A STUDY OF GOLD AND SILVER NANOPARTICLES SYNTHESISED USING

PLANT EXTRACT OF Madhuca longifolia FOR ENHANCEMENT IN SELECTED BIOEFFICACIES

A SYNOPSIS

SUBMITTED FOR PARTIAL FULFILLMENT FOR THE AWARD

OF DEGREE OF DOCTOR OF PHILOSOPHY IN CHEMISTRY

Submitted by

MUKTI SHARMA M.Sc., M.Phil.

Dr. SHALINI SRIVASTAVA Dr. N. GANESH Supervisor Co-Supervisor Professor Head & Sr. Scientist Department of Chemistry J.N. Cancer Hospital & Research Center, Bhopal

Dr. Sahab Dass Prof. Ravinder Kumar Prof. & Head Dean Department of Chemistry Faculty of Science

DAYALBAGH EDUCATIONAL INSTITUTE (Deemed University)

DAYALBAGH, AGRA OCTOBER, 2015

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INTRODUCTION Nanotechnology is one of the most interesting and open inquisitive field for research in modern science. It has great potential in the development of excellent procedures and products for ecofriendly development of sustainable environment and human mankind (Masurkar et al., 2011). Nanotechnology has experienced a rapid growth because nanostructures exhibit physical and chemical properties that are distinctly different from the bulk solid (Nagaraj et al., 2011) in size, distribution, morphology and demonstrates completely new or improved properties (Khadri et al., 2013). These unusual properties have attracted immense attention of researchers from almost every field of science particularly in material science and medicine (Gopinath et al., 2013). Among various methods of synthesis of different types of nanoparticles, physical and chemical methods are more popular. However, the involvement of toxic compounds limits their applications. Inorganic nanoparticles due to their small size have been associated with toxicity and potential side effect issues, especially in biological system. Application of plants in synthesis of nanoparticle is quite novel leading to Green Chemistry concepts which is cost effective and environment friendly, avoiding rigid experimental conditions like high pressure, energy and temperature etc (Prasad and Elumalai 2011). Green Nanotechnology uses biological organisms and plant extract is considered as an alternative to the conventional chemical and physical methods of preparation of nanoparticles. It involves the synthesis of nanoparticles in a clean, non-toxic, ecologically sound and environment friendly manner (Gavhane et al., 2009, Wang et al., 2009). Use of plant extracts for the synthesis of nanoparticles is more advantageous over other environmentally benign biological processes, eliminating the complications of maintaining cell culture. Plants are like natural laboratories where a great number of chemicals are bio synthesized (Raj and Padhi 2015). The chemical partnership of secondary metabolites presents in the herbs results into several synergistic, additive and antagonistic effects. The cocktail of bioactive secondary metabolites can be used as therapeutic armamentarium while on the other hand it may cause delay in resistance by increasing immunity. When chemicals act in synergism, the mixture has greater effect than the sum of total. A vast number of secondary metabolites are found in all plants which possess redox capacity and can be exploited for biosynthesis of nanoparticles. Nanoparticles produced by plants are more stable and the rate of synthesis is faster in comparison to microorganisms (Kumar et al., 2012). The phytochemicals involved in the plant-assisted reduction are terpenoids, flavones, ketones, aldehydes, amides, and carboxylic acids. Polyphenolics, flavones, organic acids, and quinones are water-soluble and responsible for the immediate reduction of the metal ions. Chemical partnership of various phytochemicals contain functional groups such as (-COOH, C=O, NH2, SH and OH) etc provide Powerful Synergistic Chemical Reduction to metallic salts into their corresponding nanoparticles. The therapeutic value of any plant thus depends on the active constituents present in the small or large quantity of pharmacological importance. They also provide a coating of phytochemicals on the metal nanoparticles. Coatings of phytochemicals on the freshly generated nanoparticles provide robust shielding from aggregations keeping them in nano state (stability) and add medicinal properties. The relatively high levels of the steroids, sapogenins, carbohydrates and flavonoids act as reducing agents as well as capping agents providing stability to nanoparticles (Ahmed et al., 2015). Synthesis of nanoparticles embedded with plant based products is supposed to be the most adopted method for eco-friendly and non toxic production of nanoparticles. Gold and Silver nanoparticles embedded with plant extract are an emerging interdisciplinary research area with potential applications in therapeutic science (Geethalakshmi et al., 2013). Such metallic nanoparticles contain remarkable medicinal properties due to their large surface area to volume ratio. It is a timely answer to the growing microbial resistance and antibiotics against metal ions. Gold is choice of element for the preparation of NPs and has potential applications in drug delivery, bio labeling, tumor imaging, cancer therapy and almost all medical applications diagnostics,

