nutraceuticals are of significant importance to curtail various physiological threats including hyperglycemia, hypercholesterolemia along with oncogenic modulating perspectives via distint pathways (Liu, 2013). Considering the facts, present exploration was an attempt to evaluate the therapeutic role of some locally grown vegetables and fruits. The literature regarding different features of the current work has been piled under the following headings.
2.1. Concepts of functional and nutraceutical foods 2.2. Oxidative stress and safety concerns 2.3. Phytoconstituent as antioxidant 2.4. Antioxidant capacities of plant Materials 2.5. Anticancer potential of plant materials 2.6. Glycation and advanced glycation end products 2.7. Carbohydrate hydrolyzing enzyme inhibition 2.1. Concepts of functional and nutraceutical foods "Let food be the medicine and medicine be the food”. Quality nutrition is a core element for optimal health to mitigate various physiological disorders during different stages of life from childhood to elderly age. The health and nutrition paradigm has been significant modified
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during the last few decades. During these days foods not merely considered as a vehicle to supply nutrients for proper body functioning but also a source to maintain good health. Thus, core devotion has been paid to illuminate the theraputic role of the diet. This had set the ideology of functional and nutraceuticals as the food that exerts beneficial effects beyond nutrition thereby reducing various ailments (Sohaimy, 2012). In developing economies, functional/nutraceutical foods have emergent market due to increase consumer awareness regarding diet health interaction and high medication cost during disease. The concept of functional foods was introduced in Japan during mid 80’s and refered as food that provides additional physiological benefits beyond basic body needs thus called as Foods For Special Health Use (FOSHU). Numerous terms are interchangeably used to describe the linkages between disease mitigation and health promotion with refernce to specific foods ingredients. Earlier the US Foundation for innovation in medicine introduces the concept of nutraceuticals in 1989 as any food or part of it that provide health benefit including disease prevention (Zia-ul-Haq et al., 2014). Phytoremedies have been in practise since centuries and also becoming popular in the recent era due to their natural origin and safe status. Considering the importance scientists are gradually focusing their attention to explore the phytochemicals for health managements and enhancement. Plants contain certian bioactive substances endowed with potent anioxidative properties. Phytochemicals are abundantly presents in plant based diets even responsibe for their distinct color and flavor. Various fruits and vegetables have innate theraputic worth linked to their bioactive constituents including phytochemicals e.g. (poly) phenolic compounds and carotenoids, phytoncides (natural antibioics) vitamins (mainly ascorbic acid, provitamin A, folate), minerals and dietary fiber (Murphy et al., 2012; Liu, 2013). Numerous plant sources have been tested for their antioxidative quality against several metabolic ailments. This can be examplified by vegetables like garlic, onlion, parsnip, fenugreek etc. that enhance the glutathion redox cycle and activate immune system due to sulfer containing organic compounds. These are considered active as antioxidant, anti- inflammatory, anticancer, antimicrobial and immunostimulating alongside showing potential against hyperglycemia and hypercholesterolemia. Similarly, phytochemicals, such as anthocyanins, from black grapes and from the red or violet fruits is in practice for prophylaxis of various diseases. Flavonoids are the active constituents from citrus fruits, tea,
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and grapes showing anti-inflammatory action, and strength the body against various allergies, viral attacks and tumor inducing factors (Hwang et al., 2012). In recent times, various epidemiological studies advocated that the intake of functional foods and nutraceuticals have protective effects against various physiological dysfunctions owing to their prophylactic role. Several health associating problems can be addressed by proper diet planning. Moreover, the consumption of natural foods including vegetables, fresh fruits and teas have defensive possessions against different chronic ailments such as diabetes, coronary heart disease, gastrointestinal disorders, and can also reduce the risk of colon cancer. The protections have partially been attributed to numerous components such as anthocyanins, flavonoids, vitamins and other phenolics (Serrano et al., 2007; Pasricha and Gupta, 2014). 2.2. Oxidative stress and safety concerns
Free radicals are formed not only in normal cells but also during the metabolism of
pathological cell. It is an irony that these vital elements through reactive species are affecting the human body deleteriously. Endogenous human metabolic and exogenous chemical processes in the food systems might produce highly reactive oxygen species (ROS) particularly oxygen derived radicals, that have a tendency to oxidize other biomolecules, leading to cell death and damage of tissues (Dar et al., 2012). The redox environment of the cell is a balance between the production of highly reactive oxygen and nitrogen species, and their elimination through antioxidants. ROS, RSS and RSN target proteins, sugars, DNA, RNA and lipids mainly. Oxidative modification of proteins can take place either through protein cross linkage with the products of lipid peroxidation or with the oxidative cleavage of peptides, modifications of particular amino acid mediated by free radicals (Lobo et al., 2010; Ayala et al.,, 2014). Oxidative damage of DNA has a distinctive alteration pattern that may be described structurally as well as chemically. It includes modifications of bases, base free site generation, breakage of strands, deletions, chromosomal rearrangements and cross-linking of proteins and DNA. Also, Fenton reaction is an imperative reaction that encompasses DNA damage through generation of free radicals. Glyco-oxidative damage arises during the early glycation process by the generation of oxygen free radicals. Sugar fragmentations such as glycoaldehydes are also the products of early phase glycosylation. Superoxide radicals also
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originated due to the shortening of chains that cannot be cyclized and are autoxidized. α and β-dicarbonyl compounds, renowned mutagens arises by the dissemination of consequential chain reactions (Dar et al., 2012). During lipid peroxidation free radicals or non-radical species attack on lipids containing carbon-carbon double bonds, particularly polyunsaturated fatty acids (PUFAs) that implicate hydrogen abstraction from a carbon, with the insertion of oxygen succeeding the generation of lipid peroxyl along with hydroperoxyl radicals. These highly reactive species have a tendency to react with the adjacent molecules leading to the propagation of lipid peroxidation
reactions (Ayala et al.,, 2014).
LH+OH∙ H2O+L∙ Where LH is a general lipid and L∙ is a lipid radical. Carbontetrachloride (CCl4) When added to oxygen, forms trichloromethyl radical (CCl3O2) which attacks lipid.
LH + CCl3O2∙ L∙ + CCl3OH Hydroperoxyl radical cause damage to isolated PUFA's as under LH + HO2∙ L∙ + H2O2 Glycolipids, cholesterol and phospholipids are also renowned targets of damaging and potentially disastrous peroxidative alterations. ROS, RNS and RSS are the outcome of numerous reactions associated to various maladies including neurological disorders, cardiovascular diseases, renal infirmities, cancer, diabetes mellitus, liver disorders, inflammation, autoimmune deficiency diseases, , degenerative disorders related with aging, obesity, etc. (Singh et al., 2010; Elya et al., 2014). 2.3. Phytoconstituents as antioxidant Phytoconstituents are the dynamic sources of antioxidants due to their redox potential.. Natural resources especially fruits and vegetables provide a huge and highly expanded chemical bank from which we can reconnoiter for potential therapeutic agents by screening the bioactivity-targeted including phenolics, flavonoids, carotenoids, alkaloids, terpenes, tocopherols, and tocotrienoles to stop oxidation substrate. In biological system plant-based antioxidants are regarded as more efficient than synthetic one. In living sytem phytoconstituents takes part in physiological functions and have better compatiblility within humans (Ayala et al.,, 2014).
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2.3.1. Tocopherols composition Tocopherols, the generic name of vitamin E, constitute a series of linked benzopyranols that occur in vegetables oils and plant tissues and are influential lipid-soluble antioxidants. Two classes of vitamin E are tocotrienols and tocopherols; each have alpha (α), beta (β), gamma (γ) and delta (δ) isoforms in which the most potent and abundant isoform is α-tocopherol for the biological system. Lipid peroxidation and radical mediated damage of other constituents of membrane is inhibited by tocopherols and shields the integrity of membranes by scavenging the lipid peroxyl radical much more rapidly as compare to the radical action on surrounding lipid substrate (Lu et al.,, 2015). Tree nuts and peanuts are enriched source of tochoferols along with vegetables and seeds. Except walnuts the traces of δ-tocopherol originated in almost all the tree nuts. Barreira et al., (2009) reported the tocotrienols in chestnuts, predominantly γ- tocotrienol. Kornsteiner et al., (2006) recounted several-folds of vitamin E content in selected tree nuts. Jeanes et al., (2004) revealed that tocopherols absorption is influenced by the high fat content of food matrix. Jambazian et al., (2005) found the fact that elevated intake of almonds significantly increases the level of plasma α-tocopherol. 2.3.2. Phenolic content Polyphenolic compounds are the most diverse group of secondary plant metabolites, distributed into several groups according to their structural diversity. Of these, flavonoids, tannins and phenolic acids are the predominent dietary phenolic compounds (D’Archivio et al., 2007), estimated at 500-1000 mg of total daily intakes. Reduced progression of numerous chronic ailments concomitant with phenolic consumption also accredited to their array of bio-mechanisms such as anti-inflammation, antioxidation, lowering of cholesterol and carcinogen detoxification. Fenugreek seeds are rich source of polyphenols including, flavonoids, scopoletin, coumarin, chlorogenic, P-coumaric and caffeic acid (Pasricha and Gupta, 2014; Yadav et al., 2014). Also, Eggplant provides a significant quantity of phenolics to the diet (Tiwari et al., 2009). Previously, Stommel and Whitaker (2003) optimized the extraction of Solanum melongena L for its phenolic acids using mixture of solvents and found eggplant as a rich source of phenolic compounds including 5-caffeoylquinic acid, N-caffeoylputrescine and 3-acetyl-5- caffeoylquinic acid (Singh et al., 2009). Fernandes et al., (2007) determined the phenolic compounds of edible parts of Brassica rapa var. rapa L. by HPLC–UV and HPLC–DAD
12 Chapter # 2 Review of Literature respectively and revealed the profile of phenolics comprehending kaempferol 3-O- sophoroside-7-O-glucoside, isorhamnetin 3-O-glucoside and isorhamnetin 3, 7-O-diglucoside as the chief phenolics. Numerous classes of polyphenolics have been identified in tree nuts and peanuts including flavonoids, anthocyanins, lignans, proanthocyanidins and tannins etc. However, inadequate information, regarding the polyphenolic intake from tree nuts is available. Saura-Calixto et al., (2007) assessed the daily polyphenolic intake of Spanish population (102-121 mg) from nut consumption in which bioaccessible content was 107mg. According to Urpi-Sarda et al., (2009), polyphenols from almonds were bioavailable and bioaccessible; subsequently metabolized by gut microflora and conjugating enzymes. Nut Polyphenolics varied from 103-1650 mg GAE/100 g in which pistachio and walnuts exhibited the maximum values (Wu et al., 2004; Kornsteiner et al., 2006). The presence of phenolic acids was also attributed in almonds by Milbury et al., (2006). According to (USDA, 2007), nuts polyphenolic content was in accordance to the fruits such as blueberries, plums and raisins.
2.3.3. Flavonoids Flavonoids are another assembly of antioxidants comprising flavanols, flavonols, anthocyanins, flavanones, isoflavonoids and flavones, having a common structure of diphenyl propane among all the subgroups of compounds. Some fruits usually contain both enzyme specific flavones and flavanones while flavonols and flavones are rarely found in the same fruit. Flavanone enriched plants lack anthocyanins. Phenolic hydroxyl groups that are attached to ring structure confer antioxidant properteis of flvonoids, acting as reucing agent, hydrogen donor, superoxide radical scavenger, metal chelator and quencher of singlet oxygen. Flavonoids isolated from S. melongena showed potent antioxidant activity against chromosomal aberrations induced by doxorubicin. Tiwari et al., (2009) confirmed the presence of flavonoids during the phytochemical examination of the methanolic and aqueous extracts of Solanum melongena. In addition, identification of trace quantities of flavonols in the pulp of freeze-dried eggplants was probed by previous studies (Singh et al., 2009; Kaur et al., 2014). Tree nuts and peanuts all are rich source of flavonoids except Brazil nut and macadamias. The predominent nut flavonoids were flavonols and anthocyanins having isoflavones and
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flavonones in lesser quantities. Almonds also contain a considerable flavonoid content demonstrating 15 mg/100g. So far, only in almonds, flavanones have been instigated. Only, pistachio and almonds are augmented with flavonols, primarily as quercetin and isorhamnetin (USDA, 2008). The stilbene and resveratrol were present in peanuts and pistachios in a comparable level (Tokusoglu et al., 2005). 2.3.4. Carotenoids Carotenoids are the natural pigments, synthesized by plants, microorganisms and animals. The two main classes of carotenoids are (1) caroteins (hydrocarbon carotenoids) e.g., β- carotene, lycopene and (2) xanthophyls (oxygenated carotenoids) such as lutein and zeaxanthin. Antioxidant property of these pigments is primarily due to their radical scavenging potential which excite the carotenoids, dessipate energy through rotational and viberational interactions with solvents and returns to the relax state that further allow to quench more oxidents. Only peroxyl radicals can completely destroy these pigments. Carotenoids are copious in mostly colored plant derived foods, e.g., 7.6 mg/100 g of β- carotene and 6.6 mg/100 g of lutein found in carrots and spinach. Lako et al., (2007) unveiled the β-carotene (13 mg/100 g) content in Ipomoea batatas (sweet potato). In contrast, tree nuts and peanuts comprehend least concentration of carotenoids (μg/100 g). Pistachios showed a modest exception with a mean β-carotene (0.21 mg/100 g) and lutein (4.4 mg/100 g) of dry weight, respectively (Kornsteiner et al., 2006; Bolling et al., 2010). 2.3.5. Alkaloids The presence of alkaloid content is responsible for the therapeutic significance of foods including healing wounds, ulcers, hemorrhoids and burns (Okwu and Okwu, 2004). Alkaloids are organic nitrogenous plant secondary metabolites that have complex molecular structures hence, mostly basic in nature, usually colorless and bitter in taste, often optically active substance e.g. nicotine. The most common precursors of alkaloids are amino acids. Many alkaloids are terpenoids in nature and some (e.g Solanine, the steroidal alkaloid of the potato) are the best considered from the biosynthetic point of view as modified terpenoids. Others are aromatic compounds e.g. colchicines (Kanoma et al., 2014). The phytochemical analysis of methanolic and aqueous extracts of methi leaves, stems and kasuri methi by Pasricha and Gupta (2014) revealed good amounts of alkaloids in all the samples. Smaller quantities of trigonelline and gentianine (pyridine), carpaine (piperidine)
14 Chapter # 2 Review of Literature and choline (steroidal) were also reported in Trigonella foenum-graecum L seeds (Mahmoud et al., 2012). In a previous experiment of Solanum melongena for its phytochemical inspection unveiled the presence of alkaloids, saponins and tannins in the methanolic and aqueous extracts of Solanum melongena (Tiwari et al., 2009). 2.3.6. Saponins Saponins, as an anti-nutritional factors have a tendency to decrease the nutrient uptake, such as glucose and cholesterol at the gut through intra luminal physicochemical interaction and divulge their therapeutic significance in the foods (Okwu and Okwu, 2004). Saponins are one of the groups of glycosides found in many plant species with known foaming properties. As glycosides they are hydrolysed by acids to give an agylcone (sapogenin) and various sugar and related uronin acids. The steroidal saponin and pertocyclicterpenoids have a glycosidic linkage at -C3 and have a common biogenetic origin through malvalonic acid and isoprenoid unit (Kanoma et al., 2014). The phytochemical analysis of methanolic and aqueous extracts of methi leaves, stems and kasuri methi by Pasricha and Gupta (2014) revealed moderate amounts of saponins in all the samples. Most prominent saponins were diosgenin, gitogenin and tigogenin (Mahmoud et al., 2012). In a previous experiment for the phytochemical inspection of the methanolic and aqueous extracts of Solanum melongena also displayed the presence of saponins and tannins (Tiwari et al., 2009). 2.4. Antioxidant capacities of plant materials 2.4.1. Antioxidant activities of vegetables Epidemiological studies revealed that greens are predominent source of polyphenolics responsible for their antioxidant activities (AOA). According to Lako et al., (2007), green leafy vegetables had the maximum antioxidative capacities, followed by the fresh fruits and root crops. Among those, leaves of Ipomoea batatas showed the maximum TAC and are enriched in TPP, quercetin and β-carotene. In case of T. foenum-graecum, the antioxidant property of the plant material is due to the presence of many active phytoconstituents. Pasricha and Gupta (2014) screened the methanolic and aqueous solvent extracts of two varieties of Trigonella foenum-graecum L methi and Kasuri methi for their antioxidant activities using ABTS scavenging and FRAP assay. A strong correlation was observed
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between the employed assays and phenolics content, a predominant source of antioxidant activity in both species of Trigonella. Of late, additional attention has been paid to the natural antioxidants, principally to the phenolic compounds, due to their reducing potential of toxic compounds in foods and by supplying exogenous antioxidants to the human body. Al-Mamary, (2002), endorsed that vegetable juices augmented antioxidant activity by decreasing pro-oxidant activities in a dose dependent manner. Also, tomatoes, eggplant and carrots show the virtual antioxidant efficiencies as compared to other analyzed vegetables (Tiveron et al., 2012). Carotenoids, phenolic compounds and ascorbic acid are natural antioxidants capable of scavenging the free radicals, which cause damage to human health associated with oxidative stress (Martínez-Flores et al., 2015). Tabart et al., (2009) revealed significant differences in free radical scavenging activity of several beverages according to the method used. Maximum antioxidant capacities was observed for red wine, followed by green tea, orange juice, grape juice, vegetable juice, and apple juice. According to Saeed et al., (2012) Brassica rapa, being enriched source of vitamins and anti- oxidants, have ability to protect the body from induced oxidative damage. The obtained data in their in vitro models clearly establish the antioxidative potency of Brassica rapa. In another study on dietary turnip, Fernandes et al., (2007) screened the strongest antioxidative potential by means of the DPPH radical scavenging assay. Results revealed the strongest antioxidant capacity of turnip was due to its diversified profile of phenolics. 2.4.2. Antioxidant activities of dry fruits Epidemiological studies indicate that fresh and dried fruits are rich in antioxidants with potent free radical scavenging activity imply their importance to human health. Correlation analysis between the TPC and AOA revealed that phenolics may contribute maximally to the ABTS and to lesser extent to DPPH in fresh fruits, where as in dry fruits they correlated well to DPPH activity and to a lesser extent to FRAP (Reddy et al., 2010). Spigno et al. (2007) probed the extraction kinetics and the solvent effect on phenols yield and quality of extracts. According to data, phenols yield increased for water content of ethanol from 10% to 30% and remained constant for water content from 30% to 60%, while phenols concentration of extracts decreased for water content above 50%. Antioxidant power (ABTS test) highly correlated to total phenols concentration, and was not influenced by water
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content of ethanol, suggesting that this variable influenced only the amount but not the nature of the extracted compounds. Ouchemoukh et al. (2012) determined the antioxidant activities of some selected dry fruits through antiradical activity, phosphomolybdate and reducing power assay. Apricots and raisins possessed a good reducing power, while Agen prune showed significant antioxidant activity to the phosphomolybdate. Significant correlations were determined between total phenolic concentration and antiradical activity of ethanolic extract and reducing power of aqueous extract. In a similar study, Yang et al., (2009) assessed the nine types of tree nuts and peanuts for their antioxidant activities. In this experiment, both soluble phenolic and flavonoid contents were positively correlated with highest total antioxidant activity. The + extracts of grape and grape products revealed highest ABTS , DPPH, H2O2 scavenging and reducing power activities (Keser et al., 2013). Chakraborty et al., (2008) demonstrated the antioxidant activity of the methanolic extract prepared from different stages of Cocos nucifera L. mesocarp, by DPPH, FRAP and deoxyribose assays, and suggests the potential of the mesocarp extract to be used for therapeutic purposes. 2.5. Anticancer potential of selected plant materials Epidemiological evidence still forms the basis of the supposition that antioxidants contained in fruit and vegetables can help to prevent, or affect the development of disease. Mechanistically, strong in vitro evidence exists for these roles, particularly in CVD, cancer and neurological conditions. Cancerous cell proliferation can be initiated by several factors including ROS-mediated cellular damage, dysfuncioning of immune system and gene expression regulation. Regulation of oxidative stress by dietary antioxidants dietary antioxidants can play a role in the pathological progression of this process, particularly in the case of carcinoma. Phenolic compounds and isothiocyanates are also capable of generating
H2O2 via their pro-oxidant activities (Samra et al., 2011). According to the prospective life span study of Japan, daily fruit consumption was associated with the reduced total cancer mortality. 2.5.1. Anticancer potential of vegetables Data specifically allied the consumption of yellow and green vegetables with a comparative threat of liver cancer, stomach and lung cancer due to their diversified chemical bank (Sauvaget et al., 2003). Another larger prospective study, revealed mixed overall results in
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the relative risk of developing cancer with increased fruit and vegetable consumption, reporting null findings for pancreatic, colorectal cancer, breast, lung and bladder cancer (Rohrmann et al., 2007; Vrieling et al., 2009), a small inverse effect on total cancer incidence (Boffetta et al., 2010) and positive results for total mortality and cancer of the upper aero- digestive tract (Boeing et al., 2006) in various different cohorts throughout Europe. One recent study has presented evidence that plasma levels of important, non-enzymatic antioxidants such as vitamin C, β-carotene and vitamin E are lower in patients with untreated breast cancers in comparison to matched controls (Shah et al., 2009). Additionally, Li et al., (2005) have demonstrated that high selenium (RR=0.33; 95% CI=0.16–0.68), and to a lesser degree, lycopene and α-tocopherol levels were associated with modified risk of prostate cancer in patients with the manganese superoxide dismutase polymorphism when they were divided into quartiles of pre-diagnostic antioxidant status. According to Wootton-Beard and Ryan, (2014) vegetables contain an carcinogenic preventive cocktail' which supplies both documented and presently unrecognized constituents and non-nutritive compounds that inhibit the synthesis of carcinogen, provide needed constituents for defense and diminish transformed proliferation of cell. Fenugreek inhibits carcinogenesis in Rats. A bioactive compound, diosgenin of fenugreek seeds, overwhelms total colonic development by inhibiting a growth factor bcl-2 and persuaded protein expression, thereby encouraging apoptosis and supress cell growth. Trigonelline, an alkaloid of fenugreek, also revealed the anticancer potential (Verma et al., 2010; Khoja et al., 2011). In an in vitro study, Polyphenolic extracts of carrot tissue, decreased cancerous cell (HT29 and LoVo) viability up to 20% at the highest concentration (200 μg/mL) (Przybylska et al., 2007). Leukemia cell lines treated with carrot juice extract induced apoptosis and inhibited progression through the cell cycle (Zaini et al., 2011). According to Yang et al., (2008), phenylpropanoids were the responsible cytotoxic components in carrot. The potential anti-cancinogenic outcomes of carrot extract could be due to the augmentation in T-lymphocytes, protien inactivation of responsible for the propagation of cancenogenic cells. Rabah et al. (2004) revealed that the aqueous extract from baked sweet potato showed potential cancer-preventing effects against various human derived cancer cell lines in a dose response manner. In a previous study, polyphenol-rich sweet potato (Ipomoea batatas) greens extract (SPGE) exerts substantial antiproliferative properties in a panel of prostate
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cancer derived cells (Karna et al., 2011). Mechanistically, SPGE disturbed cell cycle progression; lessen clonogenic persistence, modulated cell cycle and regulatory components of apoptosis in human prostate cancer derived cells both in vitro and in vivo. 2.5.2. Anticancer potential of dry fruits Earlier epidemiological studies have indicated that higher nut consumption appear to confer health benefits despite their high fat content and have potentially protective effect in cancer by a number of different mechanisms. The proposed mechanisms of action for the health benefits of nuts may include a favorable fatty acid profile, antioxidant activity (Wu et al., 2004), reduction in the initiation of tumour, cell differentiation and proliferation, immunological regulation and hormonal regulation. Davis and Iwahashi, (2001) assessed the in vivo effect of almonds on colon cancer and proposed that almond consumption may reduce the risk of colon cancer via at least one lipid-associated constituent. Cocos nucifera husk may also be a source of new drugs with antineoplastic and anti-multidrug resistances activities. It is of great interest for chemotherapy to identify new compounds to overcome resistance mechanism (DebMandal and Mandal, 2011). Phytoconstituents, especially polyphenolics in nuts considered to be the chief bioactive compounds due to its health promoting potential. Yang et al., (2009) evaluated nine types of tree nuts and peanuts for their antioxidant, and antiproliferative potential. In this investigation, walnuts possessed maximum total phenolics and flavonoids responsible for it’s the highest total antioxidant potential and the reduced proliferation of Caco-2 and HepG2 cells in a dose-dependent pattern after exposure to the extracts of nuts. 2.6. Glycation and formation of advanced glycation end products Non-enzymatic glycation can be induced by hyperglycemia, causing formation of reversible Schiff base between free amino group and carbonyl group of a protein and reducing sugar respectively due to nucleophilic addition reaction. This reaction takes time and once Schiff base is formed, it rearranges itself to more stabilized form called Amadori product which may be a ketoamine or fructosamine (Lapolla et al., 2005) in equilibrium as cyclic forms of pyranose or furanose for added stability. Amadori products on reaction with a primary amine like ε-amino-lysine or itself give rise to advanced glycation end products (Ho et al. 2010). Oxidative Pentosidine, N-carboxymethyllysine, crossline and glucospane are the AGEs produced by oxidative or non-oxidative pathways. The covalent bond formation to build
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sugar-protein complex via a series of reactions called Millard reaction (Ferchichi et al., 2012). 2.6.1. Advanced glycation end products AGEs are hetrogenous, complex molecules causing cross linking of proteins while exhibiting browning generates fluorescence. All of the AGEs have not yet identified and their mechanism of formation is still not clear. Due to their slow generation, it was assumed that only extracellular proteins which are long lived causes formation of AGEs. But now it is clear that molecules that are short-lived give rise to AGEs including intracellular growth factors (Goldin et al., 2006; Ferchichi et al., 2012). AGEs may be derived from food expressing its origin exogenously (Miroliaei et al., 2011). More than dozen AGEs detected in tissues are categorized as under: 1. Fluorescent crosslinking AGEs like pentosidine and cross line Non-fluorescent cross linking AGEs includes imidazolium dilysine cross-links, cross-links of arginine-lysine imidazole (ALI) and cross-links of alkyl formyl glycosyl pyrrole (AFGP). 2. Non-cross-linking AGEs like N-carboxymethyllysine (CML) and pyrraline.
CML Pentosidine MOLD Fig. 2.1. Structure of the advanced glycation end products
Pentosidine arise from cross-link formation between residues of arginine and lysine and its level is increased in diabetes and is a glycooxidation product. Crossline formation may take place in vitro or in vivo was identified from kidneys of rats having diabetes. Cross-links of imidazolium dilysine, known as glyoxal lysine dimer (GOLD) or methylglyoxal lysine dimer (MOLD) are formed by the reaction between residues of lysine and derivatives of glyoxal. Reaction between two molecules of sugars and a single residue of lysine forms AFGP and is not very significant in vivo. ALI is a cross-link of interamolecules. Pyralline is AGE which is non-cross-link and can be detected in human plasma, skin and brain plaques. Formation of
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CML is the result of amodari products by oxidative breakdown and in vivo it is major one (Reddy and Beyaz, 2006; Goldin et al., 2006). Different AGEs play different role in diabetic complications e.g. CML serum levels increased in retinopathy but not in nephropathy whereas pentosidine level rises in both (Miroliaei et al., 2011). Genetic factors may also be responsible for AGE determination.
Fig. 2.2. AGEs formation pathways incorporating the polyol pathway and by the α-oxoaldehydes, methyl glyoxal (MGO) and 3-deoxyglucosone (3-DG). N-ε-(carboxymethyl) lysine (CLM); N-ε-(carboxyethyl)lysine (CEL); deoxyglucosone-lysine dimer (DOLD); methyl glyoxal-lysine dimer (MOLD); glyoxal-lysine dimer (GOLD) (Singh et al., 2001) 2.6.2. Intracellular AGEs and their harmful effects Except glucose reducing sugars forms glycation products much faster than it (Singh et al., 2001). Glucose is less reactive and explains the reason of its selection as main metabolic sugar (Ott et al., 2014). AGE formation takes place from intracellular sugars (Singh et al., 2001; Kellow and Sevige, 2013). During hyperglycemia intracellular sugars such as fructose and fructose-3-phosphate that is the product of polyol pathway increased (Singh et al., 2001). In diabetes methylglyoxal (MGO) a dicarbonyl compound is accumulated intracellularly in increase amounts. MGO formation takes place in anaerobic glycolysis from fructose of polyol pathway and polyunsaturated fatty acids decomposition oxidatively. Increase in glycation leads to implication of diabetic complications altering activity of enzymes, decreasing ligand binding, causing modification of protein half-life and alters immunogenicity. Autoantibodies may develop against serum AGEs forming immune complexes with AGEs in diabetes. Free radicals derived from glycation cause fragmentation
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of proteins and oxidation of lipids as well as nucleic acids. Amino groups of DNA bases adenine and guanine may also form AGE by reacting with intracellular sugars. Direct reaction between amino groups of phospholipids and glucose induces peroxidation of lipids and forms AGEs (Matsuura et al., 2002; Ahmad et al., 2007). 2.6.3. Mechanisms of Actions of AGEs Effects of AGEs on tissues are mediated mainly by three mechanisms. (1) Extracellular (matrix) proteins cross linking thus affecting mechanical properties of tissues (2) Intracellular proteins cross linking altering different physiological functions (3) Binding to their surface receptors of cell RAGE to induce multiple signalling cascades intracellularly. 2.6.3.1. Cross linking extracellular matrix proteins Formation of AGEs is a process that usually affects long lived proteins chronically. Collagen type IV involved in structure of basement membrane is an example of extracellular matrix protein that is exposed to advance glycation end product due to its long turnover. Cross linking of Advanced Glycation end products with other proteins like elastin and collagen makes them stiffer and less prone to proteolytic digestion (Farrar et al., 2007). This may be the cause of increased vascular stiffness in old age and diabetics. Cross linking of AGEs with myocardial collagen attributes towards the myocardial stiffness in diabetics. AGEs also alter the LDL structure and prevent their normal clearance by endothelial cells. Instead moncytes in blood takes them up forming foam cells causing pathogenesis of atherosclerosis (Peng et al., 2008). 2.6.3.2. Crosslinking intracellular proteins AGEs are also found to be involved in cross linking of intracellular proteins thus altering their properties and functions physiologically. In cardiomyocytes ryanodine receptor and SER-CA2a cross link with AGEs altering calcium homeostasis as reported in diabetic cardiomyopathy. 2.6.3.3. RAGE dependent effects of AGEs
RAGE receptor: RAGE is member of immunoglobulin superfamily receptors. In humans its gene is located on chromosome 6 between genes that codes for major histocompatibility
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complexes of class II and class III. Binding site of nuclear factor kappa B (NF-kB) is associated with RAGE promoter thus linking inflammatory cascade with expression of RAGE. It is a receptor with multilig and site of which AGE is identified as its first ligand. Other ligands include some family members of S100 protein, aggregates of fibrillar protein, amyloid beta and protein box-1 high mobility group (Zhang et al., 2011). Therefore RAGE is considered to be involved in the pathogenesis of diseases that are induced by these ligands causing tumours, inflammation, amyloidosis and neuro-degeneration (Patel et al., 2012). RAGE comprises an extracellular domain arranged as a variable “V” type immunoglobulin domain comprising 332-aminoacids with two constant “C” type domains. Modern present day techniques reveal that C1 and V domain act together for the some ligands to bind while C2 domain remain independent.
Fig 2.3. Receptor for advanced glycation end product
RAGE consists of a transmembrane domain with a cytosolic tail made up of 43 aminoacids and is highly charged. This cytosolic tail is involved in activating intracellular signalling cascades. If this tail is absent AGE bids but on ligand binding intracellular signalling fails to elicit. Phosphorylation/activation steps which are needed for initiating signalling cascades are exhibited as RAGE actions when tail binds to mammalian diaphanous-1, a cytoplasmic molecule. RAGE receptor binds to AGEs on V domain. AGEs with diversity in their chemical structure have some general common characteristics. First all proteins modified by AGE demonstrates accumulation of net negative charge by oxidation and glycation (Fritz, 2011; Xue et al., 2011). Secondly molecules of high molecular mass are formed due to covalent cross linking of proteins modified by AGEs. This geometry of ligands is important for activation of RAGE. Techniques like X-ray crystallography and NMR have revealed that binding ability of RAGE receptor is due to VC1 domain which is due to electrostatic interactions between subunits having positive charge and ligands with negative charge.
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Fluorescent labelled receptors have shown that aggregates of receptor form receptor assemblies and do not exist as single in plasma membrane (Fritz, 2011). It is speculated that multimers of AGE are essential to preserve receptor assemblies and stability required for the activation of RAGE. 2.6.4. Protein modification to form Amadori in diabetes & its complications Proteins with glycated lysine form Amodri products which are potential marker in diabetes mellitus for hyperglycemia.HbA1C is used to detect long term glucose control in diabetes is an amodri product in the form of glycated hemoglobin. There are a number of intracellular and extracellular proteins which are amadori products, associated with diabetic complications given in table1. Liver cells of diabetic patients were identified with amadori products of histones after modification (Jobst et al., 1991) while glycated albumin was found to be related with retinopathy and early nephropathy in type 1 of diabetes (Schalkwijk et al., 2002). Raised levels of amadori products of glycated collagen in patients of diabetes type 1 with retinopathy or without it and were also associated with retinopathy independently (McCance, 1993). Amadori products from glycation of serum proteins are reported to contribute towards nephropathy in diabetes. On the other hand increased amadori glycated albumin was related with retinopathy in diabetes. Purified immunoglobulins (IgG, IgM, IgA) Table 2.1. Amadori products associated with diabetic complications Amadori products Diabetes mellitus Reference Histones Diabetes Jobst et al., 1991 Human serum albumin Type 1 diabetes with Schalkwijk et al., 2002 retinopathy & nephropathy Collagen Diabetes with retinopathy McCance, 1993 IgG,IgM,IgA Type 1&2 diabetes with Kalia et al., 2004 nephropathy Plasma proteins Type 2 diabetes Jaleel et al., 2005 (e.g.albumin,fibrin,transferrin) Albumin,Lipoproteins Diabetes with atherosclerosis Cohen et al., 2006 Phosphatidyl-ethanolamine Diabetes Sookwong et al., 2011 with raised levels of fructosamine were found in diabetic patients of type 1 type 2 with nephropathy complications as compared to normal subjects (Kalia et al., 2004). Amadori products of plasma proteins like immunoglobulin (Ig) were detected in diabetes type 2 with Amadori antibody (1-deoxyfructosyl lysine) (Jaleel et al., 2005). Amadori forms of
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lipoprotein and albumin were linked with elevated atherosclerosis in diabetic patients (Cohen et al., 2006). Phosphotidylethanolamine, a glycated lipid is found to be raised in rats having diabetes (Sookwong et al., 2011). Detection methods for Amadori specific antibodies were ELISA, Gel retardation and Western Blot. 2.7. Carbohydrate hydrolyzing enzymes inhibition Epidemiological studies associated with disease risk have suggested that habitual consumption of dietary agents offer protection against occurrence of metabolic ailments such as diabetes along with other associated co-morbidities (Wang et al., 2012; Farzaneh and Carvalho, 2015). The forfeits of a carbohydrate and fat enriched foods are further allied among the deprived societies of developing countries, where nutritional variations in combination with poverty and preeminent therapeutic hazards and malnutrition generate a double burden (Popkin, 2002). Diabetes mellitus is a foremost chronic ailment accompanying poor glycemic index and intolerance of glucose; imperative risk factors for other metabolic syndromes (Ranilla et al., 2010). Before the insulin discovery in 1922, Plant based therapies and dietary manipulations were the prime treatment for the diabetes mellitus. Even in recent times, plant-based systems persistent to play an requisite role in this area. It has been proposed that almost 1200 herbal plants in nature are capable to effectivity diminish or hold the blood glucose content (Farzaneh and Carvalho, 2015). Herbal plants with antidiabetic potentials may effectively inhibit insulin resistance and oxidative stress; in fact, the antidiabetic properties of herbal plants might be due to chemical interactions occurred among effective compounds available in infusions with the diverse biochemical molecules involved in diabetes (Perez-Gutierrez and Damian-Guzman, 2012). Currently, alternative approaches are in demand to treat diabetes (e.g. plant-derived medicines) in industrially-developed countries because of the hazardous effects related to the use of insulin and other oral hypoglycaemic means (Shobana et al., 2009). Inhibition of principal carbohydrate hydrolyzing enzymes (α-glucosidase and α-amylase) connected to postprandial hyperglycemia due to natural antioxidant, is an impending remedial approach for diabetes mellitus (Kwon et al., 2008; Ranilla et al., 2010). Inhibitors of these enzymes slowdown the digestion of carbohydrates thus, prolong the overall digestion period, lessen glucose absorption rate and consequently blunt the postprandial glucose escalation in plasma (Rhabasa-Lhoret and Chiasson, 2004; Elya et al., 2012).
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2.7.1. Biochemistry of carbohydrate hydrolyzing enzymes and their inhibitors Alpha-amylase, a prominent salivary and pancreatic enzyme hydrolyzes the large insoluble starch into absorbable molecules through the cleavage of α-D-(1-4) glycosidic linkages (Whitcomb and Lowe, 2007). The consequential yield of α-amylase action is a mixture of oligosaccharides including maltotriose, maltose and oligosaccharides of 6–8 glucose units having both α-1, 4 and α-1, 6 bonds (Sales et al., 2012). In contrast, α-glucosidase cleaves the terminal non-reducing glucose molecule by hydrolyzing the α-1, 4 glycosidic linkages of glucose residues. α-glucosidase localized at the luminal surface of small intestinal epithelium and catalyzes the end step of digestion of abundant dietary carbohydrates (Kwon et al., 2008; Kazeem et al., 2013). Acting as a key enzyme for carbohydrate digestion, both of these enzymes are renowned therapeutic tool for the modulation of postprandial increment of glucose, an earlier metabolic abnormality in type 2 diabetese (Sales et al., 2012).
Fig 2.4. Competitive inhibition of enzymatic hydrolysis of carbohydrate substrates by α-glucosidases in the small intestine (Bischoff, 1994) Owing to the competitive mechanism of action, Inhibitors such as acarbose, miglitol, and voglibose reduces the postprandial increment of glucose principally by snooping the action of carbohydrate-digesting enzymes (Fig.2.2). In addition, several other anti-α-glucosidase agents have been extracted from plants, which are of clinical importance (Yao et al., 2013). In plants, these inhibitory agents occur as part of defense mechanisms, predominantly in legumes and cereals. The α-Amylase inhibitory constituents are also renowned to prevent edible starches from being absorbed by the human body, by acting as starch blockers or so
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called slimming pills (Dineshkumar et al., 2010). Several reports described inhibitory activity in several families (Sales et al., 2012). 2.7.2. Inhibitors of carbohydrate hydrolyzing Enzymes from plants Natural sources have been investigated with respect to their modulatory effect on glucose liberation from starch in the gut and subsequent absorption through the intestinal walls (Oboh, et al., 2010). Over the thousands of years, folk herbal therapeutic system accomplished the antidiabetic potential of plants with no apparent known hazardous effects (Bhat et al., 2011). Prashanth et al., (2001) tested the effect of Indian medicinal plants on the activity of α-amylase. In their study, interesting inhibitory properties have been exposed by Embelia ribes Burm, Mangifera indica L., Punica granatum L. and Phyllanthus maderaspatensis Linn. Ranilla et al. (2010) revealed the preventive potential of Phyllantus niruri L., Ilex paraguayensis St-Hil, Smilax officinalis and Tagetes minuta for hyperglycemia allied to their digestive enzyme inhibitory activities. According to previous studies, many other plants such tea, sweet potato (Maloney et al., 2014), berry (McCue et al., 2004) and Cleistocalyx operculatus, Syzygium zeylanicum, Horsfieldia amygdalina and Careya arborea (Mai et al., 2007) also unveiled the inhibitory effect on carbohydrate-hydrolyzing enzymes. Andrade- Cetto et al., (2008) speculated that Cecropia abtusifolia, Acosmium panamense and Malmea depresa have greater tendency to inhibit α- glucosidase than arcabose. According to Koh et al., (2010), natural enzyme inhibitors isolated from white bean and wheat ominously reduce the level of postprandial glucose. Plants also from Apocynaceae, Clusiaceae, Euphorbiaceae and Rubiaceae families have therapeutic potential including as the α-glucosidase inhibitor (Elya et al., 2012). Evidence from another study also linked the phytochemical enriched plant extracts and their potential regarding carbohydrate digesting enzymes inhibition (Sunil et al., 2010).
2.7.3. Phytoconstituents with carbohydrate hydrolyzing enzymes inhibitory activity
Plant infusions containing phytonutrients act as the most potent origin for treatment of diabetes (Grover et al., 2002; Mentreddy, 2007; Suryanarayana et al., 2004). Ponnusamy et al., (2011) declared the numerous hypoglycemic features of the herbal infusions; linked not only to their inhibitory potentials on glucose transporters, α-glucosidase, and α-amylase but
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also to their strong antioxidant activities. Previously, several epidemological studies authenticated a positive correlation between enzymatic inhibition and antioxidant potential linked to diverse range of plant-derived constituents, primarily glycosides, alkaloids, galactomannan gum, hypoglycans, polysaccharides, peptidoglycans, steroids, guanidine, terpenoids and glycopeptides (Mai et al., 2007; Wang et al., 2010). However, a number of vegetables and herbal extracts also have exhibited inhibitory activities against α-glucosidase and α-amylase, and therein may have potential as dietary antidiabetic agents for the control of postprandial hyperglycemia (McCue et al., 2004). Yao et al., (2013) attributed that α- glucosidase and a-amylase are inhibited to a certain extent by total flavonoids within different extract concentrations which is in accordance to present results. In accordance to present results, Kwon et al., (2008) reported that phenolic enriched extracts from several eggplant samples (e.g., White pulp and Graffiti skin) have high α-glucosidase inhibitory activity combined with low two folds a-amylase inhibitory activity.
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The current research work was carried out in Bioassay Section, Protein Molecular Biology Lab, Department of Biochemistry, and Enzyme Research Lab/Clinical Biochemistry Lab, Department of Biochemistry, University of Agriculture, Faisalabad Pakistan. However, in vitro cancer study was performed in the Department of Food Science, University of Massachusetts, Amherst, MA, USA. The materials and protocols followed are elaborated herein. 3.1. List of chemicals All the chemicals and reagents used were of analytical grade such as gallic acid (99%), catechin (99.9%), folin-ciocalteu reagent, DPPH (2, 2, Diphenyl-1-picrylhydrazyl) and homologous series of C4-C28 n-alkanes. The absolute methanol, sodium phosphate buffer, streptomycin solution, ammonium thiocyanate, ferrous chloride, butylatedhydroxytoluene, potassium ferricyanide, trichloroacetic acid, ferric chloride, cyclophosphamide monohydrate, fetal bovine serum, triton-X 100, absolute ethanol, hydrochloric acid, sodium hydroxide, sodium nitrite, sodium carbonate, aluminum chloride, D-glucose, sodium azide (NaN3),
potassium dichromate (K2Cr2O7), n-hexane, chloroform, n-butanol, potassium iodide, iodine, dimethylsulfoxide, ethyl acetate, hydrogen peroxide, silica gel (Sigma Aldrich Chemical Co. USA and Merck USA. All other reagents used were also of analytical grade. 3.2. Instruments used The instruments used for analysis during the study along with their company identification detailed are as below. • Grinder, Model CB 222, Cambridge United Kingdom. • Atomic absorption spectrophotometer(Model Thermo Electron S-Series) • Electric water bath (Memmart, Germany) • Microkjeldhal (Memmart, Germany) • Muffel Furnace (Controller BC-170, Nabertherm) • Incubator, Sanyo, Germany. • Laminar air flow, Dalton, Japan. • Magnetic stirrer, Gallen Kamp, England. • pH meter (WTW inolap multi, 720, Germany) • Microplate stirrer, New Jersey, USA.
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• Orbitrary shaker, Gallen Kamp, England. • Oven, Memert GmbH, D-91126, Germany. • Rotary evaporator, Heidolph, model LABORATA 4000, Schwabach, Germany • Ultra low freezer, Sanyo, Germany. • UV detection lamp 254 and 365 nm, Fisher Scientific Ltd, Pandan Crescent, UE Tech Park, Singapore. • UV/VIS spectrophotometer ( Hitachi, U-2001,model 121-0032 spectrometer,Japan) • 96 micro well plate readers BioTek, model µ-QuantTM, Winooski, VT, USA. • Autoclave , Omron, Japan. • Blender, Mamrelax, Fait Common, France. • Centrifuge, H-200 NR, Kokusan, Japan. • EI-MS MAT 312 spectrometer, Scientific Instrument Services, Inc., Ringoes, NJ, USA. • Fluorescence spectrophorometer 3.3. General procedure Methods: 3.3.1. Collection of selected plants. 3.3.2. Grinding and sample size/extraction. 3.3.3. Proximate analysis of plants. 3.3.4. Mineral analysis of selected plant species. 3.3.5. Qualitative and quantitative content analysis. 3.3.6. Antioxidant activity of all prepared samples through different methods. 3.3.7. Brine shrimp toxicity assay of prepared samples through different methods. 3.3.8. Antithrombolytic activity of prepared samples by different methods. 3.3.9. Anticarcenogenic activity of selected plant materials. 3.3.10. Antiglycation activity. 3.3.11. Carbohydrate hydrolyzing enzyme assay 3.3.12. Statistical analysis of all data obtained. 3.3.1. Collection and identification of plant materials In the present study the following plants species were used for the analysis (Table 3.1). Selected vegetables were purchased at the peak of their season, washed with distilled water to remove the dust and soil particles, cut into small pieces and shade dried for several days.
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Rest of the samples were commercially available as dried fruits. All the selected samples were purchased from the local market of Faisalabad, Pakistan and identified and authenticated from Department of Botany, University of Agriculture Faisalabad, Pakistan. Table 3.1. Scientific and vernacular name of selected vegetables Sr. No Scientific Name Vernacular Name Family Name 1 Ipomoea batatas Sweet potato Convolvulaceae 2 Trigonella foenum-graecum Methi Fabaceae 3 Daucus carota Carrot Apiaceae 4 Solanum Melongena Bringle Solanaceae 5 Brassica rapa rapa Turnip Brassicaceae 6 Phoenix dactylifera Dates Arecaceae 7 Juglans Wal nut Juglandaceae 8 Prunus dulcis Almond Rosaceae 9 Pistacia vera Pista Anacardiaceae 10 Arachis hypogaea Peanut Fabaceae 11 Vitis vinifera L. Reisins Vitaceae 12 Cocos nucifera Coconut Arecaceae 13 Prunus armeniaca Apricot Rosaceae 3.3.2. Grinding and sample size/extraction The selected vegetables were washed with distilled water and shade dried while other samples were commercially available as dried fruits. All samples were milled to a fine powder using 60 mesh sizes, mixed thoroughly and stored at -20 or -80 °C. The -20 °C samples were used for routine analysis. 3.3.2.1. Extraction of free phenolic compounds Total phenolic extraction of selected plant species is shown in the flowchart (Fig. 3.1). Soluble free phenolics of samples were extracted using the method (Yang et al., 2009; Chandrasekara and shahidi, 2011) with slight modifications. Briefly, 25 g of plant materials were blended for 10 min in 250 mL of chilled 70% methanol (1:10, w/v) using a Waring blender. The mixture was then homogenized in a Virtis High Speed Homogenizer for 5 min and filtered under vacuum. The methanol/water mixture was washed using 50 mL hexane by centrifugation at 2500g for 10 min. The hexane fraction was removed and the extract was washed twice. Finally, the methanol/water mixture was evaporated using a rotary evaporator at 45 oC to dryness. Later on the same process was repeated with ethanol/water mixture. The extracts were stored at -40 oC until use.