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therapy, hygiene and biocompatibility. Gold nanoparticles are very fine and stable particles (Thanighaiarassu et al., 2014, Priya et al., 2015). Silver nanoparticles are striving towards the edge level utilities in every aspect of science and technology including medical fields. Silver is having a long history of use as a disinfectant and is able to interact with disulphide bonds of the glycoprotein/ protein contents of microorganisms (Kumar et al., 2015). One of the potent applications of silver is in management of plant diseases. Silver displays multiple modes of inhibitory action against plant pathogens. Silver nanoparticles have gained boundless interests because of their unique properties such as chemical stability, good conductivity and catalytic properties. For biomedical applications; silver is added to wound dressings, topical creams, antiseptic sprays and fabrics. It displays a broad effect against microorganisms through the disruption of their unicellular membrane thus disturbing their enzymatic activities. Silver nanoparticles could induce rapid healing in animal model through their antimicrobial activities, capacity to decrease wound inflammation (Jia et al., 2008, Rai et al., 2009, Sadeghi and Gholamhosempoor et al., 2015). Gold and silver nanoparticles find significant exploitations in biomedical field due to their following special features and benefits

FIG 1. SALIENT FEATURES OF GOLD AND SILVER NANO PARTICLES

Source: (Boisselier et al., 2009) The rationale behind the hypothesis is based on the reduction capabilities of cocktail of phytochemicals present in plants to chemically reduce Au (III) salts to Au (0) or Ag (I) salts to Ag (0) nanoparticles. It is expected to occur through the oxidation of phytochemicals having hydroxyl to carbonyl groups of phytochemicals.

FIG 2. POWERFUL SYNERGISTIC CHEMICAL REDUCTION OF PHYTOCHEMICALS

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Recent work carried out on the synthesis of gold and silver nano particles using different plant extract followed by the assessment of their bio efficacies has been summarised in the table. TABLE1. GOLD AND SILVER NANOPARTICLE EMBEDDED WITH PLANT EXTRACT AND THEIR BIO-EFFICACIES

Nanoparticles have advantages of their unique interaction with light compared to the bulk materials due to their surface Plasmon resonance. Surface Plasmon resonance is explained as in the presence of the oscillating electromagnetic field of the light, the free electrons of the metal nanoparticles undergo a collective coherent oscillation with respect to the positive metallic lattice and start resonating at a particular frequency of the light (Kelly et al., 2003, Link et al., 2003, Jain et al., 2006). With advent of 21st century, a wave of environmental awareness and health consciousness is developed regarding the side effects of hazardous chemical substances. Economic constraints and environmental evaluation pushed the chemical industry to adopt new ecofriendly technologies. Recent phytochemical researches have directed attention that if phytochemical based therapy is effective; its efficacy can be further enhanced for designing new generation of pharmaceuticals. Experimental breakthrough in Green nanotechnology has been recognized providing Enhanced Therapeutic Bio-efficacy with reduced side effects.

Name of plant Bio-efficacy Nano particle References Madhuca longifolia Not reported Gold Fayaz et al., 2011 Moringa oleifera Antimicrobial Silver Prasad et al., 2011