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3.3.2.2. Extraction of bound phenolic compounds Bound phytochemicals of selected plant species were extracted by the method reported previously (Yang et al., 2009). The solid residues (10g) from the soluble-free extraction were collected, flushed with nitrogen gas, sealed, and hydrolyzed directly with 40 mL of 4M NaOH at room temperature for 1 h with shaking. The mixture was then neutralized with concentrated hydrochloric acid to pH 7.0 and extracted with hexane to remove lipids. The final solution was extracted five times with ethyl acetate. The ethyl acetate fraction was evaporated to dryness and stored at -40 °C until use.
Fig 3.1. Flow chart of phytochemical extraction of selected plant materials 3.3.3. Proximate analysis of plants materials The dried plants powder was analyzed for its moisture content, crude proteins, carbohydrates, crude fats, crude fibers and ash content by the following methods. 3.3.3.1. Estimation of crude protein Crude protein was determined by micro Kjeldahl apparatus according to Ayuba et al. (2011). Protein percentage of plants was determined by the following formula: Crude protein (%) = 6.25 × Nitrogen (%) A known weight of each fat-free samples of the selected plants were first wrapped in Whatman filter paper (No. 1) and put in a Kjeldhal digestion flask. The 10mL of concentrated sulphuric acid was added. In one gram of catalyst (a mixture of sodium sulphate (Na2SO4) copper sulphate (CuSO4) and selenium oxide (SeO2), ratio 10:5:1
32 Chapter # 3 Materials and Methods was added into the flask to facilitate digestion. Antidumping granules were added. The flasks were then put on Gallenkamp digestion apparatus for 2 hours until liquid turned light green. The digested samples were cooled and diluted with distilled water to 100 mL in standard volumetric flask. A 10 mL aliquot of each diluted solution with 10mL each of 45% sodium hydroxide were put into the Manhan distillation apparatus and distilled into 10mL of 2% boric acid containing 4 drops of bromocresol green/methyl red indicator. Each distillate was then titrated with standardized 0.01N hydrochloric acid to grey colored end point to obtain nitrogen. The percentage crude protein in the original sample was calculated using the formula. Crude protein (%) = (a-b) x 0.01 x 14.01 x C x 100x 6.25 / d x e Where: a = Titre value of the digested sample (b) = Titre value of the blank sample c = Volume to which the digested sample was made up to with distilled water (100 mL) d = Volume of sodium hydroxide used for distillation (10 mL) e = Weight of dried fat-free sample 3.3.3.2. Estimation of crude fats Crude fats of dried plants powder were determined by the method described by Ayuba et al. (2011). For the estimation of crude fats dried plants powder were taken in a thimble. The thimble was placed in extraction tube of soxhlet apparatus. It was placed in a water bath and the temperature was so adjusted that continuous drops of water fell on the plant sample that was placed in extraction tube. The process of extraction was carried out with petroleum ether (Boiling point is 40-60oC) for 16 hours. Then the sample was removed from the extraction tube and the solvent was allowed to evaporate by placing the flask in boiling water bath. The extract was placed in hot air oven at 105oC for 30 minutes and it was completely dried. It was allowed to cool in desiccators. The weight of dried extract was recorded. Crude fat percentage was calculated with the help of following formula: Crude fat (%) = Wt of fat in sample (g) × 100/ Wt of sample 3.3.3.3. Estimation of crude fibers Crude fibers of dried plants were determined according to the protocol described by Ayuba et al. (2011). In 1L beaker, 3g of dried and fat free sample were taken and 200 mL of
1.25% H2SO4 were added to it and level of beaker was marked. This was allowed to boil for 30 min with constant stirring and also the level of water was maintained. These contents
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were filtered while warmed giving 2 to 3 washings with hot water until it became alkali free. The residue was carefully transferred in a crucible and dried in hot air oven at 100oC for 3 to 4 h until constant weight was obtained. These contents were heated on an oxidizing flame until the smoke ceased come out. Then this sample was put in a muffle furnace at 550oC for 4 hours until grey color ash was obtained. This was allowed to cool in desiccators and weighed. Percentage of crude fibers was calculated by the following formula: Crude fiber (%) = 100(crucible wt before ashing – crucible wt after ashing)/ Wt. of sample 3.3.3.4. Determination of ash contents Ash contents of dried plant samples were determined by the method as described by Ayuba et al. (2011). Dried plant samples were placed in a crucible and heated on an oxidizing flame until the smoke was stopped. The crucible was placed in a muffle furnace for 6 h at 550 oC until ash was obtained. This sample was allowed to cool and weighed. Ash content was calculated with help of following formula. Ash (%) = wt of ash in sample (g) × 100/ wt of sample (g) 3.3.3.5. Determination of total carbohydrates Total carbohydrates were calculated by difference method as follows: Total carbohydrates = 100- (Total moisture + Total protein + Total fat + Total ash) 3.3.3.6. Total energy Total energy was calculated according to the following equations: Energy (Kcal) = 4× (g protein + g carbohydrate) + 9 × (g lipid) Energy (KJ) = 17 × (g protein + g carbohydrate) + 37 × (g lipid) 3.3.4. Mineral Analysis Mineral contents of all the selected plant samples were determined by atomic absorption spectrometry, flame photometry and spectrophotometry according to the methods of AOAC (2003). The selected plants materials were digested in diacidic mixture of nitric acid and perchloric acid (1:1), and analyzed on atomic absorption spectrophotometer (Perkin Elmer, A Analyst 300) for Minerals (Iron (Fe), Zinc (Zn), Calcium (Ca), Chromium (Cr), Manganese (Mn) and Magnesium (Mg). Na and K analysis of the sample were done by the method of flame photometry by using the same wet digested food sample solutions. The results are presented in µg/100g of the original sample.
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3.3.5. Phytochemistry of Plants 3.3.5.1. Qualitative analysis i). Test for alkaloids: A 2 mL of the extract and 0.2 mL of dilute hydrochloric acid were taken in a test tube. Then, 1mL of Mayer’s reagent was added. A yellowish buff precipitate was indicative of the presence alkaloids (Biswas et al., 2011). ii). Test for tannins The extract 0.5 g was boiled in 10 mL of water in a test tube for 5 min. The mixture was filtered and then in 5 mL of filtrate solution. A few drops of 0.1% ferric chloride was added and observed for brownish green or a blue-black coloration (Menghani et al., 2011). iii). Test for terpenoide (Salkowski’s test) Chloroform (1 mL) was added to 200 µL of the extract followed by a few drops of concentrated sulfuric acid. A reddish brown precipitate produced immediately indicated the presence of terpenoides (Misra et al., 2011). iv). Test for flavonoids A few drops of concentrated hydrochloride acid were added to a small amount of plant extract. Immediate development of a red colour indicates the presence of flavonoids (Biswas et al., 2011). v). Test for saponins The extract 0.5 g was added in 5mL of distilled water. The solution was shaken vigorously and observed for the formation of an emulsion (Menghani et al., 2011). 3.3.5.2. Quantitative analysis i). Alkaloid determination Sample (5 g) was weighed into a 250 mL beaker and 200 mL of 10% acetic acid in ethanol was added and covered and allowed to stand for 4 h. This was filtered and the extract was concentrated on a water bath to one-quarter of the original volume. Concentrated ammonium hydroxide was added drop wise to the extract until the precipitation was complete. The whole solution was allowed to settle and the precipitated was collected and washed with dilute ammonium hydroxide and then filtered. The residue is the alkaloid, which was dried and weighed (Sutharsingh et al., 2011). ii). Saponin determination Grounded sample (20 g) was put into a conical flask and 100 mL of 20% aqueous ethanol was added. Then the flask was heated on a hot water bath for 4 h with constant stirring at
35 Chapter # 3 Materials and Methods about 55°C. The mixture was then filtered and the residue was again extracted with another 200 mL 20% ethanol. The combined extract was reduced to 40 mL on a hot water bath at about 90°C. The concentrate was transferred into a 250 mL separator funnel, added 20 mL diethyl ether in it followed by vigorous shaking. The aqueous layer was recovered while the ether layer was discarded. The purification process was repeated 60 mL of n-butanol was added. The combined n-butanol extracts were washed twice with 10 mL of 5% aqueous sodium chloride. The remaining solution was heated in a water bath. After evaporation the samples were dried in oven, weighed and saponins content was calculated as percentage (Khan et al., 2011). iii). Flavonoid determination Colorimetric method with slight modifications was used to determine flavonoids content. Plant extract (1mL) in methanol was mixed with 1mL of methanol, 0.5 mL aluminium chloride (1.2%) and 0.5 mL potassium acetate (120 mm). The mixture was allowed to stand for 30 min a room temperature then the absorbance was measured at 415nm. Quercetin was used as standard. Flavonoid content is expressed in terms of quercetin equivalent (mg-1 of extracted compounds) (Vaghasiya et al., 2011). 3.3.6. Antioxidant activity of plant extracts Antioxidant activity of plant extracts were measured by different methods including; i. Total phenolic contents (TPC) of selected plant extracts ii. Total flavonoid contents (TFC) of selected plant extracts iii. Free radical scavenging activity by DPPH of selected plant extracts iv. Oxidation of compounds by linoleic acid of selected plant extracts v. Determination of reducing power of selected plant extracts 3.3.6.1. Total phenolic contents (TPC) of selected plant extracts Total phenolic content was determined according to the Folin–Ciocalteu method Ho et al., (2010) using gallic acid as the standard. The extract (5 mg) was dissolved in 5 ml of methanol/ water (50:50 v/v). The extract solution (500 µl) was mixed with 500 µl of 50% Folin–Ciocalteu reagent. The mixture was kept for 5 min, which was followed by the addition of 1.0 ml of 20% Na2CO3. After 10 min of incubation at room temperature, the mixture was centrifuged for 8 min (150g/5400rpm). 200 µL of the supernatant was transferred to a clear and clean 96-well plate and absorbance of each well was measured at 730nm. The total phenolic content was expressed as gallic acid equivalents (GAE) in milligrams per gram of sample.
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3.3.6.2. Total flavonoids content (TFC) of selected plant extracts To measure the TFC, One mL of extract containing 0.01 g/mL of dry matter was placed in a 10 mL of volumetric flask, Then 5 mL of distilled water was added followed by 0.3 mL of
5% NaNO2. After 5 min, 0.6 mL of 10% AlCl3was added. After another 5 min 2 mL of 1 M NaOH was added and volume was made up with distilled water. Absorbance was measured at 510 nm. TFC amounts were expressed as catechin equivalents (100-1300 mg/L) per dry matter (Barros et al., 2011). All samples were analyzed thrice and results were averaged. 3.3.6.3. DPPH radical scavenging assay of selected plant extracts The DPPH assay was carried out as described by Ho et al., (2010). The antioxidant activity of seed extracts were assessed by measuring their scavenging abilities to 2, 2-diphenyl-1-1- picrylhydrazyl stable radical. 50uL aliquot of various concentrations of the samples was added to 5 mL of a 0.004 % methanol solution of DPPH. After 30 min incubation period at room temperature, the absorbance was read against a blank at 517 nm. The assay was carried out in triplicate. I % = (A blank –A sample /A blank) ×100 Where A blank is the absorbance of the control reaction (containing all reagents except the test compound) and A sample is the absorbance of the test compound. Extract concentration providing 50 % inhibition (IC50) was calculated from the graph plotted inhibition percentage against extract concentration. The assay was carried out in triplicate. 3.3.6.4. Determination of reducing power of selected plant extracts The reducing power of extracts was determined according to the procedure described by Barros et al., (2011). A 100 μL of solution was mixed with sodium phosphate/potassium phosphate buffer (5.0 mL/0.2 M, pH: 6.6) and potassium ferricyanide (5.0 mL, 1.0 %); the mixture was incubated at 50 oC for 20 min. Then 5 mL of 10 % of tricholoroacetic acid was added and centrifuged at 1000 rpm (10 min at 5oC) in refrigerated centrifuge. The upper layer of the solution (5.0 mL) was diluted with 5.0 mL distilled water and ferric chloride (1.0 mL, 0.1 %), and absorbance read at 700 nm. The experiment was performed thrice and results were averaged and data presented as mean ± SD. 3.3.7. Brine shrimp lethality assay Brine shrimp lethality bioassay was carried out to investigate the cytotoxicity of extracts of medicinal plants. Brine shrimps (Artemia salina) were hatched using brine shrimp eggs in a conical shaped vessel (2L), filled with sterile artificial seawater (prepared using sea salt 38 g/L and adjusted to pH 8.5 using 1N NaOH) under constant aeration for 48 h. After
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hatching, active nauplii free from egg shells were collected from brighter portion of the hatching chamber and used for the assay. Twenty nauplii were drawn through a glass capillary and placed in each vial containing 4.5 mL of brine solution. In each experiment, 0.5ml of the plant extract was added to 4.5 mL of brine solution and maintained at room temperature for 24 h under the light and surviving larvae were counted. Experiments were conducted along with control, four different concentrations (10-100, 1000, 3000 µg/mL) of the plants were used to check their toxicity and the experiment was repeated three times (Ali et al., 2013). 3.3.7.1. Lethality concentration determination The percentage lethality was determined by comparing the mean surviving larvae of the test
and control tubes. LC50 values were obtained from the best fit line plotted concentration verses percentage lethality. Methanol was used as a positive control in the bioassay. 3.3.8. Thrombolytic assay The thrombolytic activity in terms of in vitro clot lysis was carried out according to the method of (Ali et al., 2013). Briefly, 500 µl of blood was withdrawn from healthy human volunteers. In the commercially available lyophilised streptokinase vial (1 500 000 IU) 5 mL sterile distilled water was added and mixed properly. This suspension was used as a stock solution from which appropriate dilution was made. Five milliliter of venous blood was drawn from the healthy volunteers (n=10) without the history of oral contraceptive or anticoagulant therapy and was distributed (0.5 mL/tube) to each ten previously weighed sterile micro centrifuge tube and incubated at 37 °C for 45 min to form the clot. After the clot formation, serum was completely removed without disturbing the clot and each tube having clot was again weighed to determine the clot weight. A volume of 100 μL of methanol extract (10 mg/ mL) was added to each micro centrifuge tube containing pre weighed clot. As a positive control, 100 μL of streptokinase and as a negative control 100 μL of distilled water were separately added to the control tube numbered. All the tubes were then incubated at 37 °C for 90 min and observed for clot lysis. After incubation, fluid released was removed and tubes were again weighed to observe the difference in weight after clot disruption. Difference obtained in weight taken before and after clot lysis was expressed as percentage of clot lysis. % of clot lysis = (wt. of released clot /clot wt.) × 100 The ability of the extracts to dissolve clot in percentage of weight loss were compared with that of standard and blank.
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3.3.9. Measurement of inhibition of cell proliferation by plants extracts 3.3.9.1. Cell lines and culture Human non-small-cell lung cancer-derived cell line H1299 and human colorectal adenocarcinoma-derived cell line HT29 were purchased from American Type Culture Collection (Manassas, VA). H1299 were maintained in RPMI-1640 medium with 10% fetal bovine serum and HT29 cells were maintained in McCoy’s 5A medium with 10% fetal bovine serum. Both types of cells were maintained in 5% CO2 humidified atmosphere at 37oC. Cell cultures used for experiments were at 60–70% confluence, which were usually achieved 24 or 48 h after plating. Plant samples were dissolved in dimethyl sulfoxide (DMSO). For treatment, the cells were incubated with 5–250 µg/mL plant extract for different periods of time at 37oC. 3-(4, 5-Dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) (Sigma, St Louis, MO) assay was performed by following the manufacturer’s instruction. 3.3.9.2. In vitro anti-proliferative assay Assay was carried out by MTT (3-(4, 5-Dimethylthiazol-2-yl) - 2, 5-diphenyltetrazolium bromide, a tetrazole) protocol of to evaluate the anti-proliferative effect of plant samples. For this purpose, a sufficient number of exponentially growing cells were used to avoid confluence of the culture during the treatment. The cell line HT29 was seeded at 2500 cells/well and allowed to adhere for 24 h. The H1299 cells were seeded at 1500 cells/well and media was replaced with 200 µl of fresh medium before the treatment with sample. In order to evaluate the optimum concentration at which the sample inhibited the cell proliferation in both the cell lines, cells were treated with concentration ranging from 5 to 250 µg/ml. DMSO was used as a solvent for the dilution of extracts which was also used as a experimental control. After 72 h treatment, cell growth was evaluated by MTT assay (Peiju et al., 2011). MTT solution of 100 µl (5 mg/ml of fresh media) was added to each well and the plates were incubated for 1 h at 37oC in dark. The media was aspirated and 100 µl of DMSO was added to each well immediately to solubilize the formazan crystals. The optical density was measured at 490 on ELISA reader (ELX800TM, absorbance microplate reader, Biotek Instrument, VT, USA) at a wavelength of 490 nm. Each sample was performed in triplicate, and the entire experiment was repeated thrice. The graphic depiction of results presented the percentage inhibition by respective treatments.
39 Chapter # 3 Materials and Methods
3.3.10. Antiglycation activity
• Non-enzymatic protein glycation
• Nitro-blue tetrazolium (NBT) reductive assay
• Girard-T assay
3.3.10.1. Non-enzymatic protein glycation Experiments were performed as the method of Hsieh et al., (2009) with slight modifications. Briefly, bovine serum albumin (BSA, 20 mg/ml, 10 ml) was mixed with glucose (500 mM, 5 ml) and 0.02% sodium azide in phosphate buffer (200 mM, pH 7.4). The sample with different concentration dissolved in phosphate buffer (200 mM, pH 7.4, 5 ml) was added to the reaction mixture, and then the mixture was incubated for 30 days at 37oC to obtain glycated materials. Aminoguanidine was used as a positive control. Some of the glycated materials were taken from the system for the following experiments in 0, 3rd, 6th, 12th, 18th, 24th and 30th day after incubation. 3.3.10.2. Spectrophotometric analyses of nitro-blue tetrazolium (NBT) reductive assay The procedure of NBT reductive assay followed the method of Zhang et al. (2011) with slight modifications. The glycated material (0.5 ml) and NBT reagent (0.3 mM, 2.0 ml) in sodium carbonate buffer (100 mM, pH 10.35) were incubated at room temperature for 15 min, and the absorbance was read at 530 nm against a blank. 3.3.10.3. Spectrophotometric analyses of Girard-T assay Dicarbonyl compounds were tested spectrophotometrically by Girard-T assay (Zhang et al. (2011). Briefly, the glycated material (0.4 ml) was incubated with Girard-T stock solution (500 mM, 0.2 ml) and sodium formate (500 mM, pH 2.9, 3.4 ml) at room temperature for 1 h. Absorbance was measured at 294 nm with UV-2550 spectrophotometer (Shimadzu, Kyoto, Japan) against a blank which contained all the reagents except the Girard-T stock solution. A calibration curve was prepared using glyoxal as a standard which was treated in a similar manner. 3.3.10.4. AGEs analysis The glycated material (0.5 ml) was diluted to 10 ml with distilled water and the fluorescence of the aterial was measured at 370 nm excitation and 440 nm emission using an F-5301 spectrofluorometer (Shimadzu, Kyoto, Japan) (Zhang et al. (2011),.
40 Chapter # 3 Materials and Methods
3.3.11. Enzyme Assay 3.3.11.1. α-Glucosidase assay The α-glucosidase inhibitory activity was measured as described by Kwon et al., (2008), and Dong et al., (2012) with slight modifications. Briefly, a volume of 60 µl of sample solution and 50 µl of 0.1 M phosphate buffer (pH 6.8) containing α-glucosidase solutions (0.2 U/ml) was incubated in 96 well plates at 370C for 10 min. After pre-incubation, 50 µl of 5mM p- nitrophenyl-a-D-glucopyranoside (PNPG) solution in 0.1 M phosphate buffer (pH 6.8) was added to each well and incubated at 370C for another 20 min. Then the reaction was stopped
by adding 160 µl of 0.2 MNa2CO3 into each well, and absorbance readings (A) were recorded at 405 nm by micro-plate reader (SpectraMax, M2/M2e, Molecular device Co., Sunnyvale, CA, USA) and compared to a control which had 60 µl of buffer solution in place of the extract. The α-glucosidase inhibitory activity was expressed as inhibition % and was calculated as follows: Inhibition (%) = (Acontrol _ Asample)/Acontrol × 100 The concentration of inhibitors required for inhibiting 50% of the α-glucosidase activity under the assay conditions was defineds the IC50 value. 3.3.11.2. α-Amylase inhibition assay The α-amylase inhibitory activity was measured according to methods described by Kim et al. (2005) and Dong et al. (2012) with some modifications. A 40 µl of sample solution (0.2 mg of sample or acarbose in 20 mM sodium phosphate buffer, pH 6.9 with 0.006 M sodium chloride) was premixed with 200 µl of α-amylase solution (1.0 U/ml in the pH 6.9 buffer), and incubated at 25oC for 10 min. After pre-incubation, 400 µl of a 0.25% starch solution in the pH 6.9 buffer was added to each tube to start the reaction. The reaction was carried out at 37 0C for 5 min and terminated by addition of 1.0 ml of the DNS reagent (1% 3, 5- dinitrosalicylic acid and 12% sodium potassium tartrate in 0.4 M NaOH). The test tubes were then incubated in a boiling water bath for 5 min and cooled to room temperature. The reaction mixture was then diluted after adding 10 ml distilled water and absorbance was measured at 540 nm using a UV–Vis spectrophotometer (mini 1240, Shimadzu, Japan). The control had 200 µl of buffer solution in place of the α-amylase solution. The % inhibition was calculated by: Inhibition (%) = (Acontrol _ Asample)/Acontrol × 100
41 Chapter # 3 Materials and Methods
3.3.12. Statistical analysis The mean±SE was calculated for the triplicate experiments through Microsoft Excel 7.0. The statistical software Minitab version 13.1 was used for computation and analysis of the proximate, antioxidant and antiglycatin activities. Differences were analyzed, by considering P<0.01 statistically significant.