Cymbopogan citrates Antimicrobial Silver Masurkar et al., 2011

Ixora coccinea Antimicrobial Gold Nagaraj et al., 2011

Ananas comosus Antioxidant Silver Ahmad and Sharma 2012

Rhododendron dauricum Antioxidant Silver Mittal et al., 2012

Neem Antimicrobial Silver Gavhane et al., 2012

Trianthema decandra L. Antimicrobial Gold ,Silver Geetalakshmi et al., 2013

Azhadirachta indica Antimicrobial Silver Lalitha et al., 2013

Olive Antifungal Silver Khadri et al., 2013

Alternanthera sessilis Antimicrobial Silver Niraimathi et al., 2013

Shorea roxburghii Antioxidant Silver Subramanian et al., 2013

P. hysterophorus Anti- bacterial Silver Kalaiselvi et al., 2013

Zataria multiflora Antioxidant Silver Goodarzi et al., 2014

Rosmrinus Antioxidant Silver Goodarzi et al., 2014

Terminalia sps Anti-inflammatory Silver Rafie et al., 2014

Piper longum Antioxidant, Silver Reddy et al., 2014

Mentha piperita Antifungal Gold Thanighaiarassu et al., 2014 Brassica rapa Antifungal Silver Narayanan et al., 2014 Aloe vera Antifungal Silver Medda et al., 2014 Mentha arvensis Antioxidant Silver Kumar et al., 2015 Euphobria milii Muscle relaxant Gold Islam et al. 2015 Salix alba Antifungal Gold Islam et al. 2015

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Thus, Green nanotechnology provides an unprecedented process for the production and stabilization of gold nanoparticles in a singular green process, opening the gate of wide applications for Gold nanoparticles embedded with phytochemical in a myriad of applications in Nanomedicine. PLANT UNDER STUDY

Source: www.google.co.in. Pharmacological importance A vigorous survey of literature on Madhuca longifolia plant indicates that it has been used as Folk medicine and Home Remedy involving the use of plant-derived remedies on an empirical basis, Folk medicine consists of the healing practices and ideas of body physiology and health known to some person and transmitted informally as general knowledge. Similarly, a home remedy is a treatment to cure a disease or ailment that employs certain spices, vegetables, or other common items. The bark, flowers, leaves and seed oil have great medicinal values such as anti burn, anti inflammation, dermatological, laxative, tonic, and wound healing property. The bark is also used for, chronic bronchitis, gums, ulcer, bleeding. It is a good remedy for itching, swelling, fractures and snake bite poisoning, internally employed in diabetes. It is useful in burning sensation in respiratory diseases. The root powder is useful in diarrhea and other chronic fluxes. Leaves extract have been used as tonic for energy and fungal infections. Leaves are used as expectorant in bronchitis. Flowers

Madhuca longifolia: Madhuca comprises a number of species. It has an economical and industrial importance belongs to the Sapotaceae family. It is commonly known as Indian butter tree Mahua, Mohwa, Mauwa, Madhuda, Ippa, Illupei, Ewpa Tuppe, poonam, Ilupa, Madgn. Habit and Habitat: It is a common tropical tree that grows naturally in deciduous forest, all over the world, especially Asian and Australian countries .It is a plant of Indian origin. It is a large, shady tree doting much of the central Indian Landscape, both wild and cultivated. It is cultivated in warm and humid regions for its oleaginous seeds. This tree has lots of nutrional value. It is one of the single largest sources of natural fat. The fat which is obtained from its fruit oil is used in cooking, frying and manufacturing chocolates. Large numbers of Mahua trees are found in the state of Uttar Pradesh, Madhya Pradesh, Orissa, Jharkhand, Gujarat, Andhra Pradesh, Maharashtra, Bihar, West Bengal, and Karnataka (Patel 2010, Siddiqui 2010 and Srirangam et al.,2010). Plant height: 12-15meter, Leaves: 10-30 cm long thick and leathery, Flower: Small fleshy, dull or pale white in colour, Fruits: 2-6 cm long fleshy and greenish, Seeds: Yellow brown or reddish spherical or Ovoid.

Classification Kingdom: Plante Family: Sapotaceae Subfamily: Caesalpinieae Order: Ericaleac Genus: Madhuca

Species: longifolia

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have been traditionally used as cooling agent, cough, tonic, and aphrodisiac, astringent, demulcent and for the treatment of worms. Fruits are largely employed as a lotion in wounds and tonsillitis. Distilled juice of the flower is considered as a tonic. Seed has been found for reducing swelling. Seed cake is reported to have insecticidal and pesticide property and used as organic manure in crops. Seeds are also used in making soap. WHO reports that "Inappropriate use of traditional medicines or practices can have negative or dangerous effects and for that further research is needed to ascertain the efficacy and safety". Home remedies may or may not have medicinal properties that treat or cure the disease in question as they are typically passed along by people based on their experiences. A special attention has been paid to the scientific study on Madhuca longifolia recently. The following table presents the recent work carried out on phytochemical investigations with reference to pharmacological aspects. TABLE: 2. MEDICINAL IMPORTANCE AND ITS PHYTOCHEMICALS AT A GLANCE:

OBJECTIVES There is a misconception about Madhuca longifolia tree about its use as liquor which is harmful. Madhuca has tremendous therapeutic potential but is not explored scientifically. It has several pharmacological activity and potential to provide health to the society. It is hidden from the eyes of the researchers and botanists. Presence of allelochemicals reported so far like polyphenolics, flavonoids, terpenoids etc in the plant Madhuca longifolia, which are known to exhibit important pharmacological activities, has motivated us to carry out an exhaustive work on the establishment of Ant oxidative, Anti-inflammatory and Wound healing bio efficacy in different parts of this plant followed by their phytochemical characterization. Main focus of the study would be on the synthesis and characterization of gold and silver nanoparticles using plant extracts of the plant Madhuca longifolia for the evaluation of any enhancement in the target bio efficacies.

Plant Part Use Chemical constituents References

Bark Stomach ache, Tonsillitis, Diabetes, Anti oxidant, Wound healing

Flavonoids, Triterpene, Sterols, and Polyphenolics compound, Ethylcinnamate, Sesquiterene alcohol, Terpeneol.

Prashanth et al., 2010, Shekhawat and Vijayvergia 2010, Patel et al.,2011, Verma et al., 2014, Gowda et al., 2014.

Leaves Bone Fracture, Rheumatism, Anti-inflammatory

Glucoside, Sterol, Carotene,Xanthophylls, Palmitic acid, Myricetn, Phenolic Content

Vijayabhaskar and Chaitanyaprasad 2014, Verma et al., 2014.

Seeds Piles, Emetics, Anti-inflammatory, Anti fungal.

Palmitic acid, Stearic acid, Alanine, Aspartic acid Flavonoids,Isoleucin,Leucin, Lysin , Methionine, Saponin

Chakme et al., 2011, Patel et al., 2012, Sidu et al., 2009, Yadav et al., 2012.

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Pharmacological studies: Bioassay for following pharmacological properties in native, gold and silver nanoparticles using plant extracts would be studied.

Bio activity in vivo Activity in vitro Activity Anti-oxidant Assay 1. Lipid Peroxidise bio assay

2. Assay for reduce Glutathione DPPH bio assay Fenton bio assay

Anti- inflammatory Assay Carragenin Induce hind paw edema Wound healing Assay 1.Excision wound

2. Incision wound

. MATERIAL AND METHODS Collection and identification of plant: The plant material of Madhuca longifolia would be collected from the nearby area and authenticated through Taxonomy Department of Botany, Dayalbagh Educational Institute. Extraction of the plant material: Plant material would be dried under shade, separated into desirable parts leaf, bark, seed and will be powered. Each part would be subjected for the Soxhlation with aqueous alcohol. In order to remove the chlorophyll content from the leaves, and fat from seeds, extraction with petroleum ether would be carried out prior to alcoholic extraction. Extracts would be concentrated under reduce pressure using Rota- vapour and dried by purging nitrogen and finally weighed. Phytochemical analysis: Aqueous alcoholic extract of each part would be analyzed using LCMS and GCMS for characterization of various phytochemical present in them. Quantitative estimation of total phenolics and flavonoids. Preparation of gold and silver nano particles using extracts: For the synthesis of gold nano particles, aqueous sodium tetra Chloro Aurate (NaAuCl4. 2H2O) solution would be added to each extract in the optimized ratio, while for silver nanoparticles aqueous solution silver nitrate would be added to each extract in the optimized ratio. Within a particular time span in ultrasonic bath colour change observed, is the sign of formation of gold and silver nanoparticles. Characterization of gold and silver nanoparticles embedded with plant extract would be carried out using X- ray diffraction, UV-VIS spectral analysis, Scanning Electron Microscopy, TransmissionMicroscopy, Atomic force Microscopy and Zeta sizer. Assessment of Antioxidant bio efficacy: In vitro studies Fenton assay The hydroxyl radical scavenging activity of test samples would be evaluated according to the Fenton reaction (Halliwell et al., 1992). The reaction mixture containing dilution series of test samples would be incubated with deoxyribose, H2O2, FeCl3, EDTA and ascorbic acid in phosphate buffer saline. The reaction would be terminated by Thiobarbituric acid and Trichloroacetic acid. The absorbance would be measured at 532 nm using Spectrophotometer. DPPH (2, 2-diphenyl-1-picrylhydrazyl) assay Free radical scavenging activity of test samples would be determined using DPPH assay (Shimada et al., 1992). The reaction mixture containing different dilution series of test samples would be incubated with 2, 2-diphenyl-1-picrylhydrazyl solution in MeOH. The absorbance of mixture would be recorded at 550nm using Spectrophotometer. The extent of discolouration would indicate the amount of DPPH scavenged.