42 CHAPTER # 4 RESULTS AND DISCUSSION
Dietary supplementation of phytonutrients is an emerging trend that provides multifaceted defensive mode against various maladies thereby enhancing the overall health status. In the developing countries like Pakistan there is abundance of fruits and vegetables nevertheless have not yet been prodded meticulously for their health promoting properties. Purposely, current research project was designed to focus on the antioxidant and antiglycation properties as well as the biochemical analysis of different edible species those are locally available and readily consumed. The in vitro antioncogenic perspectives of their extracts were also determined using colon and lung cancer cell lines. Subsequent data were analyzed statistically to estimate the level of significance. The results and discussion regarding different parameters of current project has been compiled under the following lay out. Layout of the results and discussion • Proximate analysis of selected plants materials • Mineral analysis of selected plant materials • Qualitative and quantitative Phytochemistry of plant extracts. • Extraction and % age yield of extracts from selected plants materials. • Antioxidant potential of plant extracts • Brine shrimp lethality assay • Antithrombotic activity • Antiproleferation assay • Antiglycation Activity • Enzyme assay 4.1. Extraction and % age yield of extracts from selected plant materials Extraction efficiency and the mass transfer of plant-based materials following the traditional solvent extraction techniques are typically influenced by extraction type, choice of right solvents, agitation, temperature and material ratio (Thomas et al., 2012). Owing to the previous facts that drying at high temperature may decompose the bioactive constituents of plants materials linked to their compromised biological potential (Larrauri et al., 1997); all the green materials were air dried under shade. The plants material was ground to get
43 Chapter # 4 Results and Discussion maximum yield (%) of extracts. According to Mukhopadhyay et al. (2006) diminutions in particle size break certain structures to release of bound phytonutrients and facilitate the penetration of solvent by reducing the distance from the inner surface. Effectiveness of extracting solvent is another parameter to dissolve endogenous components of plant materials. Previously, methanol as well as ethanol has been proven as an effective organic solvent for phytochemicals extraction (Dalla-Valle et al., 2007; Gorinstein et al., 2007; Yang et al., 2009). So, in the current study, soluble and free non-nutrient phytochemicals from selected plant material were extracted with methanol as well as ethanol. Present study mainly focuses on the soluble free phytochemical extracts for biological activities due to highest % yields of analyzed samples as compare to bound form which shows insignificant extent of extract yield. The % yields of soluble phytochemicals of selected plant materials with different organic solvents are shown in (Fig 4.1).
120 Methanol Ethanol 100
80
60
40
20
Extraction yield (%)Extraction 0
Samples
Fig 4.1. Percentage yield of extracts from selected plant materials using methanol/ethanol as solvent
Among the selected vegetables highest extraction yield (%) for carrot (60.65%) and the lowest for fenugreek (29.77%) was observed. No difference was observed in the extraction yield of rest of the samples in both the organic solvents. Methanolic extraction yield (%) of vegetables varied ranging from 29.77–60.65% and the yield (%) of ethanolic extract was ranging from 26.75–59.6%. Overall trend for the selected vegetable extracts yield (%) in both the solvent medium was almost same which was as follows; carrot> turnip> sweet potato> eggplant> fenugreek.
44 Chapter # 4 Results and Discussion
Moreover, in comparison of selected dry fruits for their bioactive compound, the maximum yield (%) of raisins, dates and apricot respectively was observed for both solvents. Yield (%) of ethanolic extraction varied from 70.21% for dates to 11.59% for almond. Methanol extract of dried fruits produced yield (%) ranging from 15.75 to 85.7%. These differences in extract yields may be due to differences in extractable components of plants, their chemical composition, agroclimatic conditions and soil nature (Hsu et al., 2006). Present results strengthen the evidences from previous reports that both the extraction solvents used in current study are virtuous for phytochemical constituents from plant-based materials (Rizwan et al., 2012; Ahmed et al., 2013; Rajan et al., 2013). Because methanol extracts provided higher yield than ethanolextracts, methanol extracts were used for subsequent biochemical and physiological analysis. 4.2. Proximate analysis of selected plant materials The knowledge of the proximate composition of cereals, grains fruits and vegetables being essential components in human diet is so much important especially when determining their functional food applications (Salvatore et al., 2005; Flyman and Afolayan, 2006). Recently, mediterranean diet has been playing an important role in complementing the staple agricultural foods in many countries (Hadjichambis et al., 2008; Tardío, 2011). However, there is a paucity of information on the nutrient contents of green food, particularly the ones traditionally consumed (Schauss, 2010). Apart from the former knowledge focusing on the therapeutic potential of bioactive compounds, very few data are available on the nutritional composition of some of the most widely-consumed edible plants. Therefore, the revalorization of traditionally consumed green material is an important strategy to improve the diversity of available foods and their composition, to facilitate health intervention research and programming, which today is receiving the focus of renewed attention (Burlingame et al., 2009). Although some reports exist on some of the nutrients in a few of the foods (Bhargava et al., 2008; Andersson et al., 2011), without common control samples between studies it is impossible to compare nutrient concentrations because inter laboratory analytical uncertainty could be confused with a true difference in composition (Phillips et al., 2006). Additionally, different growing conditions can affect the concentration of nutrients in the same plant species (Bhargava et al., 2008). Biodiversity of food composition is of increasing interest for sustainable food supplies (Burlingame et al., 2009; Charrondière et al., 2013).
45 Chapter # 4 Results and Discussion
4.2.1. Proximate analysis of selected vegetables The proximate analysis of selected vegetables indicate that as usual, there was a variation in crude protein content ranging from 7.29± 0.03% to 28.07±0.03 % (Table 4.1). Fenugreek (28.07±0.03%) showed the maximum protein while turnip and carrot were at lowest position respectively. In sweet potato and eggplant similar percentage of crude protein (12.21±0.11% and 13.85±0.03%) was observed respectively. According to the National Research Council of United States, crude protein less than 20% indicates low protein content of that feed stuff. However, present study was in the harmony with the finding of Sumayya et al. (2012) and Singh et al. (2013). Singh and Usha (2003) reported that fenugreek seed was rich in protein with a well-balanced amino acid pattern which was in accordance to current results. Carbohydrate content in tested vegetables varied from 51.84±0.02 to 20.93±0.02%. The maximum carbohydrate was observed in carrot whereas it was the minimum in fenugreek. The principle function of carbohydrates is to serve as a major source of energy for the body. In developing countries diets 60 to 80% of energy is derived from carbohydrates. The findings are in consonance with Sumayya et al. (2012) and Singh et al. (2013). The crude lipid contents were recorded similar in fenugreek, carrot and eggplant (2-3%), while S. potato and turnip showed slightly less fat contents among the samples. Results of the lipid contents of present investigation were in line with that reported by Dike (2010). Table 4.1. Proximate composition of selected vegetables (%) Scientific Vernacul CP (%) Carb (%) Fat (%) Fiber (%) Ash (%) name ar Name I. batatas S. potato 12.21±0.11a 35.74±1.09b 0.88±0.01a 5.88±0.10a 8.83±0.16c T. foenum Fenugreek 19.07±0.03b 20.93±0.02a 2.50±0.01a 16.50±0.06b 13.00±0.01d graecum D. carota Carrot 7.66± 0.05a 51.84±0.02c 2.00±0.01a 18.5± 0.02c 10.00±0.02c S.Melongena Eggplant 13.85±0.03ab 45.65±0.01b 3.50±0.02a 18.50±0.02c 8.50± 0.02c B. rapa Turnip 7.29± 0.03a 40.71±0.04b 1.00±0.02a 12.5± 0.03b 8.50± 0.02c Values are mean ± SD of carefully conducted triplicate experiments. Superscripted alphabets within a column indicate significant (p<0.05) observed at 95% confidence interval (CI). Recent research indicated that all the tested vegetables were good source of dietary fiber ranging 10.00±0.02 to 13.00±0.01 g/100g. Certain reports suggested that consumption of high fibrous diet have certain health promoting effects, including regulation of the intestinal function, improved glucose tolerance in diabetics, and the prevention of chronic diseases
46 Chapter # 4 Results and Discussion such as colon cancer (EFSA, 2010). Fenugreek and carrot had slightly higher ash content (13.00±0.01 and 10.00±0.02/100 g respectively), as compare to other samples which showed the similar percentage of ash content. Macronutrients are responsible for energy production in the human organism. In this regard, it should be highlighted that all the edible plants studied are foods with a very moderate energy value (Fig. 4.2), most providing around 186.56–321 kcal/100 g. 4.2.2. Proximate analysis of selected dry fruits The results of proximate analysis of tested dry fruits showed variant concentrations of biochemical and other contents (Table 4.2). The results obtained from carbohydrate analysis, dates (94.04±0.03%), raisins (93.45±0.03%) and apricot (86.49±0.01%) had prominent level of carbohydrates but all these were lowest in fat content (0-1%), respectively among the selected species. These results resemble to those obtained by (Dimitrios et al. 2008; Gary and Arianna 2010; Ghrairi et al., 2013). Dimitrios et al. (2008) explain that carbohydrates existing in raisin are in the form of glucose and fructose; it easily passes into the blood without digestion because of their nature as rapid sugars. This is of nutritional importance, especially for certain ailments and in situations demanding immediate energy. These rapid sugars especially glucose needed for the central nervous system, the kidneys, the brain, the muscles (including the heart) to function property (Srilakshmi, 2006). Except dates, raisins and apricot all the tested fruits were high in fat percentages ranging from 34.00±0.03% (almond) to 55.50±0.01% (walnut). While analyzing the protein contents, a significant difference was reported among the selected fruit species ranging from 1.46± 0.03 g/100g to 27.71±0.03 g/100g. Peanuts (27.71±0.03%), almond (23.70±0.08%), walnut (21.51±0.03%) and pistachio (21.51±0.03%) were superior while raisins (2.55± 0.03%), apricot (4.01± 0.03%) and dates (1.46± 0.03%) were observed inferior in protein content among the tested fruits species. These results were analogous to those of Dimitrios et al. (2008) and Ghrairi et al. (2013). Considering the resulted achieved from fiber analysis coconut (23.00±0.02%) and almonds (18.00±0.02%) exhibited the highest content followed by pistachio (11.50±0.05%), Walnut (8.50± 0.01%) and peanut (6.00± 0.01%) which were moderate in fiber. Among the samples, apricot, dates and raisins have the lowest fiber content, 3.00± 0.02%, 2.50± 0.01% and 1.50± 0.01, respectively. Considering the results varied ash content was observed in all the tested samples ranging from 1.50± 0.02 to 5.50± 0.01.
47 Chapter # 4 Results and Discussion
Table 4.2. Proximate composition of selected dry fruits (%) Scientific Vernacular CP (%) Carb (%) Fat (%) Fiber (%) Ash (%) name Name P. dactylifera Dates 1.46± 0.03a 94.04±0.03d 0.05± 0.00a 2.50± 0.01a 2.00± 0.03a Juglans Wal nut 21.51±0.03c 12.99±0.02a 55.50±0.01d 8.50± 0.01a 1.50± 0.02a P. dulcis Almond 23.70±0.08c 20.30±0.02b 34.00±0.03b 18.00±0.02c 4.00± 0.01a P. vera Pistachio 21.51±0.03c 20.99±0.02a 42.50±0.04c 11.50±0.05b 3.50± 0.05a A. hypogaea Peanut 27.71±0.03d 21.79±0.05a 41.50±0.01c 6.00± 0.01a 3.00± 0.02a V. vinifera L. Raisins 2.55± 0.03a 93.45±0.03d 0.00± 0.00a 1.50± 0.01a 2.50± 0.01a C. nucifera Coconut 14.22±0.00b 11.78±0.02a 45.50±0.01c 23.00±0.02d 5.50± 0.01a P. armeniaca Apricot 4.01± 0.03a 86.49±0.01c 1.00± 0.05a 3.00± 0.02a 5.50± 0.01a
Values are mean ± SD of carefully conducted triplicate experiments. Superscripted alphabets within a column indicate significant (p<0.05) observed at 95% confidence interval (CI). Maximum ash content was reported in apricot coconut, and almond. Pistachio peanuts, raisins were moderate while dates and walnut were lowest in case of ash content. According to the results, all the tested fruits species had significantly high energy values as compare to studied vegetables (Figure 4.2). The order for the calories reported by current research was as follows: Walnut> Peanuts> Pistachio> Coconut> Almond> Raisins> Dates> Apricot. The values were varied ranging from 371.00± 0.02 to637.49± 0.08(Kcal/100g
1000
800 600 400 200 0 Energy (Kcal/100g) Energy
Samples Fig 4.2. Energy values of the selected plant species. 4.3. Mineral Analysis of the selected plant materials Human beings are encouraged to consume vegetables and fruits, due to their excellent nutritional potential associated to health enhancing properties (Akinyele and Shokunbi, 2015a). The upshots of modern research shifted the scientists focus towards the chemical
48 Chapter # 4 Results and Discussion
composition of plant materials, particularly the content of minerals and trace elements due to their vital role in human wellbeing (Demirel et al., 2008; Kalpna et al., 2011). Trace amounts of some metals such as manganese, iron, copper, zinc, cobalt and selenium are essential micro-nutrients and have a variety of biochemical functions in all living organisms (Sharma, et al., 2012; Zaccari et al., 2015). While these elements are essential, they can be toxic when taken in excess. However, some elements like lead, cadmium and arsenic are non-essential metals as they are toxic, even in traces. Metals such as aluminum, cadmium and lead are found throughout the environment and are present virtually in all food at the extremely low levels (Demirel et al., 2008). These heavy metals interfere with protenious stuctures by covalent linkages; lead towards the protien denaturation and decline their biological activities. The ingestion of heavy metals (Cd, Ni, Pb) can cause depletion of some essential nutrients in the body, which in turn causes a decrease in immunological defences, intrauterine growth retardation, psychosocial dysfunctions, disabilities associated with malnutrition and a high prevalence of upper gastrointestinal cancer (Akinyele and Shokunbi, 2015a). The elemental composition of food items differs from one group to another due to their different particulate nature and structural matrix (Akinyele and Shokunbi, 2015 b). 4.3.1. Mineral analysis of selected vegetables The mineral analysis of the vegetable species showed significant variability among different macro and microminerals (Table 4.3). K, Na, Ca and Fe were present in significant amount in all the investigated plant species ranging from 906.60-2845.80 (mg/100g), 260.20-1788.60 (mg/100g), 110.20-373.10 (mg/100g) and 37.26-107.00 (mg/100g), respectively. P, Mg, Cu and Zn were moderately high in all the samples with different ranges 24.60-51.70 (mg/100g), 14.50-31.20 (mg/100g), 3.36-12.50 (mg/100g) and 3.50-18.10 (mg/100g) respectively. Ni, Cr, Mn, Pb and Co contents were present in negligible concentrations within the vegetables. Fenugreek was superior in Cu, Zn, Fe, Ca, P and Mg mineral elements among the edible samples. The trend was seems to be same as reported by Gala and Gujar, (2014). High level of Na and K in carrot and turnip was in accordance to Ekholm et al. (2007). The lowest values of Cu, Zn, Fe, K and Ca were recorded in sweet potato. In turnip P was observed in lowest concentration. According to current data carrot, eggplant and turnip were also
49 Chapter # 4 Results and Discussion
moderately rich source of minerals (Ca, K, Mg, Fe, Cu and Zn) that was in accordance to previous studies (Sharma et al., 2012; Zaccari et al., 2015). The remaining nutrients including Ni, Cr, Mn, Pb, Ni and Co had negligible concentration levels. It has been reported that for many plant species Cr proved to be toxic at 5 mg/L (Hussain et al., 2009). In this regard, all the studied vegetables have very low concentration of Cr as compared to that of reported level for toxicity in plants. In case of the Pb concentration, the suggested concentration in plant species is 2 - 6 mg/L (Hussain et al., 2009). However, the plant species under investigation carries very lesser level of Pb, which further clarifies their use as food supplement (Akinyele and Shokunbi, 2015b). 4.3.2. Mineral analysis of selected dry fruits Table 4.4 shows selected mineral compositions of studied dry fruits. Among the minerals, maximum Calcium (Ca) concentrations were found in dry fruits ranging from 15.40 to 261.50 mg/100g. However, the magnesium (Mg) concentration changes between 29.50 mg/100 g (apricot) and 184.20 mg/100 g (peanuts) are significantly different from those reported by Simsek et al. (2004). Currently, high potassium (676.60–1592.00 mg/100 g DW) and bit lower sodium (130.10–455.30 mg/100 g DW) was in accordance to Emine et al. (2011) and Gary and Arianna, (2010). Copper levels ranged from 3.90 to 11.90 mg/100g in which elevated concentrations were found in walnut. According to several food composition tables and other authors, Cr content reflects the wide variability in nuts varying between 4.00 and 16.05 mg/100g; the higher concentrations were detected in apricot, almonds and dates. Normal dietary intakes of Cr in adults range from 13 to 61 mg/day, depending on age, country and gender (He and MacGregor, 2008). Iron concentrations ranged from 23.80 to 50.0 mg/100g in present samples which is lower from the value 7.30 to 75.60 mg/g reported by Cabrera et al. (2003). Present results were superior from the reported value of Fe in nuts 1.6–10.4 mg/100 g (He and MacGregor, 2008). Zinc levels determined in present samples ranged between 4.40 and 15.10 mg/100g. Foods differ widely in their Zn content in which nuts are relatively good sources of Zn (Sharma et al., 2012). Total dietary Zn intakes are influenced greatly by food choices. Frequently, Zn intakes are correlated with protein intake, but the exact relationship is influenced by protein source. In recent study, Ni levels in dry fruits ranged from 13.00 to 26.50 mg/100g, in disaccordance to 10.00 to 64.00 mg/100g (Cabrera et al., 2003). Total dietary Ni intakes of humans vary greatly with amounts and
50 Chapter # 4 Results and Discussion
portions consumed of foods of animal origin (nickel-low) and plant origin (nickel-high). Conventional diets, however, often provide less than 150 mg/day (Ekholm et al., 2007). Toxic compounds including heavy metals find their way into food during manufacture, storage or transportation; these include largely heavy metals (Akinyele and Shokunbi, 2015a). Lead levels ranged from 2.50 to 23.60 mg/100g in the current study in accordant to Cabrera et al. (2003) who estimated the range 0.14 to 0.39 mg/g in selected samples. Plant susceptibility to Pb accumulation is also affected by the duration of exposure, climate, physicochemical characteristics of the soil, the plant species in question, or the anatomical part of the plant considered (Ghrairi et al., 2013). Keeping in view the results, all the studied fruits and vegetables are rich source of micro and macro elements exhibiting the least toxic risks regarging heavy metals.
51
Table 4.3. Mineral composition of selected vegetables (mg/100g)
Name Cu Pb Co Zn Fe Cr Ni Na K Ca P Mg Mn
Sweet potato 3.36 ±0.1 0.08±0.5 0.80±0.2 3.50±0.1 37.26±0.2 0.17±0.1 0.80±0.1 506.10±0.5 906.60±0.01 135.10±0.2 45.40±0.2 27.60±0.1 0.29±0.2 Fenugreek 12.50±0.2 1.20±0.2 1.90±0.1 18.10±0.2 107.00±0.2 3.70±0.1 4.60±0.2 1203.30±0.5 2119.40±0.1 373.10±0.2 51.80±0.5 31.20±0.2 0.14±0.2 Carrot 6.20±0.3 1.00±0.0 1.80±0.1 7.30±0.1 55.90±0.0 1.40±0.2 1.80±0.1 1788.60±0.4 2089.60±0.3 155.20±0.3 37.20±0.1 14.50±0.2 3.16±0.1 Bringle 10.40±0.2 1.40±0.1 0.70±0.1 8.20±0.2 56.40±0.0 1.80±0.1 1.10±0.0 325.20±0.4 2109.50±0.1 110.20±0.1 24.60±0.1 16.10±0.1 0.45±0.1 Turnip 12.00±0.4 0.50±0.1 3.50±0.1 12.40±0.2 63.80±0.1 3.90±0.5 5.70±0.0 260.20±0.01 2845.80±0.4 135.30±0.1 30.70±0.3 25.30±0.1 0.35±0.1 Values are mean ±SD, n=3
Table 4.4. Mineral composition of selected dry fruits (mg/100g) Name Cu Pb Co Zn Fe Cr Ni Na K Ca P Mg Mn Dates 9.90±0.0 18.10±0.1 57.70±0.2 12.70±0.1 37.20±0.3 16.00±0.2 14.40±0.1 455.30±0.5 955.20±0.2 41.50±0.1 65.60±0.2 46.80±0.1 0.27±0.2 Wal nut 11.90±0.1 19.70±0.2 66.80±0.1 14.50±0.2 50.00±0.1 7.60±0.1 26.50±0.1 162.60±0.6 676.60±0.5 163.19±0.2 20.40±0.3 142.10±0.1 3.47±0.1 Almond 7.00±0.0 2.50±0.1 17.20±0.3 14.20±0.1 35.40±0.1 16.10±0.3 15.70±0.2 260.20±0.5 985.10±0.2 261.50±0.1 474.00±0.2 248.00±0.2 2.59±0.2 Pistachio 9.80±0.0 18.40±0.1 18.60±0.0 4.40±0.1 45.70±0.3 10.40±0.3 13.00±0.0 357.70±0.2 1293.50±0.3 110.30±0.1 485.70±0.2 120.20±0.1 1.29±0.1 Peanut 11.40±0.0 14.50±0.1 35.30±0.0 8.80±0.2 30.10±0.4 9.20±0.1 18.70±0.0 227.60±0.3 865.70±0.4 64.30±0.3 335.70±0.1 184.20±0.2 0.78±0.1 Raisins 3.90±0.2 14.10±0.2 51.90±0.1 11.70±0.1 48.70±0.2 4.00±0.2 22.40±0.1 195.10±0.1 1293.50±0.2 30.20±0.1 40.50±0.2 76.20±0.1 0.15±0.1 Coconut 8.10±0.0 23.60±0.3 36.80±0.2 11.80±0.3 33.80±0.1 6.80±0.3 29.60±0.1 357.70±0.5 1383.10±0.1 15.40±0.1 115.20±0.1 32.90±0.1 0.09±0.1 Apricot 8.50±0.0 16.30±0.1 20.90±0.1 15.10±0.3 23.80±0.2 16.20±0.5 34.30±0.2 130.10±0.4 1592.00±0.01 49.50±0.2 65.50±0.1 29.50±0.1 0.30±0.1
Values are mean ±SD, n=3
52 Chapter # 4 Results and Discussion
4.4. Preliminary phytochemical analysis Phytochemicals are compounds, derived from plants hypothesized to confer disease protective characteristic (Berma et al., 2013). Generally, the plant chemicals protect plant cells from hazards of environment like stress, pollution, pathogen attack, drought and UV exposure. Now their role to protect human health is well defined. They exhibit extensive potential as antioxidants (Khan et al., 2013) antimicrobial agents (Das, 2012), anticancer (Amadi et al., 2013), boosts immune system, decrease platelets aggregation and modulates hormone metabolism. The results of the preliminary phytochemical screening are presented in table 4.5. Preliminary screening revealed the presence of alkaloids, saponins, tannins, flavonoids and β-carotene in both vegetables as well as in dry fruit samples. Phytochemical constituents in all the edible samples vary significantly. The presence of these phytoconstituents was deep-rooted by several studies (Tiwari et al., 2009; Sumayya et al., 2012; Amadi et al., 2013). Table 4.5. Qualitative analysis of phytoconstituents of selected plant materials Sr Common Scientific Alkaloids Tannins Saponins Flavonoids β-carotene # Name Names
1 I. batatas S. potato +ve +ve +ve +ve +ve 2 T. foenum- Fenugreek +ve +ve +ve +ve +ve graecum 3 D.s carota Carrot +ve +ve +ve +ve +ve 4 S. Melongena Bringle +ve +ve +ve +ve +ve 5 B. rapa rapa Turnip +ve +ve +ve +ve +ve 6 P. dactylifera Dates +ve +ve +ve +ve +ve 7 J. uglans Wal nut +ve +ve +ve +ve +ve 8 P. dulcis Almond +ve +ve +ve +ve +ve 9 P. vera Pista +ve +ve +ve +ve +ve 10 A. hypogaea Peanut +ve +ve +ve +ve +ve 11 V. vinifera L. Reisins +ve +ve +ve +ve +ve 12 C. nucifera Coconut +ve +ve +ve +ve +ve 13 P. armeniaca Apricot +ve +ve +ve +ve +ve
53 Chapter # 4 Results and Discussion
4.5. Quantitative analysis of phytochemicals 4.5.1. Quantitative analysis of phytochemicals of vegetables Among the selected vegetables highest amount of alkaloids were obtained from fenugreek (0.07 mg/g) while least were determined for eggplant (0.01 mg/g). In remaining plants the order of crude alkaloids content (mg/g) lie as given carrot> turnip >s. potato ranging from 0.04 to 0.02 (mg/g). Flavonoids, polyphenolic compounds with common benzopyrone ring structure are found to have definite role in biological system as antioxidants. This phytoconstituent was estimated quantitatively (mg/g) and maximum quantity was found in fenugreek (68.25 mg/g) followed by turnip (38.35 mg/g) and s. potato (37.68 mg/g). Carrot and eggplant had least concentration of flavonoids 17.89 mg/g and 9.37 mg/g respectively. Order of flavonoids content varied from fenugreek> turnip>s. potato> carrot> eggplant. Plants saponins known as natural antibiotics (Raju and Rao, 2012) were also present in fenugreek (38.78 mg/g) in high amount while least value was estimated for eggplant (11.63 mg/g). In term of quantity for saponins the difference in selected vegetables was as follows: fenugreek>carrot> turnip> s. potato> eggplant. Tannins have also been recognized due to their therapeutic potential. Lowest amount of tannins was reported in carrot (7.45 mg/g) while fenugreek once again at the top of the list with highest value (37.42 mg/g) as shown in Table 4.6. Remaining samples were in the trend as follows: turnip>s.potato>eggplant ranging from 11.91 to 16.64 (mg/g). Fruits and vegetables contain different types of carotenoids in different extents (Fesco and Boudion, 2002); act as potential antioxidants and a key to preserving health and avoiding different ailments (Southon and Faulks, 2003; Krinsky and Johnson, 2005). Dietary β carotenes have a potential in vitamin synthesis within the human body (Low and Van, 2008; Omodamiro et al., 2013). The results of present study conferred that all the selected vegetables are rich source of β-carotens ranging from 0.01-0.02 (mg/g); overall order was as follows: Carrot>fenugreek>sweet potato>eggplant>turnip. In a previous reports, Fesco and Boudion, (2002) suggested that carrots, sweet potatoes and leafy vegetables contain high levels of β-carotene, usually exceeding 8000 I.U. per 100 g and can therefore cover the recommended daily intakes (5000 to 25000 I. U. ). Current findings strengthen the evidences of Kornsteiner et al. (2006) that carotenoids are abundant in most colorful plant foods; with 7.6 and 6.6 mg/100 g β- carotene and lutein found in carrots.