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In vivo studies Lipid Peroxides assay (LPO) Mice in all groups would be sacrificed 21 days after the experimental schedule. The liver would be dissected, weighed and stored at -80°C until analyses would be completed. The liver would be homogenized in saline KCl (0.15M) to give a 10% homogenate (W/V). The crude homogenate would be assayed for LPO (Ohkawa et al., 1979). The reaction mixture contains crude homogenate, sodium dodecyl sulphate acetic acid and thiobarbituric acid (pH7.0). The whole mixture would be heated at 95°C for one hour in a water bath and the absorbance of colour would be measured at 532 nm. The malondialdehyde, an indicator of lipid peroxidation formation, would be evaluated by measuring thiobarbituric acid reactive substances using the standard of 1, 1, 3, 3-tetraethoxyprone (TEP) in different concentrations. Assay for reduced Glutathione The level of down regulation of reduced glutathione in liver of experimental mice would be investigated (Beutler 1984) to determine the antioxidative effect of test samples against the oxidative stress. The liver would be dissected, weighed and homogenized in saline (0.154 M, KCl) to give a 10% homogenate (w/v). The crude homogenate would be centrifuged at 2000 rpm for 15 minutes and supernatant would be collected. Phosphate EDTA buffer (0.9 ml) and DTNB solution (0.050 ml) would be added to supernatant (0.050 ml) making the solution 1.0 ml. The reaction mixture would be incubated at room temperature for 20 min and the optical density would be measured at 410 nm. The GSH levels would be monitored by the reduction of 5, 5’-dithio-bis-(2-nitrobenzoic acid) DTNB to 5-thio-2-nitrobenzoate (TNB) (Brown & Jeffries1975). Anti-inflammatory bio efficacy: Carrageenan-induced rat hind paw oedema method The hind paw oedema would be produced by injecting 0.1 ml of carrageenan (prepared as 1% suspension in normal saline) locally into the plantar of the right hind paw of rats. Extracts would be given at different dose. Extracts would be administered 1hr prior to the injection of carrageenan. The linear paw circumference would be measured at hourly interval by plythesmometer. Anti-inflammatory activity will be measured as the percentage reduction in oedema level when drug would be present. Increase in the paw oedema volume would be considered as the difference between 1 and 3 hr. Percent inhibition of oedema volume between treated and control groups would be calculated as follows: % Inhibition = (1-Vt/Vc) X 100 Where Vt and Vc are the relative changes in the oedema of the test and control respectively (Winter et al., 1962). Wound healing bio efficacy: Excision wound In the excision wound, model rats would be anaesthetized for creation of the wounds. A full thickness of appropriate diameter the excision wound would be created by using toothed forceps, a surgical blade and pointed scissors. The entire wound would be left open. The extract would be topically applied once in a day. The progressive changes in wound area would be monitored planimetrically by tracing the wound margin on graph paper on wounding day. The animals would be divided into 3 different groups. Time of Wound closure and Epithelialisation time would be noted (Hunt 1990). Incision wound The animals would be anesthetized by injecting ketamine intraperitoneally. The back of the rats would be shaved. Two 6 cm long paravertebral straight incision would be made, 1 cm lateral to the vertebral column on either side through the entire thickness of the skin. Wounds would be closed with intermittent sutures, 1 cm apart, with black silk thread and curved needle. Animals would be treated once daily with the drugs from day 0 (day of wounding) to day 9, sutures would be removed on day 7. On day 10, breaking strength of the wound would be measured by applying tearing force in the form of continuous water flow technique (Lee 1968).

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