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Agoreyo et al. (2012) revealed that alkaloids, tannins and saponins could be responsible for many pharmacological activities. They quantify alkaloids, tannins and saponins in two varieties of S. melongena and reported the following trend 0.99-1.16 mg/100g, 11.33- 12.82mg/100g and 5.34-11.34mg/100 g, respectively. Several studies were also linked the presence of saponins and glycoalkaloids with the protection of plant from microbial pathogens as defensive molecules (Tiwari et al., 2009; Amadi et al., 2013). Phytochemical screening of the acetone extracted seed samples of Trigonella foenum-graecum by Shaikh et al., (2013) disclosed the presence of flavonoids, phenols, tannin, terpenoid, saponin and proteins but the absence of alkaloid which was in discordance to present study Table 4.6. Quantitative analysis of phytoconstituents (mg/g) of selected vegetable
Scientific Name Common names Alkaloids Tannins Saponins Flavonoids β-carotene
I. batatas S. potato 0.02±0.01 13.51±0.61 21.94±0.23 37.68±0.58 0.01±0.25 T. foenum-graecum Fenugreek 0.07±0.51 37.42±0.52 38.78±0.25 68.25±0.52 0.01±0.25 D. carota Carrot 0.04±0.72 7.45±0.21 28.34±0.54 17.89±0.45 0.02±0.46 S. Melongena Eggplant 0.01±0.35 11.91±0.17 11.63±0.58 9.37±0.65 0.01±0.27 B. rapa rapa Turnip 0.03±0.37 16.64±0.25 23.82±0.19 38.35±0.45 0.01±0.24 Values are mean ±SD, n=3 4.5.2. Quantitative analysis of phytochemicals of selected dry fruits The analyses of different dry fruits samples revealed fascinating outcomes. The quantitative results of phytochemical analysis exposed high preponderance of secondary metabolites especially flavonoids, tannins, alkaloids and saponins except β-carotene which was in least concentration among the selected samples (Table 4.7). The quantitative results revealed that pistachio (0.04 mg/g) was at the top followed by walnut (0.03 mg/g) with highest concentration of alkaloids. Apricot (0.03 mg/g), almond (0.02 mg/g) and dates (0.02 mg/g) also had appreciable alkaloids. Least alkaloid content was observed in peanuts (0.01 mg/g), coconut (0.01mg/g) and raisins (0.01 mg/g). The tannins content varied as follows: almond> pistachio> walnut> peanuts> apricot> coconut> dates> raisins ranging from highest (32.78 mg/g, walnut) to lowest (7.45 mg/g, Raisins). Higher concentration of saponins was observed in peanut (9.51 mg/g) followed by dates (9.32 mg/g), almond (8.34 mg/g) and pistachio (7.89 mg/g) respectively. Walnut also reported a good source of saponins by our results having the value (7.15mg/g). Lower concentration was observed in raisins (4.34mg/g), apricot (3.82mg/g) and coconut (3.36mg/g).
55 Chapter # 4 Results and Discussion
Flavonoids have been identified in all tested samples at significant level. Almonds had the highest total flavonoid content at 38.63 mg/g while raisins were at the lowest order having 7.89 mg/g among the fruits. Remaining samples were in a trend: apricot>pistachio>dates>peanut>walnuts>coconut ranging from 7.89 to 22.35 mg/g. Bolling et al. (2010) gave the confirmation about the presence of flavonoids in all the nuts ecxept macadamias and Brazil nuts. In their study, almonds also had an appreciable content of flavonoids with 15 mg/100 g, respectively. According to USDA (2007) flavanones have only been found in almonds. Almonds and pistachios are the only nuts that contain flavonols, mainly as isorhamnetin and quercetin. Pistachios have the highest isoflavone content of nuts at 3.63 mg/100 g, 100-fold greater than the levels of other nuts; in contrast, peanuts contain 0.26 mg isoflavones/ 100g (USDA, 2008). Table 4.7. Quantitative analysis of phytoconstituents (mg/g) of selected dry fruits Common Scientific Name Alkaloids Tannins Saponins Flavonoids β-carotene names P. dactylifera Dates 0.02±0.22 12.53±0.09 9.32±0.25 12.46±0.25 0.007±0.32 Juglans Wal nut 0.03±0.23 23.42±0.07 7.15±0.32 10.31±0.31 0.007±0.05 P. dulcis Almond 0.02±0.54 32.78±0.24 8.34±0.07 38.63±0.19 0.003±0.02 P. vera Pistachio 0.03±0.36 31.25±0.15 7.89±0.08 19.37±0.12 0.008±0.14 A.hypogaea Peanut 0.01±0.24 23.27±0.06 9.51±0.10 10.84±0.05 0.003±0.01 V. vinifera L. Raisins 0.01±0.28 7.45±0.07 4.34±0.21 7.89±0.05 0.002±0.02 C. nucifera Coconut 0.01±0.08 15.31±0.31 3.63±0.05 9.37±0.04 0.002±0.01 P. armeniaca Apricot 0.03±0.25 16.64±0.04 3.82±0.03 22.35±0.21 0.004±0.01
Values are means ±SD, n=3s Chen and Blumberg (2008) also reported the highest total flavonoid concentrations in almonds at 15, and pistachios and hazelnuts at 12 mg/100 g, respectively. No flavonoids have detected in Brazil or macadamia nuts. It is worth noting that some nuts can contribute total flavonoids to the diet in amounts comparable to some fruit and vegetables; e.g., almonds have a similar quantity of total flavonoids as red delicious apples at 15 and apricots at 13 mg/100 g; these results were in accordance to the values given by USDA (2007) but in contrast our reported values for almond, pistachio and apricot; superior than their values. Phytochemical analysis of present samples revealed that the least level of β-carotene for all the samples exhibiting least values. Kornsteiner et al. (2006) reported that carotenoids are abundant in colorful foods, tree nuts and other dry fruits contain very low amounts of
56 Chapter # 4 Results and Discussion
carotenoids found in μg/100 g concentrations that were in the support of current study. Pistachios present a modest exception with mean β-carotene content of 4.40 mg/100 g dry weight (Kornsteiner et al., 2006) which was higher from our reported value. Ouchemoukh et al. (2012) reported the concentration of carotenoïds of apricots (10.70 mg/100g) and raisins (2.20 mg/100g) which also differs from current results. 4.6. Antioxidant potential of plant materials 4.6.1. Antioxidant concentration of selected plant materials Natural products are under exploration due to high costs and potentially hazardous side effects of commercially available synthetic antioxidants and drugs. Herbal remedies are important sources of natural antioxidants and possible candidates for drug development. By the middle of the 19th century approximately 80% of all remedies were derived from herbs (Gilani and Rahman, 2005). It is widely accepted that green sources indicate the presence of wide variety of biologically active constituents (Alves and Rosa, 2007). The beneficial effects of medicinal plants, which are rich sources of phenolic compounds, are often assumed to be acting by antioxidant mechanisms and enzyme inhibitory properties such as inhibition of α-amylase, α-glucosidase and pancreatic lipase due to their ability to bind the proteins (Tang and Halliwell, 2010; Exteberria et al. 2012; Gulati et al. 2012). However, phenolic compounds are extensively metabolized in vivo due to a large number of enzymatic reaction (Manach et al., 2004) and therefore their bioactivities are still unclear. The selected plant materials were analyzed to study their antioxidant activities. Preliminary phytochemicals screening revealed that the plants under investigation showed richer source of alkaloids, flavonoids and saponins. 4.6.1.1. Total phenolic contents Polyphenols are the large group of phytochemicals that are gaining acceptance as being responsible for the health benefits associated with fruits and vegetables. Because of their chemical structure, plant polyphenols can scavenge free radicals and inactive other pro- oxidants, and also interact with a number of biological relevance (Yilmaz, 2006; Cooper et al., 2008). A number of possible health-related activities of biological system such as anti- cancer, anti-inflammatory, antifungal antimicrobial as well as antioxidants have been attributed to phenolic compounds including phenolic acids and flavonoids (Yilmaz, 2006). Considering that the phenolic compounds are potent antioxidants in fruits and vegetables, we investigated further the total phenolic content in both selected vegetables and dry fruits.
57 Chapter # 4 Results and Discussion
i. Total phenolic contents of selected vegetables The total phenolic contents of selected vegetables are shown in Table 4.8 gives the total phenolic content (TPC) of the selected vegetables. Phenolic contents were expressed as milligrams of gallic acid equivalents per gram of dry weight of the edible portion of vegetables. The total phenolic content (TPC) was markedly higher in fenugreek (16.45±.01 mg/g) among all the vegetables which signifies it highest antioxidant activity. Previously, similar studies of Aqil et al. (2006) and Ramya et al. (2011) have been reported the TPC value of methanol (74.33±5.13 mg/g GAE) and ethanolic (4.9 mg/g) fenugreek leaf extracts which differ from our value. Eggplant had a moderate (6.96±.02 mg/g) while carrot, sweet potato and turnip were showed minimum and almost same TPC content respectively. Among the vegetables, overall trend for the TPC content was as follows: fenugreek> eggplant> carrot>s. potato>turnip. Phenolics have also attracted the researchers for their use in different biological activities (Patel et al., 2012). Moreover, amounts of phenolics reported for methanolic extract have high mean value of 3.87 g/100 g DW than aqueous extract of 2.93 g/100 g DW mean value. These findings are in line with current study where high phenolics are obtained by methanolic extract. Differences between the results may be due to differences in ecological conditions such as climate, temperature, location, diseases and pest exposure within the species, choice of parts tested, time of taking samples, and methods of determination (Singh et al., 2014). ii. Total phenolic content of dry fruits Nuts are rich source of several therapeutically active constituents, especially polyphenols (Carvalho et al., 2010). Total phenolic content of dry fruits have been presented in Table 4.9. All dry fruit extracts exhibited significant level of total phenols ranging from 8.44±.02 to 1.46±.05 mg/g. Walnut, pistachio, dates and peanuts exhibit the highest antioxidant activity. Maximum phenolic content (8.44±.02 mg/g) was observed in walnut whereas coconut showed least but significant phenolic content among all the extracts. Apricot, almonds and raisins were modest in phenolic content. Previously, walnut and almonds have been reported to be rich in tannins (Amarowicz and Pegg, 2001); these phenolic compounds might account for the values obtained as tannin (Hwang et al., 2001). Highest total phenolic content of methanolic extracts of walnut is in agreement to several other studies showing walnuts (seeds) as a good dietary source of phenolics (Chen and Blumberg, 2008; Yang et al., 2009). Ouchemoukh et al. (2012) reported that ethanolic extract of raisins had more concentration of
58 Chapter # 4 Results and Discussion
total phenolics as compare to aqueous extract of figs which was in accordance to our value. In a similar study, Mishra et al. (2010) reported the concentration of total phenolic compounds of methanolic extract of raisins (0.83 g/100 g) that was superior to our reported value. However the discordance in total phenolic content of fruits between different studies could be due to varietal, seasonal, agronomical differences, genomics, moisture content, method of extraction and standards used, etc. (Kalpna et al., 2011). 4.6.1.2. Total flavonoid content (TFC) Flavonoids are the main class of polyphenols; widely distributed in plant-based foods, consist of two aromatic rings linked by 3 carbons typically in an oxygenated heterocycle ring. Based on differences in the heterocycle C ring, flavonoids are categorized as flavonols, flavones, flavanols, flavanones, anthocyanidins, and isoflavonoids. Naturally occurring flavonoids are mostly conjugated in glycosylated or esterified forms but can occur as aglycones (Pereira et al., 2011). It is estimated that flavonoids account for nearly 60% of the phenolics in our diet, and the remaining 30% are from phenolic acids. They are known to be antioxidants and free radical scavengers (Tapas et al., 2008). The antioxidant activity of plants has also been correlated to the total flavonoid content. i. Total flavonoid content of selected vegetable extracts Total flavonoid content of vegetable extracts is shown in Table 4.8. The amount of total flavonoid content in methanolic extracts of different vegetables ranged from 0.16±0.53- 6.56±0.01 mg/g. Higher amount of flavonoid were found in fenugreek extract while lowest amount was tested for extract of Turnip. Depending upon flavonoid contents order of different vegetables lie as fenugreek>sweet potato>eggplant>carrot>turnip. These results suggested that the antioxidant activities might be due to their high level of flavonoid content ii. Total flavonoid content of selected dried fruits extracts Among the studied dry fruits (Table 4.9), walnuts (3.78±0.21 mg/g) contained the highest flavonoid content, followed by pistachio and dates, respectively. Peanuts, apricots and raisins have moderate phenolic content. The lowest content was observed in almonds and coconut sample. The trend varied ranging from 3.78±0.21 mg/g to 0.09±0.20 mg/g. The order of flavonoid content was observed in current study as follows: walnuts> pistachio> dates> peanuts> apricots> raisins> almonds> coconut. In a previous study, Walnuts had the highest flavonoid content followed by pistachios, almonds and peanuts, (Yang et al., 2009). Ouchemoukh et al., (2012) reported that the highest concentrations of flavonoids for
59 Chapter # 4 Results and Discussion ethanolic and aqueous extracts of figs. The aqueous extract of raisins presented the lowest concentration (Vallejo et al., 2012). 4.6.2. Antioxidant activities of plant materials A variety of methods specific to their chemical properties have been used to quantify antioxidant activity (AOA) in plant foods. Since DPPH and FRAP radical scavenging assays are widely used due to their simplicity, stability, accuracy and reproducibility we conducted these methods to quantify AOA in the present study. 4.6.2.1. Reducing power assay Reducing power is one of the mechanisms of antioxidant activity, determined by measuring the reductive ability of antioxidant through the conversion of ferric ion into ferrous form present in the plant extracts (Ouchemoukh et al., 2012). When ions conversion takes place, colour is changed from yellow to bluish green. The colour intensity is indicator for reducing potential. Plant extract showing high antioxidant activities will give increased absorbance and more intense will be the colour (Oktay et al., 2003; Sultana et al., 2007). It is a fast and simple assay which gives reproducible results. i. Reducing power assay of selected vegetables Among the tested vegetables, the strongest antioxidant property as depicted by the reducing power assay was observed in fenugreek (3.52±0.03), which could be due to the presence of total phenolics and flavonoids with respects to other extracts followed by the eggplant which was on the second highest position. On the other hand, sweet potato and turnip gave lowest activities. Carrot extract showed moderate potential among the samples. The antioxidant activity of the extracts was in the order of fenugreek >eggplant> carrot>s. potato>turnip, varied ranging from 1.28±.06 - 3.52±0.03 in the ascending order (Table 4.8). The results of present study revealed that total reducing power was in proportional with the TPC of the extracts. Sathisha et al. (2011) hypothesised that the reducing power of a compound may serve as a significant indicator of its potential antioxidant activity. Previous reports on the relationship between the TPC and antioxidant capacity demonstrated a linear correlation between TPC and the antioxidant capacity (Abdel-Hameed, 2009; Nisha et al., 2009). The reducing power of antioxidants have been attributed to various mechanisms, such as prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued proton abstraction, and radical scavenging activities (Mishra et al., 2010). Present results were in accordance with other investigators who have
60 Chapter # 4 Results and Discussion
also reported that antioxidant properties are concomitant with the development of reducing power (Topcu et al., 2007; Ozsoy et al., 2008; Riberio et al., 2008; Nisha et al., 2009). ii. Reducing power assay of selected dry fruits The reducing power of the selected dry fruit samples increased with concentration and the values obtained for all the extracts were excellent. The effect of methanolic extract on the reducing power was compared to that of BHA in this study (Table 4.9). Dose response studies indicated that before reaching a threshold level, there was a positively linear relationship between the reducing power and the concentration of different dry fruits. The absorbance values were above 0.65 for all extracts, once more with the exception of walnut (2.21±0.00). The extracts obtained with almonds, peanuts and raisins showed similar values, while coconut showed lowest value. Dates, pistachio and apricot observed as moderate in reducing potential. It has been reported that the reducing properties are generally associated with the presence of reductones, which have been shown to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom (Riberio et al., 2008; Nisha et al., 2009). Results of current study demonstrated that significant stronger reducing power of dry fruits could be due to high amount of total phenolic. The order of reducing capacity of ethanolic extracts ranging from 0.83±0.08 to 2.21±0.00 varying from one fruit to another. These results verify the earlier studies that walnut and almonds showed highest reducing potential while raisins showed least results as compare to other samples (mishra et al., 2010). 4.6.2.2. DPPH radical scavenging activity Antioxidant properties, especially radical scavenging activities, are very important because of the deleterious role of free radicals in foods and in biological systems. Hydrogen-donating ability to free radicals is an index of the primary antioxidants; leading to nontoxic species and therefore to inhibition of the propagation phase of oxidation (Adedapo et al., 2009). DPPH is an organic stable free radical and has been widely used to test the scavenging ability of samples from the diverse nature that gives maximum absorption between 515-528 nm. Thus, the free radical scavenging capacity of the extracts against common free radicals (DPPH) in vitro were further determined. i. DPPH radical scavenging activity vegetables DPPH radical scavenging effects of selected vegetable extracts at various concentrations are presented in Table 4.8. Antioxidant activity of vegetable extracts were determined for their
IC50 (mg/mL) relating concentration with percentage inhibition. Extract of fenugreek was
61 Chapter # 4 Results and Discussion
found to be most potent with lowest IC50 0.18±0.01 mg/g while turnip was found to be least
effective (IC50 = 0.89±0.012 mg/g). In term of antioxidant activity their order of effectiveness followed fenugreek>eggplant>carrot>BHT>sweet potato>turnip. The sequence of effectiveness of radical scavenging by DPPH method showed that studied samples could be used as effective antioxidants. Nisha et al. (2009) worked on the same lines and revealed that eggplant fruit demonstrated better antioxidant activities which may be attributed to the higher phenolic content. It was also ratified that the crude methanolic leaves extract of T. foenum-graecum possessed lowto moderate free radical scavenging activity at variable dilutions (Azam et al., 2014). From the research study conducted earlier, the seeds extract of the plant possessed highest free radical scavenging activity (Tejaswini et al., 2012). Khalaf et al. (2008) showed that the IC50 for free radical scavenging activity of methanolic T. foenum-graecum leaf extracts was 444.10
μg/mL, which is higher than the current IC50 for T. foenum-graecum leaf.
ii. DPPH radical scavenging activity of dry fruits
The results of antioxidant activities regarding dry fruits are given in Table 4.9. methanolic extract of coconut and almonds had the lowest antiradical activity, whereas extract of walnut
followed by pistachio were found to be the most potent DPPH radical scavengers with IC50 values 0.98±0.48 mg of extract/g and 1.55±0.04 mg of extract/g, respectively. This can be related to the richness in their total phenolic compounds compared to the other fruits. Raisins, apricot and date sample show moderate capacity. All these fruits showed a capacity to trap radical DPPH and that this property differs from a fruit to another, ranging from 0.98±0.48 to 5.05±0.03 mg/g extract. The ranking of the selected fruits in order to their scavenging capacity, based on absolute numbers and not necessarily on statistical differences was walnut> pistachio >peanut> raisins> apricot> dates> almond> coconut. Ouchemoukh et al. (2012) reported that ethanolic extract of raisins (92.20%) showed the maximum scvanging potential as compare to methanolic ectract (89.20%) which was in disaccordance to present values. In the same study apricot shows a moderate scvanging capability in both the mediums which was in the agreement of our study. Reddy et al. (2010) reports the antiradical properties of methanolic extracts of dry fruits with the maximum activity in walnuts. Another study Deve et al. (2014) reported that phenolics and flavonoids in nuts and peanuts comprise a generous portion of the total antioxidant activity, suggesting that the combination of
62
Table 4.8. Antioxidant potential of selected vegetables Sr. No Scientific Name Common Name TPC(mg/g) TFC(mg/g) Antioxidants Activity
DPPH(IC50 mg/g) R. Power 1 Ipomoea batatas Sweet potato 2.94±.07a 1.53±0.41a 0.77±0.02a 1.39±0.13b 2 Trigonella foenum-graecum Fenugreek 16.45±.01d 6.56±0.72d 0.18±0.01a 3.52±0.03d 3 Daucus carota Carrot 3.46±.04a 0.16±0.53a 0.24±0.004a 1.84±0.20c 4 Solanum Melongena Eggplant 6.96±.02b 0.21±0.31a 0.22±0.004a 2.46±0.06c 5 Brassica rapa rapa Turnip 2.51±.01a 0.07±0.50a 0.89±0.012ab 1.28±.06a Values are mean ± SD of carefully conducted triplicate experiments. Superscripted alphabets within a column indicate significant (p<0.05) observed at 95% confidence interval (CI). Table 4.9. Antioxidant potential of selected dry fruits Sr. No Scientific Name Common Name TPC(mg/g) TFC(mg/g) Antioxidants Activity
DPPH(IC50 mg/g) R. Power 6 Phoenix dactylifera Dates 4.40±.05a 1.99±0.60ab 3.354±0.00d 1.89±.03c 7 Juglans Wal nut 8.44±.02c 3.78±0.21c 0.980±0.48ab 2.21±0.00d 8 Prunus dulcis Almond 1.68±.01a 0.73±0.70a 4.254±0.00d 1.03±0.00a 9 Pistacia vera Pistachio 6.9±.05b 2.32±0.50ab 1.548±0.04b 1.98±0.11b 10 Arachis hypogaea Peanut 3.06±0.01a 1.80±0.23ab 2.538±0.01c 1.43±0.06a 11 Vitis vinifera L. Raisins 2.21±0.05a 0.88±0.50a 3.062±0.00c 1.20±0.04a 12 Cocos nucifera Coconut 1.46±.05a 0.09±0.20a 5.050±0.03d 0.83±0.08a 13 Prunus armeniaca Apricot 2.22±0.02a 1.66±0.71a 3.097±0.01c 1.83±0.07b Values are mean ± SD of carefully conducted triplicate experiments. Superscripted alphabets within a column indicate significant (p<0.05) observed at 95% confidence interval (CI).
63 Chapter # 4 Results and Discussion
phytochemicals and synergistic mechanisms in the nut matrix may be responsible for their potent antioxidant activities. 4.7. Toxicological screening of plant materials against brine shrimp nauplii Plants are important source of potentially useful structures for the development of new chemotherapeutic agents (Ullah et al., 2013). Brine shrimp lethality bioassay (BSLB) has been used extensively in the primary screening of the crude extracts as well as the isolated compounds to evaluate the toxicity towards brine shrimps (Artemia salina), which could also provide an indication of possible cytotoxic properties of the tested materials (Sahgal et al., 2010). Furthermore it has been established that the cytotoxic compounds generally exhibit significant activity in the BSLB, and this assay can be recommended as a guide for the detection of antitumor and pesticidal compounds because of its simplicity and low cost (Wu, 2014). Based on the broad bioactivities, methanolic extracts of selected samples were also screened for their cytotoxic effect against the brine shrimps nauplii. 4.7.1. Brine shrimp lethality bioassay of vegetables The lethal concentrations of crude methanolic extracts of tested vegetables regarding brine
shrimp lethality bioassay (LC50 and LC90) are given in Table 4.10. Based on the results, the brine shrimp lethality of the tested samples were found to be concentration-dependent and maximum mortalities were observed at a concentration of 3000 µg/mL in all the extracts. The
LC50 values of the plant extracts were within the range of 204.26 to 514.01µg/mL, whereas
LC90 ranges between 494.02 to 1567.35µg/mL. Among the extracts Trigonella foenum-
graecum showed more cytotoxic effects with LC50 value 204.26 µg/mL; however Ipomoea batatas and Brassica rapa rapa were the least toxic among the tested vegetables having
LC50 value 494.02 and 514.01µg/mL respectively. Solanum melongena and Daucus carota were moderate in larvicidal properties. The observed lethality of the extracts to brine shrimps indicated the presence of potent cytotoxic components of these plants. The metabolites either affected the embryonic development or slay the eggs (Mazid et al., 2008). Therefore, the cytotoxic effects of the plant extracts enunciate that it can be selected for further cell line assay because there is a correlation between cytotoxicity and activity against the brine shrimp nauplii using extracts (Manilal et al., 2009). It was found that no investigation has been carried out for cytotoxic activities till now among current studied vegetables.
64 Chapter # 4 Results and Discussion
Table 4.10. Cytotoxic activity of methanolic extracts of vegetables against brine shrimp nauplii at different concentrations 95% Confidence Interval Sr. Common Conc. (µg/mL) Scientific Name Lc50 Lc90 No Name (µg/mL) (µg/mL) 1 3000,1000,100,10 Ipomoea batatas Sweet potato 494.02 1558.26 2 3000,1000,100,10 T. foenum-graecum Methi 204.26 969.49 3 3000,1000,100,10 Daucus carota Carrot 409.89 1667.58 4 3000,1000,100,10 Solanum Melongena Bringle 332.26 1271.37 5 3000,1000,100,10 Brassica rapa rapa Turnip 514.01 1567.36 Positive control: cyclophosphamide (larvae are died 100%), negative control: artificial sea water brine shrimp ( nauplii are live) 95% confidence interval=Estimation of possible values for the population parameter. LC50= lethal concentration at which 50 percent of shrimps died. LC90= lethal concentration at which 90 percent of shrimps died. 4.7.2. Brine shrimp lethality bioassay of dry fruits The brine shrimp lethality test was carried out on the eight dry fruits. Table 4.11 is a
summary of lethal concentration LC50 and LC90 values. The LC50 values of the plant extracts
were within the range of 248.74-1100.15 µg/mL, whereas LC90 ranges between 924.40- 9376.32 µg/mL. The degree of lethality was directly proportional to the concentration of the extract. Maximum mortalities were observed at a concentration of 3000 µg/mL. Five fruits exhibited toxicity levels between LC50 248.74 µg/mL and 1000 µg/mL and were classified as the slightly toxic among the samples. These were Juglans (248.74 µg/mL), Pistacia vera (594.29 µg/mL), Arachis hypogaea (887.24 µg/mL), Prunus dulcis (901.08 µg/mL) and Vitis vinifera L. (994.69 µg/mL). Whereas Phoenix dactylifera (1052.83µg/mL), Cocos nucifera (1083.62 µg/mL) and Prunus armeniaca (1100.15 µg/mL) have high values for lethality
concentration LC50 exhibiting lower toxicity against brime shrimp nauplii. However, current study classified all the studies samples as non-cytotoxic edibles. According to our literature survey and best of knowledge, no investigation has been carried out for cytotoxic activities till now among studied fruits. Hence, the present study supports brine shrimp lethality bioassay as a reliable method for the assessment of bioactivity of also displayed potent cytotoxic potential in our observations. The in vitro cytotoxicity displayed by the plant extracts tested is an initial indicator of in vivo antitumour activity (Wu, 2014). This step is necessary to eliminate cytotoxic compounds with little value. Therefore, further isolation of the highly active fractions may lead to the discovery of new cytotoxic compounds.
65 Chapter # 4 Results and Discussion
Table 4.11. Cytotoxic activity of methanolic extracts of dry fruits against brine shrimp nauplii at different concentrations
95% Confidence Interval Sr. Common Lc50 Lc90 No Conc. (µg/mL) Scientific Name Name (µg/mL) (µg/mL) 6 3000,1000,100,10 Phoenix dactylifera Dates 1052.83 6684.32 7 3000,1000,100,10 Juglans Wal nut 248.74 924.40 8 3000,1000,100,10 Prunus dulcis Almond 901.08 7043.84 9 3000,1000,100,10 Pistacia vera Pista 594.29 2764.85 10 3000,1000,100,10 Arachis hypogaea Peanut 887.24 5486.53 11 3000,1000,100,10 Vitis vinifera L. Reisins 994.69 7764.91 12 3000,1000,100,10 Cocos nucifera Coconut 1083.62 8324.30 13 3000,1000,100,10 Prunus armeniaca Apricot 1100.15 9376.32 Positive control: cyclophosphamide (larvae are died 100%), negative control: artificial sea water brine shrimp ( nauplii are live) 95% confidence interval=Estimation of possible values for the population parameter. LC50= lethal concentration at which 50 percent of shrimps died. LC90= lethal concentration at which 90 percent of shrimps died. 4.8. Thrombolytic activity of plant materials Herbal drugs are wide-spoken as green medicine for their safe and dependable health care
paradigms. In order to discover new sources of herbs, natural foods and their supplements having immunomodulatory effects has emerged in recent times (Licciardi and Underwood, 2011; Apu et al., 2012). A number of plants source especially several fruits and vegetables for their supplements having antithrombotic effect (anticoagulant and antiplatelet) have been studied by various researchers; also revealed that consuming such food leads to prevention of coronary events and stroke (Rahman et al., 2013). Thrombolytic agents are used to dissolve the already formed clots in the blood vessels (Ansari et al., 2012; Shilpi et al., 2013). Currently available thrombolytic agents are streptokinase, tissue plasminogen activator and urokinase cause serious bleeding complications along with reinfarction and reoccolution, and therefore safe and effective thrombolytic agents that can lyse a blood clot are needed (Jahan et al., 2015). In the present study an attempt has been made to develop an in-vitro clotlytic model to check the thrombolytic activity of different natural extracts, using a known thrombolytic agents, streptokinase as a standard and positive control and distilled water used as a negative control.
66 Chapter # 4 Results and Discussion
4.8.1. Thrombolytic activity of methanolic extracts of selected vegetables As a part of discovery of cardio-protective drugs from natural sources the extracts of tested vegetables were assessed for thrombolytic activity (Licciardi and Underwood, 2011; Apu et al., 2012; Rahman et al., 2013). The results of the studied vegetables regarding their antithrombotic potential are summarized in Table 4.12. The values of clot lysis percentage of methanolic extracts were directly proportional to concentration, time of incubation and amount of extract. Addition of streptokinase, a positive control to the clots and subsequent incubation for 90 minutes at 37°C, showed 64.95% lysis of clot. On the other hand, distilled water was treated as negative control which exhibited a negligible percentage of lysis of clot (1.56%). In the present study, clot lysis percentage for methanolic extracts ranging from 5.68-21.94 % in which Trigonella foenum-graecum exhibited maximum thrombolytic activity (21.94 ± 0.38%) among the studied samples. However, least thrombolytic activity was demonstrated by the Brassica rapa rapa (5.68 ± 0.14 %) followed by Ipomoea batatas (6.02 ± 0.05 %) respectively. Table 4.12. Thrombolytic activity of methanolic extracts of selected vegetables Sr no. Scientific Name Common Name Clotlysis (%age) 1 Ipomoea batatas Sweet potato 6.02 ± 0.05 2 Trigonella foenum-graecum Fenugreek 21.94 ± 0.38 3 Daucus carota Carrot 9.73 ± 0.11 4 Solanum Melongena Egg plant 13.06 ± 0.06 5 Brassica rapa rapa Turnip 5.68 ± 0.14 6 Control (-ve) Water 1.56±0.00 7 Control (+ve) Streptokinase 64.95±0.07 Values are mean ± SD of three consecutive experiments.
4.8.2. Thrombolytic activity of methanolic extracts of selected dry fruits A diet rich in fruits and vegetables is known to decrease the risk of cardiovascular disease. However, the information regarding the antithrombotic activity (antiplatelet, anticoagulant, and fibrinolytic) of fruits and vegetables is scarce (Saravanan et al., 2013; El-Abhar and Schaalan, 2014). Treatment of clots with Juglans extracts provided the highest clot lysis percentage (32.50± 0.06%) followed by Prunus dulcis (25.54 ± 0.09%), Pistacia vera (19.66 ± 0.12%) and Arachis hypogaea (17.7 ± 0.20%) respectively. The relatively lower percentage of clot lysis by Phoenix dactylifera (9.85 ± 0.19%), Vitis vinifera L. (8.23 ± 0.17%) and Prunus armeniaca (8.00±0.14%) was observed. However, Cocos nucifera (6.4 ± 0.35%) did
67 Chapter # 4 Results and Discussion not show any significant clot lysis effect. Percent clot lysis obtained after treating the clots with different organic extracts and appropriate controls is shown in Table 4.13. Table 4.13. Thrombolytic activity of methanolic extracts of selected dry fruits Sr no. Scientific Name Common Name Clotlysis (%age)
6 Phoenix dactylifera Dates 9.85 ± 0.19 7 Juglans Wal nut 32.50± 0.06 8 Prunus dulcis Almond 25.54 ± 0.09 9 Pistacia vera Pistachio 19.66 ± 0.12 10 Arachis hypogaea Peanut 17.7 ± 0.20 11 Vitis vinifera L. Reisins 8.23 ± 0.17 12 Cocos nucifera Coconut 6.4 ± 0.35 13 Prunus armeniaca Apricot 8.00±0.14 14 Control (-ve) Water 1.56±0.00 15 Control (+ve) Streptokinase 64.95±0.07 Values are mean ± SD of three consecutive experiments. In conclusion, from the screening of a variety of dietary extracts, these fruits and vegetables possessed interesting results regarding blood clotlytic activity in vitro, however, in vivo clot dissolving property and active components for clot lysis are yet to be investigated (Rahman et al., 2013; Jahan et al., 2015). Further investigations are necessary to advance in knowledge of the active compounds of these fruits and vegetables and their mechanisms of action.
4.9. Antiproliferative activity of selected plant materials In the past few years, food-borne phenolics have attracted increased attention because of their potential to afford protection against a variety of disorders, including different types of cancer (Zia-Ul-Haq et al., 2014; Zhang and Liu, 2015) and cardiovascular diseases (Wootton- Beard and Ryan, 2014). Some authors investigated the synergistic effects of flavonoids on cell proliferation in cancer cell lines (Campbell et al., 2006). They found that aglycon flavonoids, such as quercetin, kaempferol, and naringenin, inhibited cancer cell proliferation of cell lines in a dose-dependent manner without cytotoxicity. Galeone et al., (2006) concluded that the uniquely large data set from southern European populations show an inverse association between the frequency of the use of allium vegetables and the risk of several common cancers and are favorably correlated with cancer risk in Europe. A limited number of in vitro studies of phenolic extracts of plants have suggested that more attention should be given to their effects on human cancer cells. Furthermore, to date, screening studies have been mainly
68 Chapter # 4 Results and Discussion
restricted to pure compounds. It is therefore of considerable interest to ascertain whether polyphenol extracts of plants possess the same antiproliferative activity as single components. 4.9.1. Antiproliferative activity of selected vegetables As a first step towards the identification of vegetables containing antiproliferative activity towards cancer cells, the inhibitory effects of extracts from commonly consumed vegetables on different tumor cell lines derived from colon (Ht-29) and lung cancer (H1299) were examined. The antiproliferative effect was in a dose-dependent manner, and the maximum inhibition was observed at the concentration of 200 µg/mL of all the tested samples. A considerable difference was observed in the sensitivity of these cell lines toward the vegetable extracts (Fig. 4.3). Tumor cells derived from colon cancer were most sensitive to the extracts while cells from lung cancers were much less affected by the tested extracts. For example, three of the tested vegetable extracts inhibited the proliferation of colon cancer cells by more than 50%, while only one extract was active against lung cancer cells. Fenugreek had the highest antiproliferative capacity with the lowest cell viability percentages against both type of cells (18.84±1.03% for Ht-29 and 25.64±2.83% for H1299 cells, respectively). Verma et al., (2010) investigated the alcoholic whole plant extracts of Trigonella foenum graecum L. in vitro cytotoxicity against different human cancer cell lines including lung and colon cancer cells. Colon 502713 and HT-29 cell lines were inhibited by the extracts of Trigonella foenum graecum L. cell lines and showed more than 70% cytotoxic inhibition which corroborated the present results.
The exposure of phytochemical extracts of Carrot and Turnip also decreases the count of living Ht-29 cells exhibiting the percentages 33.17±3.71% and 42.95±4.03%. These results are in accordant to an in vitro study of Przybylska et al., (2007) which bared the fact that polyphenolic extracts of carrot tissue, decreased cancerous cell (HT29 and LoVo) viability up to 20% at the highest concentration (200 μg/mL). Their perceived health benefits were related to their phytochemicals (such as chlorogenic acid), vitamins and fiber (Wootton-Beard and Ryan, 2014). Treatment of leukemia cell lines with carrot juice extract induced apoptosis and inhibited progression through the cell cycle (Zaini et al., 2011).
69 Chapter # 4 Results and Discussion
A)
S.potato 150 Fenugree 125 k Carrot 100 75
( %) 50 25 Cell proliferation 0 0 50 100 150 200 250
Concentration of phytochemical extracts (µg/mL)
150 S.potato Fenugreek 125 Carrot Egg plant 100 Turnip 75 50 25 Cell prpliferation (%) 0 0 50 100 150 200 250 B) Concentration of phytochemical extracts (µg/mL)
Fig 4.3. Percent inhibition of (A) Colon cancer and (B) Lung cancer cell (H1299) proliferation by methanolic extracts of selected vegetables (mean ± SD, n=3)
4.9.2. Antiproliferative activity of selected dry fruits
According to current data, minor inhibitory activities were observed for sweet potato and turnip extracts showing the percentages, 64.85±3.24% and 64.04±3.78%, respectively. Against lung (H1299) cell line, carrot showed 49.34±4.24% growth and 53.36±2.65% in case of turnip extract. Phytochemical extracts of eggplant (76.36±3.65%) and sweet potato (81.27±2.74%) showed least but clear cytotoxic activity towards H1299 cell lines. We therefore, can conclude that plant extracts showed selective in vitro cytotoxicity against different cancer cell lines and all the selected vegetables were found to be highly effective against various cancerous cells. The antiproliferative activities of dry fruit extracts toward the growth of Ht-29 human colon and H1299 human lung cancer cells in vitro are presented in Figs. 4.4. According to the data, all the fruits were more sensitive to Ht-29 as compare to
70 Chapter # 4 Results and Discussion
H1299. The phytochemical extracts of almond, peanut and coconut in Ht-29, raisins and apricot in Ht-29 while walnut and pistachio in both the cell lines exhibited less than 50% cell viability (Fig 4.4 A). Walnuts and almonds contained the highest antiproliferative activities toward Ht-29 cells exhibiting the viability percentages 29.96±7.30%, 38.39±2.24% followed by peanuts (44.33±3.59%), coconut (44.63±5.86%) and pistachio (48.12±2.83%), respectively. The phytochemical extracts of apricot and raisins exhibited a weaker antiproliferative activity toward colon cancer cells with cell viability 61.21±4.03% and 65.55±1.17% respectively. Highest antiproliferative effect of walnuts and raisins toward lung cancer-derived cell line (H1299) with the lowest viable values (40.88±3.05% and 40.80±7.44%, respectively) followed by apricot (45.61±1.67%) and pistachio (48.96±5.49%) was observed. The phytochemical extracts of peanuts, almond and coconut also directed significant antiproliferative activities having viable percentages 53.07±6.13%, 52.15±7.63% and 58.22±4.78%, respectively (Fig. 4.4 B). However, the lowest antiproliferative consequences of dates extracts were detected for both the cell lines Ht-29 (68.48±3.69%) and H1299 (57.91±5.35%), respectively. The inhibition of Ht-29 and H1299 cell proliferation was observed in a dose dependent manner after exposure to the phytochemical extracts of walnuts, peanuts, and almonds; these nuts demonstrated greater antiproliferative activity among the selected samples. The inhibition of cancer cell proliferation by nut extracts can be partially explained by the total phenolic contents in the nuts tested, suggesting that a specific phenolic compound or a class of phenolics in nuts was responsible for their antiproliferative activities. The antiproliferative activities of resveratrol is proposed by the direct inhibition of ribonucleotide reductase, which supplies proliferating cells with deoxyribonucleotides required for DNA synthesis. In addition, it is hypothesized that bound phytochemicals reach the colon in an undigested form which could be metabolized by the microflora (Yang et al., 2009). For example, diferulic acids from cereal brans are ester-linked to cell wall polysaccharides and cannot be absorbed in this form. It has been demonstrated that esterase from human and rat colonic microflora are able to release the dietary diferulates (Wootton- Beard and Ryan, 2014). Tree nuts and peanuts possess numerous phytochemicals that may offer beneficial effects and lower the risk of chronic diseases. Consumption of flavonoids was shown to be inversely associated with morbidity and mortality resulting from CHD. Generally, phytochemicals
71 Chapter # 4 Results and Discussion present in nuts include phenolics, carotenoids, and phytosterols. Phenolics in nuts contain phenolic acids, flavonoids, and stilbenes. Walnuts are a good source of phenolics with (Chen and Blumberg, 2008). A)
Dates Walnut 150 Almond Pistachio
125 Peanuts Raisins 100 Coconut Apricot 75
50
25 Cell proliferation %
0 0 50 100 150 200 250 Concentration of phytochemical extracts (µg/mL)
B)
150 Dates Walnut 125 Almonds Pistachio %) 100 Peanuts Raisins 75 Coconut Apricot 50 proliferation ( 25 Cell 0 0 50 100 150 200 250 Concentration of phytochemical extracts (µg/mL)
Fig 4.4. Percent inhibition of (A) Colon cancer and (B) Lung cancer cell (H1299) proliferation by methanolic extracts of selected dry fruits (mean ± SD, n=3) 4.10. Antiglycation activities of selected plant materials Maillard reaction is considered as a complicated series of reactions which involve reducing- sugars and proteins and finally give rise to end-products better known as AGEs. They can be part of diabetes aggravating its complications and other neurological diseases as in Alzheimer's disease. AGEs also take part in atherosclerosis, vascular stiffening,
72 Chapter # 4 Results and Discussion
inflammatory arthritis, osteoarthritis, and cataracts. In this way, AGE inhibitors and its breakers may offer a therapeutic potential in these diverse diseases. In recent years AGE inhibitors have attained significance and the underlying mechanism is the basis of attenuation of glycoxidation, reactive 1, 2-dicarbonyl compounds oxidative stress, reactive nitrogen species and reactive oxygen (Ho et al. 2010).
Sequence of events that leads to protein damage induced by glucose may be divided in category of three stages that overlaps and form early Amadori products, Intermediate (Cross linking of protein and formation of carbonyl groups) and final one with post Amadori adducts. Initiation of first stage takes place with advancing Millard reaction. Measurement of browning is the approach used to determine protein AGE adducts (Miroliaei et al., 2011). It was performed to monitor the extent of plasma glycation. Glycation developed alterations of colour in plasma with significant degree of modification during 5 weeks of incubation. At relevant wave length the lower absorbance for the treated samples of different plants extract is the indicator of their efficacy on alleviation of glycation reaction that tends to have pronounced effect after specific period of incubation. A remarkable decrease of glycation reaction (% inhibition) reveals the potency of extract to act as natural inhibitor attenuating Millard reaction during the initial stage. Different plants were treated for two temperatures 37°C and 50°C (above physiological temperature). Raised temperature enabled us to accelerate the glycation level in vitro that takes decades to be accomplish in vivo (Matsuura et al., 2002).
Accordingly, the finding of AGEs inhibitors from natural foods for reasons of safety seems feasible. Indeed, numerous dietary plants and their constituents exhibit an anti-glycation capacity, even more strongly than aminoguanidine (Ahmad et al., 2007; Peng et al., 2008). Several studies have demonstrated that the anti-glycation capacities of tested dietary plants correlated significantly with their phenolic content (Peng et al., 2008). Furthermore, since an oxidative reaction is involved in the formation of AGEs and in AGEs-induced cell damage, compounds with both anti-glycation and antioxidant properties may most effectively protect against glucose-induced cytotoxicity. To date our selected plants are reported first time for antiglycation activities, although the work on antiglycation activities is carried on widely. Ho et al. (2010) evaluated and compared the antiglycation activities of herbal infusions including a known antiglycation beverage green tea for the determination of antiglycation activities.
73 Chapter # 4 Results and Discussion
4.10.1. Effect of selected plant materials on the reduction of NBT
Measurement of early-stage glycation product (Amadori product) is routinely used to evaluate metabolic control in diabetic patients. The two parameters commonly used are HbA1c and glycated serum proteins. Glycated serum proteins are the products of the nonenzymatic reaction between serum glucose and the free amino groups of proteins in serum; measurement of the resulting early glycation product expresses metabolic control during the 2 weeks before testing because that is the half-life of albumin and other proteins (Jeppsson et al., 2002; Hoelzel et al., 2004; Lapolla et al., 2005). Among the various methods proposed for measurement of glycated serum proteins, scientists generally prefer to measure fructosamine (Lapolla et al., 2005), applying a colorimetric method based on the ability of ketoamine linkages to reduce the dye nitro blue tetrazolium (NBT) and to produce a compound which absorbs at 525 nm.
4.10.1.1. Effect of selected vegetable extraxts on the reduction of NBT
Schiff base and Amadori products are formed during the early stages of non-enzymatic glycation of proteins. Amadori products can reduce NBT in alkaline solution to yield a coloured monoformazan dye with a maximum absorption at 530 nm (Baker et al., 1994). Glycated-BSA caused a rapid reduction of NBT to a significant degree with increasing incubation time. The reduction of NBT by Amadori products was inhibited by incubating glucose/BSA system with different concentrations of biological extracts and aminoguanidine. Our results revealed that all the green materials showed stronger inhibitory effects in a dose dependent manner during the 18th day of incubation and quantitative results are shown in (Table 4.14). Fenugreek bared solidest inhibitory effect (24.70±0.00 %) on NBT reduction at 1 mg/mL in comparison to other vegetables at this concentration. Sweet potato also exhibited a sturdier inhibitory effect ranging from 11.73 to 41.85 % among the tested samples at concentration 1-5(mg/mL). Nevertheless, eggplant (6.74-29.34%) and turnip (3.28-28.00%) were much less efficient in reduction inhibition respectively. The ability of the tested samples to inhibit NBT reduction was in the order of turnip, (3.28-28.00) < eggplant (6.74-29.34) < carrot (8.10-31.70) < aminoguanidine (10.79-31.89) < sweet potato (11.73- 41.85) < fenugreek (24.70-47.67).
74 Chapter # 4 Results and Discussion
Table 4.14. Effect of methanolic extracts of different vegetables on the reduction of NBT induced by glycated BSA Inhibitory effect (%) Common Name Concentration(mg/mL) 1 2 3 4 5 Sweet potato 11.73±0.01a 26.51±0.02b 30.18±0.01b 37.11±0.02c 41.85±0.01d Fenugreek 24.70±0.00a 35.51±0.01b 39.69±0.00b 44.06±0.01c 47.67±0.00d Carrot 8.10±0.00a 10.96±0.02a 14.46±0.00b 19.04±0.07c 33.70±0.05d Eggplant 6.74±0.09a 9.43±0.03b 11.39±0.02b 17.45±0.07c 29.34±0.08d Turnip 3.28±0.07a 11.32±0.02b 17.09±0.08bc 22.43±0.01c 28.00±0.00d Aminoguanidine 10.79±0.00a 21.61±0.00b 30.48±0.00c 37.11±0.00d 31.89±0.00c
BSA (20 mg/mL) was incubated with glucose (500 mM) in phosphate buffer (0.2M, pH 7.4) at 37 C for 18 d. Values are mean ± SD of carefully conducted triplicate experiments. Super and subscripted alphabets within a column and row indicate significant (p<0.05) observed at 95% confidence interval (CI) between plant investigated and concentration applied, respectively. 4.10.1.2. Effect of selected dry fruit materials on the reduction of NBT The inhibitory effects of dry fruit extracts on the formation of early-stage glycation product (Amadori product) by NBT reduction assay are presented in Table 4.15. Our results revealed that all the selected edible materials of this group exposed stronger inhibitory effects in a dose dependent manner; exhibiting the maximum inhibitory values during the 18th day of incubation at the concentration of 5(mg/mL). Among the fruits, coconut (49%) followed by walnut and raisins (46%), pistachio, peanut and apricot (43%) displayed robust inhibitory effect on NBT reduction at 5 mg/mL in comparison to aminoguanidine (38%) at the same concentration. However, dates (37%) and almonds (35%) were less proficient in reduction inhibition than aminoduanidine respectively. The ability of the tested samples to inhibit NBT reduction was in the order of coconut (29-49%)> walnut(19-46%) > raisins (23-46%), pistachio (24-43%) > peanut (19-43%) apricot (15-43%) > aminoguanidine (10-38%) > dates (9- 37%) and almonds (11-35) respectively.
75 Chapter # 4 Results and Discussion
Table 4.15. Effect of methanolic extracts of different dry fruits on the reduction of NBT induced by glycated BSA Inhibitory effect (%) Common Name Concentration(mg/mL) 1 2 3 4 5 a b b b b Dates 9.40±0.43a 21.45±0.45b 31.13±0.05c 35.94±0.23d 37.61±0.02d c b bc d d Wal nut 19.88±0.04a 25.61±0.04b 35.44±0.00c 41.25±0.01cd 46.45±0.03d b a a a a Almond 11.82±0.09a 15.30±0.01a 25.17±0.02b 30.45±0.07c 35.55±0.01d c b c cd c Pistachio 24.02±0.01a 27.74±0.02a 36.52±0.01b 39.45±0.01c 43.16±0.00d c b c d c Peanut 19.16±0.00a 26.32±0.02b 37.63±0.02c 41.61±0.02d 43.36±0.02d c c cd d d Reisins 23.17±0.05c 30.19±0.02b 39.54±0.04c 45.97±0.01d 46.69±0.03d d c c cd d Coconut 29.18±0.01a 31.68±0.07b 35.78±0.01b 39.93±0.00c 49.03±0.023d a d d d c Apricot 15.19±0.08a 38.88±0.06b 42.75±0.04d 42.37±0.11d 43.27±0.05d a b c c b Aminoguanidine 10.79±0.00a 21.61±0.00b 30.48±0.00c 37.11±0.00d 38.89±0.00d
BSA (20 mg/mL) was incubated with glucose (500 mM) in phosphate buffer (0.2M, pH 7.4) at 37 C for 18 d. Values are mean ± SD of carefully conducted triplicate experiments. Super and subscripted alphabets within a column and row indicate significant (p<0.05) observed at 95% confidence interval (CI) between plant investigated and concentration applied, respectively. 4.10.2. Effect of selected plant materials on the formation of α-dicarbonyl
Intermediate glycated products which can currently be estimated are glyoxal, methyl glyoxal and deoxyglucosones. Some studies have shown that in diabetic rats, methyl glyoxal formation is high in kidneys, lens, and plasma, and that it increases proportionally to glucose concentration (McLellan et al., 1994). Methyl glyoxal can form cross-links and modify some enzymes of the glycolysis pathway, microtubular proteins, and collagen (McLellan et al., 1994). Furthermore, in vivo studies have demonstrated that methyl glyoxal levels are five to six times higher in type 1 diabetics and two to three times higher in type 2 diabetic patients, with respect to controls. In one of formar studies, in type 2 diabetic patients with bad metabolic control, attaining good control with optimized insulin therapy was not successful in normalizing either glyoxal or methyl glyoxal levels after 6 months, unlike HbA1c and fructosamine values, thus emphasizing that these products, like AGE, need more stable, prolonged, good metabolic control for their normalization (Lapolla et al., 2005). There are several methods for measuring glyoxal and methyl glyoxal (Kusunoki et al., 2003). In our study we tested Dicarbonyl compounds spectrophotometrically by Girard-T assay.
76 Chapter # 4 Results and Discussion
4.10.2.1. Effect of selected vegetables on the formation of α-dicarbonyl
In the intermediate stage of glycation, through oxidation and dehydration reactions, the amadori products subsequently degrade into a variety of α-dicarbonyl compounds such as glyoxal, methylglyoxal and deoxyglucosones. These compounds are more reactive than the parent sugars with respect to their ability to react with amino groups of proteins to form AGEs (Lapolla et al., 2005). In addition to direct glycosylation reactions, the α-dicarbonyl compounds can also be generated through monosaccharides autoxidation (Wells-Knecht et al., 1995). The autoxidation of glucose could rapidly generate α-dicarbonyl compounds within 18-24 d of incubation. Conversely, the generations of α-dicarbonyl compounds in the BSA glycation system were increased slowly at the beginning. After incubating glucose with BSA for 18 d, the α-dicarbonyl compounds were formed quickly (Fig 4.5). Many studies have identified that dicarbonyl compounds can induce rapid cross-linking of proteins and yield stable AGEs (Meade and Gerrard, 2002; Lapolla et al., 2005). It is reported that approximately 45– 50% of the AGEs originate from dicarbonyl compounds (Ruggiero-Lopez et al., 1999). However, the development of the fluorescence of AGEs was obviously inhibited when extracts of vegetables and aminoguanidine were added to the incubation system.
According to our research, during the 18th days of incubation, the content of dicarbonyl compounds was increased and then leveled off. The maximum inhibitory effects of vegetable extracts on the formation of α-dicarbonyl compounds are shown in Table 4.16. Except carrot and turnip, all the selected vegetables together with aminoguanidine inhibited the formation of dicarbolnyl compounds; especially the inhibitory effect of fenugreek was 59.97%, slightly greater than aminoguanidine, a renowned antiglycation agent at a concentration of 5mg/mL. At a concentration of 1mg/mL, fenugreek showed the strongest inhibitory effects on the a-dicarbonyl compounds generation in the BSA glycation systems, followed by sweet potato (15.30%), eggplant (13.80%), carrot (10.28%) and turnip (9.50%) showed the lowest inhibitory effect. The inhibitory capacity of the selected vegetables on the α-dicarbonyl compounds formation was in the order of fenugreek (29.74-59.97%) > sweet potato (15.30-53.99%) >eggplant (13.80-51.56%) > aminoguanidine (13.27-48.77%) > carrot (10.280-42.21%) > turnip, (9.48-40.82%) at 2 mg/mL but it is towards increasing trend in a dose response relationship.
77 Chapter # 4 Results and Discussion
Table 4.16. Effect of methanolic extracts of different vegetables on the formation of α- dicarbonyl compounds in glucose/BSA system Inhibitory effect (%) Common Name Concentration(µg/mL) 1 2 3 4 5 b cd d d c Sweet potato 15.29±0.01 a 33.43±0.01 b 40.91±0.01 c 43.97±0.01 c 53.99±0.00 d d d bc d d Fenugreek 29.74±0.01 a 35.81±0.0 b 36.52±0.01 b 44.21±0.01 c 59.97±0.00 d a a b b a Carrot 10.28±0.02 a 22.10±0.01 b 32.91±0.01 c 36.78±0.01 c 42.21±0.00 d b b bc d b Eggplant 13.79±0.01 a 28.39±0.00 b 34.80±0.01 bc 43.47±0.01 c 51.56±0.01 d a a a a a Turnip 9.48±0.03 a 20.55±0.02 b 29.50±0.00 c 32.43±0.01 c 40.82±0.02 d b a a a b Aminoguanidine 13.26±0.01 a 19.47±0.00 b 26.88±0.01 c 30.40±0.01 c 48.78±0.01 d BSA (20 mg/mL) was incubated with glucose (500 mM) in phosphate buffer (0.2M, pH 7.4) at 37 C for 18 d. (means ± SD, n=3). Super and subscripted alphabets within a column and row indicate significant (p<0.05) observed at 95% confidence interval (CI) between plant investigated and concentration applied, respectively. 4.10.2.2. Effect of selected dry fruits on the formation of α-dicarbonyl Many studies have identified that dicarbonyl compounds can induce rapid cross-linking of proteins and yield stable AGEs (Meade and Gerrard, 2002). It is reported that approximately 45–50% of the AGEs originate from dicarbonyl compounds (Ruggiero- Lopez et al., 1999). In order to evaluate the inhibitory effect of dry fruits on AGEs formation, BSA was also incubated with glucose in the presence or absence of sample extracts; aminoguanidine was used as a positive control. After 30 days of incubation at 37oC, the fluorescence of BSA/glucose system increased many fold (Fig. 4.6). According to current data, during the first 18 days of incubation of dry fruit extracts with glucose/ BSA model, the content of dicarbonyl compounds was increased and then levelled off. All together with aminoguanidine inhibited the formation of dicarbolnyl compounds at concentration of 5 (mg/mL) at 18th day of incunation. The dose-dependent inhibitory effects of methanolic extracts of selected dry fruits on the formation of dicarbonyl compounds are shown in Table 4.17. Except apricot (36.00%) and peanuts (38.90%), the other edible samples were found to have more inhibitory activities than that of aminoguanidine in the BSA glycation systems at 5 (mg/mL) concentrations. All samples, except pistachio (24.66%), displayed weaker inhibitory effects in the BSA glycation systems at 1 (mg/mL). Coconut exhibits the strongest inhibitory effects on the dicarbonyl compounds generation in the BSA glycation systems, followed by pisthachio, walnut, dates, raisins, almond, aminoganidine, peanuts and apricot; varied ranging in a decreasing trend from 23-51%, (24-48%), (15-46%), (9-43%), (21-42%), (12-40%), (13-40%), (15-38%) and (10-40%).
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Chapter # 4 Results and Discussion
S. Potato/Glucose/BSA Fenugreek/Glucose/ BSA 2500 2500
2000 2000
1500 1500
1 1000 1000 1 2 2 3 3 500 4 500 Fluorescence intensity Fluorescence Fluorescence Intensity Fluorescence 4 5 5 0 0 0 10 20 30 40 0 10 20 30 40 Incubation Time (Days) Incubation Time Period (Days)
2500 Carrot/Glucose/BSA 2500 Eggplant/Glucose/BSA
2000 2000 1500
1500
1 1000 1 1000 2 2 3 3 500 500 4 Fluorescence Intensity Fluorescence 4
Fluorescence Intensity Fluorescence 5 0 5 0 0 10 20 30 40 0 10 20 30 40 Incubation Time Period (Days) Incubation Time Period (Days)
2500 Turnip/Glucose/BSA 2500 Aminogunidine/Glucose/BSA
1 2000 2 2000 3 4 1500 1500 5 1 1000 1000 2 3 500 500 Fluorescence Intensity Fluorescence
4 Intensity Fluorescence 5 0 0 0 10 20 30 40 0 10 20 30 40 Incubation Time Period (Days) Incubation Time Period (Days)
Fig 4.5. Fluorescence intensity of albumin in various glycation systems of selected vegetables at different concentrations (Concentration of samples = 1,2,3,4,5 mg/mL)
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Table 4.17. Effect of methanolic extracts of different dry fruits on the formation of α-dicarbonyl compounds in glucose/BSA system
Inhibitory effect (%) Concentration (mg/mL) Scientific name Common Name
1 2 3 4 5 a b c cd bc Phoenix dactylifera Dates 9.16±0.02 a 25.89±0.01 b 35.34±0.01 c 41.38±0.02 d 43.18±0.01 d b a bc d c Juglans Wal nut 15.29±0.01 a 19.52±0.01 b 34.95±0.01 c 42.38±0.01 d 46.26±0.01 d a b c c b Prunusdulcis Almond 12.27±0.01 a 25.92±0.01 b 35.52±0.00 c 38.85±0.01 c 40.86±0.00 d d c c d cd Pistaciavera Pistachio 24.66±0.00 a 27.98±0.01 a 38.91±0.01 c 45.28±0.01 d 48.49±0.01 d b b b a a Arachishypogaea Peanut 15.15±0.01 a 26.42±0.01 b 30.02±0.01 c 32.57±0.00 c 38.90±0.01 d cd b c c b Vitisvinifera L. Reisins 21.22±0.02 a 25.25±0.00 a 37.07±0.02 c 40.66±0.01 cd 42.09±0.00 d d d d d d Cocosnucifera Coconut 23.81±0.00 a 32.76±0.01 b 45.85±0.01 c 46.77±0.01 c 51.07±0.01 d a a a a a Prunusarmeniaca Apricot 10.24±0.01 a 17.54±0.01 b 25.11±0.00 c 30.84±0.00 cd 36.00±0.00 d b a a a b Aminoguanidine 13.26±0.01 a 19.47±0.00 b 26.88±0.01 c 30.40±0.01 c 40.77±0.01 d BSA (20 mg/mL) was incubated with glucose (500 mM) in phosphate buffer (0.2M, pH 7.4) at 37 C for 18 d. (means ± SD, n=3). Super and subscripted alphabets within a column and row indicate significant (p<0.05) observed at 95% confidence interval (CI) between plant investigated and concentration applied, respectively.
80 Chapter # 4 Results and Discussion
1
2000 2000 Dates/Glucose/BSA 2 Walnut/Glucose/BSA 3 1500 4 1500 5
1000 1000 1 2 500 500 3 4 5 Fluorescence Intensity Fluorescence 0
Fluorescence Intensity 0 0 10 20 30 40 0 10 20 30 40 Incubation Time Period (Days) Incubation Time Period (Days)
2000 1 Almond/Glucose/BSA 2000 Pistachio/Glucose/BSA 2 1500 1500 3
1000 1000 1
Intensity 2 500 500 Fluorescence Fluorescence 3 4
Fluorescence Intensity Fluorescence 0 0 0 10 20 30 40 0 20 405 Incubation Time Period (Days) Incubation Time Period(Days)
2000 Peanut/Glucose/BSA 2000 Reisins/Glucose/BSA
1500 1500
1 1000 1 1000 2 500 500 3 2 4 5 0 Intensity Fluorescence 0 Fluorescence Intensity Fluorescence 0 10 20 30 40 0 10 20 30 40 Incubation Time Period(Days) Incubation Time Period (Days)
2000 Coconut/Glucose/BSA 2000 Apricot/Glucose/BSA 1500 1500
1000 1 1000 1 500 500 2 2
Fluorescence Intensity Fluorescence 0
Fluorescence Intensity Fluorescence 0 0 10 20 30 40 0 10 20 30 40 Incubation Time Period(Days) Incubation Time Period (Days)
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Chapter # 4 Results and Discussion
2000 Aminogunidine/Glucose/BSA 1 1500 2 1000
500
Fluorescence Intensity Fluorescence 0 0 10 20 30 40 Incubation Time Period (Days)
Fig 4.6. Fluorescence intensity of albumin in various glycation systems of selected dry fruits at different concentrations (Concentration of samples = 1, 2,3,4,5 mg/mL).
4.10.3. Effect of methanolic extracts of selected plant materials on the formation of AGEs 4.10.3.1. Effect of methanolic extracts of different vegetables on the formation of AGEs The inhibitory effect of vegetables against AGEs formation was evaluated in vitro, the fluorescence intensity was measured and employed aminoguanidine, a well-known AGEs inhibitor. All the vegetables extracts exhibited good inhibitory effects on AGEs formation in BSA glycation systems (Table 4.18). The ability of the extracts to inhibit AGEs generation decreased in the order of Fenugreek>S.potato>Eggplant>Aminoguanidine>Carrot>turnip. All the extracts along with aminoguanidine presented stronger inhibitory effects during 18th day of incubation in the glycated BSA system except carrot and turnip which exhibited their best inhibitory effect during 21day of incubation. The strongest inhibition of AGEs by fenugreek (65.55%), sweet potato (48.85%), eggplant (46.55%), carrot (37.24%) and turnip (33.38%) was exposed at the concentration of 1 mg/mL in contrast to aminoguanidine (45.43%) best acticity at 5 mg/mL. Lunceford and Gugliucci (2005) indicated that the inhibition of AGEs formation by measuring AGEs fluorescence generated on BSA by glycation with methylglyoxal. They concluded that the antiglycation of natural extracts was mainly due to the inhibition of the second phase of the glycation reactions, namely the free-radical mediated conversion of the amadori products to AGE. It has been proposed that no oxidation reaction is involved in the formation of amadori rearrangement products, whereas oxidation plays a role in the formation of fluorescence and the molecular crosslinking, which are characteristic features of AGEs (Fu et al.,
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Chapter # 4 Results and Discussion
1994). Several researches also indicated the inhibitory effect of plant extracts, including ginseng (Bae and Lee, 2004), Luobuma tea (Yokozawa and Nakagawa, 2004), Finger millet (Eleusine coracana) and Kodo millet (Paspalum scrobiculatum) (Hegde, et al., 2002) on the formation of glycated proteins.
4.10.3.2. Effect of methanolic extracts of different dry fruits on the formation of AGEs In order to evaluate the inhibitory effect of dry fruits on AGEs formation, BSA was incubated with glucose in the presence or absence of methanolic extracts of dry fruits; aminoguanidine was used as a positive control (Table 4.19). After 24th days of incubation at 37 0C, the fluorescence of BSA/glucose system was comparatively increased by 11.98-fold (Fig. 2 c). However, the development of the fluorescence of AGEs was obviously inhibited when sample extracts and aminoguanidine were added to the incubation system. The ability of the samples to inhibit AGEs formation was in the order of Walnut (49.46%)> Pistachio (46.81%)> Coconut (45.83)> Aminoguanidine (45.43%)> raisins (42.64%)> Peanuts (36.14%)> Apricot (35.70%)> Dates (32.53%)> Almond (27.83%). Especially, walnut followed by pistachio and coconut displayed higher antiglycative activities than aminoguanidine. Except pistachio and aminoguanidine at 18th day of incubation; all the other tested samples were found to exhibit their best inhibitory antiglycative activities at 24th day of incubation in glycated BSA model. Dates, walnut, peanuts, raisins and apricot at 1mg/mL, almond at 3 mg/mL, pistachio at 4 mg/mL while coconut and aminoguanidine displayed their strongest inhibitory effects at the concentration of 5 mg/mL respectively. Different plant extracts are studied for their antiglycation potential due to their cost effectiveness, ease to use and availability. Garlic and its components like S-allyl cysteine has attracted more attention as possible cost-effective, nontoxic candidates for delaying diabetes complications and aging (Ahmad and Ahmed, 2006). Sheikh et al. (2004) determined effect of garlic extract by Thiobarbuchuric acid (TBA) method with varying concentration. In term of inhibitory activity maximum inhibition of 79.1% was reported at 1g/dL concentration. This study proved the antiglycation potential of garlic and thus can eventually save from advanced glycation end products. In another study by Sovia et al. (2013) Curcuma longa with its active compounds, curcuminoids were determined for antiglycation activity by TBA method in vitro.
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Table 4.18. Effect of methanolic extracts of different vegetables on the formation of AGEs in glucose/BSA system
Sample Conc. Inhibitory effect (%) (mg/mL) Incubation Time period (Days) 6 12 18 24 30 36 S.potato cd d d d d d 1 31.10±7.23 a 41.18±6.45 b 48.85±3.42 d 45.99±5.39 d 40.92±7.23 c 45.75±5.59 d
c bc c c d cd 2 28.94±1.03 a 34.69±5.53 b 40.69±3.29 c 41.77±6.41 d 44.66±1.17 d 43.46±5.39 d
b c d d d c 3 25.34±1.85 a 38.24±1.84 b 46.74±1.96 d 44.545±2.88 d 40.53±2.87 c 42.08±1.39 d
b b b b d b 4 20.77±2.94 a 29.78±3.23 b 37.41±2.11 c 37.29±1.71 d 41.84±1.85 d 36.64±2.14 c
a a a a a a 5 5.42±1.15 a 9.72±1.78 b 30.53±5.67 d 29.18±1.56 d 22.75±1.15 c 22.54±3.29 c Fenugreek d d d d c c 1 42.72±1.72 a 49.11±4.83 b 65.55±3.79 d 57.53±2.15 d 53.95±1.43 c 55.03±3.79 d
c cd c d d c 2 36.50±1.42 a 43.69±2.53 b 57.42±3.06 d 55.18±3.44 d 55.89±2.30 d 53.35±1.43 c
b c b c a a 3 19.46±2.30 a 36.80±2.12 b 49.94±1.31 d 51.07±1.29 d 49.67±1.38 d 43.63±3.06 c
b b b b c a 4 16.10±3.87 a 19.12±1.87 b 42.13±1.41 c 43.78±1.84 c 53.72±1.67 d 45.17±1.93 c
a a a a b d 5 1.51±0.38 a 13.18±1.99 b 28.26±1.92 c 25.89±2.67 c 51.79±1.79 c 57.25±1.54 d Carrot d c a cd cd c 1 29.34±6.48 c 21.72±2.42 b 16.51±5.84 a 37.24±1.64 d 32.88±6.48 c 30.14±1.12 c
c d b c cd d 2 24.93±2.28 a 27.69±3.35 b 24.40±1.14 a 35.85±1.29 d 34.34±5.17 d 39.35±1.72 d
d d bc b b b 3 27.71±6.40 a 36.75±3.95 d 33.91±1.39 d 28.81±3.14 c 25.85±6.30 b 22.53±4.37 b
b b d d d c 4 14.59±2.38 b 10.87±1.30 a 41.91±3.79 d 39.10±1.04 d 35.83±1.16 c 30.11±1.34 b
a a d a a a 5 7.48±1.81 a 8.08±4.43 a 51.15±5.23 d 25.80±2.17 c 19.94±1.81 b 13.72±1.22 b Eggplant d d d d d d 1 17.92±1.94 a 33.15±1.55 b 46.55±7.93 d 43.64±1.58 d 41.09±4.93 d 37.23±5.60 c
b b c c c d 2 13.54±4.33 a 14.89±2.61 a 24.48±2.30 b 34.82±2.50 c 35.31±4.81 c 39.39±6.77 d
84
cd c c b d c 3 15.20±1.34 a 23.03±3.32 b 26.24±1.16 c 30.38±5.50 c 40.14±4.88 d 32.27±3.33 c
b b b b b b 4 13.34±4.49 a 12.85±3.22 a 16.57±4.37 b 27.10±2.53 d 27.82±2.49 d 21.74±1.51 c
a a a a a a 5 5.37±2.15 a 4.35±1.43 a 5.29±2.10 a 15.02±2.15 d 14.08±1.13 d 7.14±1.60 a Turnip d d cd c c c 1 14.82±3.47 a 23.72±1.02 b 30.27±5.23 d 33.39±5.17 d 32.13±1.61 d 36.55±3.53 d
d d d d d d 2 13.03±0.82 a 25.83±2.01 b 34.26±3.79 c 36.69±5.46 c 46.18±1.91 d 46.01±2.93 d
c c c c c c 3 10.38±3.80 a 19.65±1.52 b 26.84±3.44 d 31.32±2.93 d 34.70±2.80 d 32.79±3.27 d
b b b b b b 4 8.96±0.94 a 9.06±2.47 b 17.69±0.10 d 24.51±1.19 d 28.71±1.19 d 26.14±3.80 d
a a a a a a 5 2.37±1.21 a 4.65±0.61 b 7.17±1.70 b 13.97±2.64 d 13.09±1.50 d 10.55±1.13 c Aminoguanidine d a a a a a 1 8.08±0.56 a 14.72±0.53 b 23.55±0.62 d 20.44±1.01 d 11.27±0.67 b 23.59±1.14 d
c b ab b b b 2 4.39±0.72 a 25.31±0.94 b 27.97±1.18 d 32.91±0.45 d 25.92±0.82 b 38.670±1.18 d
c c c c c c 3 3.92±0.03 a 29.08±0.61 b 37.59±0.89 c 45.06±0.28 cd 49.45±0.95 d 52.964±0.59 d
a cd cd d d d 4 1.19±0.30 a 32.97±1.04 b 42.55±1.09 c 49.88±0.89 cd 59.81±0.33 d 57.45±1.06 d
b d d c c c 5 2.88±0.39 a 35.30±1.05 b 45.43±0.56 d 42.71±0.83 d 41.75±1.00 d 45.08±0.85 d
Glucose (500 m M) was incubated with or without BSA (20 mg/mL) in phosphate buffer (0.2M, pH 7.4) at 37 ºC for 36 d. (mean ± SD, n=3). Super and subscripted alphabets within a column and row indicate significant (p<0.05) observed at 95% confidence interval (CI) between plant investigated and concentration applied, respectively.
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Table 4.19. Effect of methanolic extracts of different dry fruits on the formation of AGEs in glucose/BSA system Sample Conc.(mg/mL) Inhibitory effect (%) Incubation Time period (Days) 6 12 18 24 30 36 d d d d a c Dates 1 14.78±4.27 a 23.95±4.67 b 29.78±1.68 c 32.53±4.25 d 12.05±2.84 a 31.17±1.70 c cd d cd b d 2 12.61±1.24 a 20.74±4.72 b 27.33±1.17 c 30.24±3.84 c 20.93±1.54 b 36.37±2.55 b c c cd d c 3 10.03±1.65 a 18.19±1.33 b 22.77±2.19 b 29.60±1.21 c 29.89±2.15 c 32.30±1.42 ab b b bc c c 4 5.56±1.78 a 6.98±2.51 a 12.66±2.06 a 26.72±1.15 d 26.98±1.61 d 25.62±1.52 a a a a c a 5 2.79±0.35 a 2.14±2.21 a 2.81±1.49 a 15.54±1.60 c 26.97±1.60 d 12.53±3.43 a d d d b b Walnut 1 6.49±1.03 a 30.38±4.99 c 39.20±1.93 c 49.46±1.93 d 14.44±1.80 b 19.16±1.09 b c c c a a 2 13.50±2.19 a 20.98±2.92 b 28.42±1.81 c 32.46±1.47 d 9.94±1.94 a 10.20±1.81 b b a a d c 3 14.90±1.98 a 16.29±1.64 b 6.82±0.51 a 11.48±1.47 a 35.53±2.45 s 28.86±2.41 c a b b b b 4 20.88±0.42 c 5.91±3.57 a 12.32±1.70 b 21.39±2.64 d 24.17±1.98 d 22.35±1.19 d d a c c c d 5 23.99±1.42 c 4.08±1.61 a 24.04±1.28 c 27.70±0.96 c 21.58±1.38 c 33.99±3.62 d a c a a a a Almond 1 2.63±0.51 c 30.51±1.97 d 9.25±1.97 b 10.95±0.89 b 14.78±1.05 c 4.93±0.56 a b a d b d b 2 17.42±1.44 a 19.57±1.02 b 22.71±2.19 c 18.71±1.45 b 27.92±1.45 d 21.85±3.29 c b b c c d d 3 14.56±1.22 a 23.51±0.19 b 17.59±1.76 a 27.83±1.80 c 27.37±1.19 c 31.51±2.59 d c d c d d b 4 19.03±1.34 a 37.63±1.36 b 15.64±1.43 a 46.23±1.54 d 28.31±1.02 b 19.47±1.02 a d c c c c a 5 31.86±2.06 d 27.98±1.41 b 16.04±1.67 a 27.23±1.59 c 25.07±1.35 c 14.85±1.55 a a a a d d b Pistachio 1 2.08±0.34 a 10.42±1.15 b 31.36±1.70 c 42.93±1.05 d 38.87±1.21 c 21.63±2.41 c b b b d c a 2 12.58±0.59 a 19.43±1.30 b 39.19±1.15 c 40.75±1.49 d 28.59±1.36 b 14.63±1.32 a c d b c c c 3 18.30±0.54 a 36.60±1.16 d 38.62±1.42 d 36.62±1.18 d 30.39±1.28 c 26.76±1.36 b cd c d a a c 4 22.42±1.93 a 28.32±1.16 cd 46.81±1.33 d 24.98±1.61 b 23.48±1.96 c 25.39±1.17 c d a d b b d 5 25.47±1.33 c 3.34±0.78 a 47.89±1.58 d 31.54±1.85 c 16.20±1.22 a 29.00±1.91 d d d c d a c Peanuts 1 14.64±2.23 a 25.89±1.43 b 30.13±3.01 c 36.14±1.38 c 32.67±1.05 d 33.85±1.29 d c a a c d d 2 12.94±0.88 a 12.27±1.75 a 18.94±1.79 b 23.20±1.43 b 40.19±0.74 d 35.75±1.58 c b a ab b a c 3 10.28±1.51 a 12.90±1.82 a 22.89±0.81 b 20.40±1.19 b 30.89±1.08 cd 32.25±1.43 d b b d a d b 4 11.11±1.95 a 14.33±0.91 a 34.74±1.34 c 15.11±1.03 a 43.18±1.40 d 18.37±1.46 a a a b b a a 5 7.91±1.06 a 10.38±0.13 b 20.54±0.77 22.62±1.86 c 29.75±1.77 d 8.95±0.89 a
86
d d d d d Raisins 1 26.16±1.06a 38.26±1.80 d 40.41±1.27 d 42.65±1.22 d 40.85±1.01 d 25.15±1.72 a b c cd d d d 2 14.74±0.60 a 30.35±0.64 c 37.86±1.27 c 41.88±0.90 d 43.32±1.05 d 26.38±0.72 b d b c b c c 3 19.42±0.63 a 21.14±1.91 a 30.60±1.13 c 34.66±0.97 d 34.45±2.16 d 17.97±1.37 a b a b b b b 4 13.82±1.30 b 8.64±1.04 a 24.81±1.08 c 31.98±5.32 d 31.41±1.30 c 14.72±1.38 b a a a a a a 5 1.11±0.09 a 4.79±1.42 b 18.59±0.93 c 24.24±1.04 c 23.82±1.80 d 4.42±0.78 d c c a b d d Coconut 1 27.74±0.98 a 28.49±1.61 a 30.87±1.76 b 37.57±1.16 c 52.55±2.82 d 46.77±2.12 c a b a a a a 2 1.06±0.10 a 24.64±1.19 b 33.51±1.46 c 30.30±1.16 c 35.38±1.36 d 32.55±2.62 d b a c b a a 3 11.14±0.46 a 11.88±0.80 a 38.22±1.37 d 39.40±1.48 d 39.75±1.46 d 31.98±2.71 c c c c c c b 4 24.11±1.10 a 30.93±1.13 b 40.76±1.72 c 41.58±4.89 cd 45.32±1.64 d 41.40±1.87 d d d d d c b 5 31.08±1.26 a 39.88±1.13 b 44.04±0.84 c 45.83±1.08 cd 47.65±1.80 d 42.49±1.75 d d d d d c d Apricot 1 26.55±1.63 a 28.84±1.65 b 33.23±1.38 c 35.70±1.49 d 33.45±1.03 c 35.52±1.25 c c c d b d b 2 16.64±0.90 a 29.58±0.86 b 31.89±3.18 c 30.83±3.97 c 36.51±1.05 d 24.77±1.64 b b b d b b a 3 10.04±0.53 a 25.11±0.78 c 30.219±2.68 d 31.54±1.89 c 31.26±1.26 d 14.38±1.48 a a a b a c b 4 6.68±0.85 a 14.30±1.56 b 25.00±1.52 c 22.84±1.84 c 33.72±1.11 d 20.01±1.38 b a a a a a a 5 1.61±0.33 a 9.72±0.78 b 19.93±1.06 c 25.88±1.06 cd 27.97±0.89 d 11.35±1.06 a d a a a a a Aminoguanidine 1 8.08±0.56 a 14.72±0.53 b 23.55±0.62 d 20.44±1.01 d 11.27±0.67 a 23.59±1.14 c b a b b b 2 4.39±0.72 a 25.31±0.94 b 27.97±1.18 c 32.91±0.45 d 25.92±0.82 c 38.69±1.18 d a c b d c d 3 3.92±0.03 a 29.07±0.61 b 37.59±0.89 c 45.06±0.28 49.45±0.95 c 52.96±0.59 d a c c d d d 4 1.19±0.30 a 32.97±1.04 c 42.55±1.09 c 49.88±0.89 d 59.81±0.33 d 57.45±1.06 d b d d c c c 5 2.88±0.39 a 35.30±1.05 c 45.43±0.56 d 42.71±0.83 c 41.75±1.00 c 45.09±0.85 d
Glucose (500 m M) was incubated with or without BSA (20 mg/mL) in phosphate buffer (0.2M, pH 7.4) at 37 C for 36 d. (mean ± SD, n=3). Super and subscripted alphabets within a column and row indicate significant (p<0.05) observed at 95% confidence interval (CI) between plant investigated and incubation time at concentration applied, respectively.
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Chapter # 4 Results and Discussion
4.11. Carbohydrate hydrolyzing enzymes inhibition by plant materials Epidemiological studies associated with disease risk have suggested that habitual consumption of dietary agents offer protection against occurrence of metabolic ailments such as diabetes along with other associated co-morbidities (Wang et al., 2012; Farzaneh and Carvalho, 2015). Pancreatic α-amylase is involved in the breakdown of starch into disaccharides and oligosaccharides before intestinal α-glucosidase catalyzes the breakdown of disaccharides to liberate glucose which is later absorbed into the blood circulation. Inhibition of principal carbohydrate hydrolyzing enzymes (α-glucosidase and α-amylase) connected to postprandial hyperglycemia due to natural antioxidant, is an impending remedial approach for diabetes mellitus (Kwon et al., 2008; Ranilla et al., 2010). Inhibitors of these enzymes slowdown the digestion of carbohydrates thus, prolong the overall digestion period, lessen glucose absorption rate and consequently blunt the postprandial glucose escalation in plasma (Rhabasa-Lhoret and Chiasson, 2004; Elya et al., 2012).
4.12. Carbohydrate hydrolyzing enzymes inhibition through selected vegetables 4.12.1. α-amylase inhibitory activity The α-amylase inhibitory properties of phytochemical enriched extracts of selected vegetable are presented in Fig. 4.8. The result revealed that the extracts inhibited α-amylase activity in a dose- dependent manner and the maximum activity was observed at 1.6 mg/mL. However, acarbose inhibitory activity (65.63%) was significantly high in comparison to vegetables. This trend of the α-amylase inhibitory property of the polyphenol-rich extracts agreed with their flavonoid content. 4.12.2. α-glucosidase inhibitory activity The α-glucosidase inhibitory activity of the vegetable extracts was depicted in Fig 4.7. The result showed that the extracts inhibited α-glucosidase activity dose-dependently (20–640 ug/mL). All the tested extracts exhibited significantly higher inhibition percentage (ranging from 78-88% at 640ug/mL) as compare to acarbose. Least inhibitory potential was observed by T. foenum graecum
88
Chapter # 4 Results and Discussion
with 53.96%. This trend of the α-glucosidase inhibitory property of the polyphenol-rich extracts was as follows: S. melongena> D. carota> Brassica rapa> I. batatas> Acarbose> T. foenum- graecum.
100 0.2 mg/mL 0.4 mg/mL 0.8 mg/mL 1.2 mg/mL 1.6 mg/mL 80
60
40
20
Enzyme Enzyme inhibition (%) 0 Acarbose I. batatas T. foenum- D. carota S. Melongena Brassica rapa graecum Samples (A)
20 µg/mL 40 µg/mL 80 µg/mL 160 µg/mL 640 µg/mL 100
80
60
40
20
0 Enzyme Enzyme inhibition (%) Acarbose I. batatas T. foenum- D. carota S. Melongena Brassica rapa graecum Samples (B) Fig 4.7. Inhibitory effects on α-amylase (A) and α-glucosidase (B) of selected vegetables extracts Ponnusamy et al. (2011) declared the numerous hypoglycemic features of these herbal infusions; linked not only to their inhibitory potentials on glucose transporters, α-glucosidase, and α- amylase but also to their strong antioxidant activities. Likewise, Mai et al. (2007) attributed the α -glucosidase inhibitory activity of medicinal plant foods to be a function of their phenolic content. Previously, several epidemological studies authenticated a positive correlation between enzymatic inhibition and antioxidant potential linked to diverse range of plant-derived constituents, primarily glycosides, alkaloids, galactomannan gum, hypoglycans, polysaccharides, peptidoglycans, steroids, guanidine, terpenoids and glycopeptides (Wang et al., 2010). However,
89
Chapter # 4 Results and Discussion
a number of vegetables and herbal extracts also have exhibited inhibitory activities against α- glucosidase and α-amylase, and therein may have potential as dietary antidiabetic agents for the control of postprandial hyperglycemia (McCue et al., 2004). Yao et al. (2013) attributed that α- glucosidase and α-amylase are inhibited to a certain extent by total flavonoids within different extract concentrations which is in accordance to present results. In accordance to present results, Kwon et al. (2008) reported that phenolic enriched extracts from several eggplant samples (e.g., White pulp and Graffiti skin) have high α-glucosidase inhibitory activity combined with low two folds α -amylase inhibitory activity. 4.13. Carbohydrate hydrolyzing enzymes inhibition through selected dry fruits 4.13.1. α-amylase inhibitory activity Dry fruit were also investigated for the inhibition of α- amylase at different concentrations of phytochemical extracts and acarbose respectively, and the result is showed as Fig 4.9(A). α- amylase inhibitory activity increased with the concentration enhancement, which illustrated a good dose dependent response. Acarbose had reached the highest α- amylase inhibitory activity up to 65.63% at 1.6 mg/mL. However, all the tested samples were significantly lower than acarobe ranged between 21-33%, at the maximum concentration. 4.13.2. α-glucosidase inhibitory activity α-glucosidase inhibitory activity of crude extracts compounds against α-glucosidases were determined using p-nitrophenyl-α-D-glucopyranoside (p- NPG) as a substrate and these were compared with acarbose (Fig 4.8). The inhibitory percentage of dry fruits ranges from 80 to 92% higher than that of acarbose (75.63%). However, extracts derived from Juglans showed lower inhibitory activity against α-glucosidase enzyme significantly, with 61.36% value. In accordance to present results, high inhibitory percentage of samples of plant extracts are higher than acarbose because their active chemical compounds have no further fractionation and may have a synergistic effect in inhibiting α-glucosidase (Andrade-Cetto et al., 2008). Antioxidant activity plays a role in the treatment of diabetes mellitus (Raphael et al., 2002). Some plant species have been reported to possess both antidiabetic activity as well as antioxidant activity. Bhandari et al. (2008) and Shobana, et al. (2009) evaluated the inhibitory activities against α- glucosidase and α-
90
Chapter # 4 Results and Discussion
amylase of phenolic compounds extracted from several plants; they found that the phenolics
inhibited both enzymes significantly with low IC50 values for α -glucosidase, but with much
higher IC50 values α-amylase. Currently used inhibitors of α-glucosidase and α-amylase such as acarbose have strong inhibitory activities against both of the two enzymes. These drug therapies are effective but have side effects caused by the abnormal bacterial fermentation of undigested carbohydrates due to an excessive inhibition of α-amylase. Therefore, strong inhibitors of α-glucosidase with mild inhibitory activity against α-amylase have long been sought to overcome this challenge (Dong et al., 2012). In this regard, all the investigated edible samples strengthen
100 0.2 mg/mL 0.4 mg/mL 0.8 mg/mL 1.2 mg/mL 1.6 mg/mL 80 60 40 20 0 Acarbose P. Juglans P.dulcis P. vera A. V.vinifera C. P. Enzyme Enzyme inhibition (%) dactylifera hypogaea L. nucifera armeniaca Samples
(A)
20 µg/mL 40 µg/mL 80 µg/mL
100 80 60 40 20 0
Enzyme Enzyme inhibition (%) Acarbose P. Juglans P.dulcis P. vera A. V.vinifera C. P. dactylifera hypogaea L. nucifera armeniaca Samples (B) Fig 4.8. Inhibitory effects on α-amylase (A) and α-glucosidase (B) of selected dry fruits extracts this finding and exhibited strong α-glucosidase with slighter α-amylase activity. These functionalities when combined in one compound are potentially useful to manage the glucose- induced hyperglycemia and provide the biochemical rationale for further animal and clinical studies.
91
CHAPTER # 5 SUMMARY
Fundamental nexus between nutrition and health has promoted the consumers to focus on plant- based products as a remedy against various metabolic syndromes. In diet-based regimen, functional/nutraceutical foods are gaining the core attention to be a therapeutic device against maladies. Keeping in view the nutraceutical worth of phytonutrients, current research project was designed to focus on the antioxidant and antiglycation properties as well as the biochemical analysis of different edible species, locally available and readily consumed.
Prior to extraction all the samples were subjected to nutrition and mineral analysis. The proximate composition indicates that all the dried edibles species were rich source of crude protein, carbohydrate and fat ranging from 1.5-23.7%, 11.8-94.0% and 0.05-55.5% respectively, whereas, highest dietary fiber in Daucus carota and S.Melongena (18.50%) and ash content (13%) was observed for T. foenum-graecum respectively. In addition to caloric potential, significantly highest level of energy values ranging from 371.0±0.02 to 637.5±0.08 (Kcal/100g) in dry fruits while a moderate caloric values ranging from 187–321 kcal/100 g were observed for vegetables. According to mineral analysis, K, Na, Ca, Mg, Fe and P were present in significant amounts in all the investigated plant species while Cr, Mn, Ni and Pb were in lower amount. Among vegetables, Cu, Zn and P were significatly higher in T. foenum-graecum and Daucus carota. Juglans had the highest amount of Fe, Ni, Mn among the investigated dry fruits. Ca and Mg were higher in Prunus dulcis, P in Prunus dulcis. The non-nutrient phytochemicals from the selected edible portions of plant materials were extracted with methanol as well as ethanol. Maximum percentage yield was recorded for methanol extracts and used for further analysis. The qualitative and quantitative investigation of phytochemicals represent a diverse range of phyto-constituent including alkaloids, tannins, saponins and flavonoids responsible for their therapeutic potential. Maximum β-carotene was observed for vegetable as compare to dry fruit samples.
Antioxidants potential was determined by measuring total phenolics and flavonoid contents of the selected plant material. Among various samples, highest phenolic and flavonoid contents were observed in T. foenum-graecum and Juglans whereas least contents were detected in
92 Chapter # 5 Summary
Brassica rapa and Cocos nucifera responsible for their significant antioxidant capacities calculated by radical scavenging assay and through ferric reducing power assay. T. foenum- graecum and Juglans represented least IC50 value exhibiting its maximum antioxidant activity as a result of inhibition of free radical scavenging by DPPH method.
Plant extracts were screened for their cytotoxic potential against the brine shrimps nauplii, no toxicity was observed for the tested vegetables. However, for dry fruits half lethal concentrations
(LC50) ranges between 249-1100 µg/mL indicate the presence of potent cytotoxic components. In order to thrombolytic activity, all the investigated samples exhibited moderate clotlytic percentage except Juglans and Trigonella foenum-graecum having maximum potential. As a first step towards the identification of antiproliferative potential, the inhibitory effects of selected extracts against the cell lines derived from colon (Ht-29) and lung cancer (H1299) were examined. Cells derived from colon cancer were more sensitive to the extracts as compared to lung cancers cells. Among the samples, T. foenum-graecum and Juglan exhibited maximum inhibitory potential against both cell lines
Antiglycation potential of all the tested plants was quantified by three different methods at different stages of glycation. Inhibitory effect of food extracts on the formation of early and intermediary glycation products were secreened by NBT reduction assay and Girard T test respectively. The data showed that all the tested foods have considerable inhibitory effect specially T. foenum-graecum, S. Melongena and Juglans, P. vera respectively during the 18th day of incubation. The glucose-induced advanced glycation end-product (AGE) inhibition of the extracts was also carried out using in vitro glucose-bovine serum albumin (BSA-glucose) assay. The results showed maximum inhibitory effect during 18-24th day of incubation at the concentration of 5000 µg/mL. Currently, an impending remedial approach is the inhibition of principal carbohydrate hydrolyzing enzymes (α-glucosidase and α-amylase) connected to postprandial hyperglycemia. In present study, all the analyzed samples exhibited significant inhibition towards α-glucosidase as compare to α-amylase.
Our results showed that selected fruits and vegetables are rich source of phyto-nutrients exhibiting considerable antioxidant, non-toxic and antiglycation potential. In this scenario, plant-derived foods enriched with bioactive moieties are effective to tailor specific healthy diet
93
Chapter # 5 Summary for the target population. Further in vivo investigations are necessary to advance in knowledge regarding biological potential, their mechanisms of action and the structural elucidation of the active compounds of these fruits and vegetables. Keeping in view the nutraceutical worth of these analyzed edibles, current study also opens new endeavors for the applications of functional/nutraceutical foods and their bioactive moieties as a therapeutic device against maladies and also in the field of nano sciences.
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