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Department of Botany University of Peshawar 2019 PHARMACOGNOSTIC STANDARDIZATION AND BIOINSPIRED SYNTHESIS, CHARACTERIZATION AND APPLICATION OF GOLD / SILVER NANOPARTICLES OF EUPHORBIA WALLICHII HOOK. By Mr. Rehman Ullah Research Supervisor Prof. Dr. Siraj ud Din DEPARTMENT OF BOTANY UNIVERSITY OF PESHAWAR 2019 i PHARMACOGNOSTIC STANDARDIZATION AND BIOINSPIRED SYNTHESIS, CHARACTERIZATION AND APPLICATION OF GOLD / SILVER NANOPARTICLES OF EUPHORBIA WALLICHII HOOK. By Mr. Rehman Ullah The Thesis submitted to Department of Botany, University of Peshawar in partial fulfillment of the requirement for the degree of Ph.D In Botany DEPARTMENT OF BOTANY UNIVERSITY OF PESHAWAR 2019 ii iii iv In the Name of Allah The Merciful and Compassionate the Gracious and Beneficent Whose help and guidance we always solicit at every step, at every moment. v DEDICATION I dedicate this humble effort to my Mother whose love and encouragement enabled me to achieve every bit of success vi TABLE OF CONTENTS Topic Page No. 1 Introduction…………………………………........................................ 1 1.1. Pharmacognosy………………………………………………… 1 1.2. Pharmacognostic Study………………………………………… 2 1.3 Phytochemistry…………………………………………………. 3 1.4 Physicochemical assessment……………………………………. 4 1.5. Nanobiotechnology…………………………………………… 5 1.5.1. Characterization………………………………………………. 7 1.5.2.i UV-Visible spectroscopy…………. …………………. 7 1.5.2.ii Fourier Transmission Infrared (FT-IR) Spectroscopy … 8 1.5.2.iii Scanning Electron Microscopy (SEM) ……………….. 9 1.5.2.iv X-ray diffraction (XRD)………………………………. 9 1.5.2.v. Energy dispersive X-Ray Spectroscopy ……………… 10 1.6. Pharmacology……………………………………………………. 10 1.6.1. Acute toxicity……………………………………………….. 10 1.6.2. Cytotoxicity………………………………………………… 11 1.6.3. Larvicidal activity…………………………………………… 11 1.6.4. Phytotoxicity………………………………………………….. 12 1.6.5. Antimicrobial activities (Antibacterial, Antifungal)………… 13 1.6.6. Antioxidant bioassay……………………………………….. 13 1.6.7. Antileishmanial assay……………………………………….. 14 1.6.8. Analgesic activity…………………………………………… 15 1.6.9. Antispasmodic assay………………………………………….. 16 1.7. Family Euphorbiaceae…………………………………………… 16 1.7.1. Description……………………………………………………. 16 1.7.2. Ethnobotanical uses…………………………………………. 17 1.8 Euphorbia (Genus)……………………………………………. 18 1.9. Euphorbia wallichii Hook. ……………………………………… 18 2. Review of Literature…………………………………………………. 20 2.1. Pharmacognostic study…………………………………………… 20 2.2. Phytochemical investigation……………………………………… 22 2.3. Nutraceutical and Elemental analysis…………………………….. 23 2.4. Phytopharmacology……………………………………………… 25 2.4.1. Acute toxicity………………………………………………. 25 2.4.2. Analgesics………………………………………………… 26 2.4.3. Cytotoxic screening………………………………………… 27 2.4.4. Antioxidant evaluation……………………………………… 28 2.4.5. Antimicrobial activity………………………………………. 29 2.4.6. Antileishmanial activity…………………………………….. 30 2.4.7. Antispasmodic activity……………………………………… 31 2.5. Euphorbia wallichii ……………………………………………… 31 vii 2.6 Green synthesis of Nanoparticles………………………………… 32 2.7.1. Gold nanoparticles………………………………………….. 32 2.7.2. Silver nanoparticles…………………………………………. 33 3. Materials and Methods…………………………………………………. 35 3.1. Pharmacognostic Evaluation…………………………………… 35 3.1.1. Collection, identification and preservation…………………. 35 3.1.2. Macroscopic examination………..………………………… 35 3.1.3. Microscopic study………………………………………… 36 3.1.3.i. Microscopy of Powder drug………………………. 36 3.1.3.ii Leaf surface microscopy………………………….. 36 3.1.3.ii.(a) Stomatal frequency and Stomatal Index………….. 36 3.1.3.ii.(b) Vein Islet and Vein Termination Number……… 37 3.1.3.ii.(c) Palisade cell Ratio……………………………… 37 3.1.4. Preliminary Phytochemical analysis……………………… 37 3.1.4.i. Test for reducing sugars……………………………. 37 3.1.4.ii. Killere killiani test for steroidal Glycosides…………. 37 3.1.4.iii. Confirmative test for tannins………………………… 38 3.1.4.iv Detection of Alkaloid……………………………… 38 3.1.4.v. Saponins confirmation……………………………… 38 3.1.4.vi Detection of Triterpenes and Phytosterols…………. 38 3.1.4.vii Detection test proteins & amino acids……………… 39 3.1.4.viii. Detection of Oil……………………………………… 39 3.1.4.ix. Detection of flavonoids……………………………. 39 3.2. Physicochemical analysis………………………………………… 39 3.2.1. Proximate and nutritional analysis…………………………. 39 3.2.1.i. Moisture content…………………………………… 39 3.2.1.ii. Determination of fat……………………………….. 39 3.2.1.iii. Protein content “Kjeldahl method”………………… 40 3.2.1.iv. Determination of crude fiber………………………. 41 3.2.1.v. Determination of Carbohydrates content…………. 41 3.2.1.vi. Energy value……………………………………….. 41 3.2.2. Elemental analysis………………………………………….. 41 3.3. Green synthesis of Silver and Gold Nanoparticles………………. 42 3.3.1. Characterizations…………………………………………… 42 3.3.1.i. UV-Visible spectroscopy……………………………. 42 3.3.1.ii. FT-IR Spectroscopy ………………………………… 42 3.3.1.iii X-ray diffraction (XRD)………………………………. 42 3.3.1.iv Scanning Electron Microscopy (SEM)………………… 42 3.3.1.v. Energy dispersive X-Ray Spectroscopy……………….. 43 3.4. Pharmacology…………………………………………………….. 43 3.4.1. Acute toxicity……………………………………………….. 43 3.4.2. Cytotoxicity…………………………………………………. 43 3.4.3. Phytotoxicity…………………………………………………. 44 3.4.4. Antibacterial activity………………………………………... 45 3.4.5. Antifungal activity………………………………………….. 45 3.4.6. Antioxidant bioassay………………………………………... 45 3.4.7. Antileishmanial assay……………………………………….. 46 3.4.8. Analgesic activity…………………………………………… 47 viii 3.4.9. Antispasmodic assay………………………………………… 47 3.4.10 Larvicidal activity…………………………………………… 49 Objectives of the Study…………………………………………………. 50 4. Results and Discussions………………………………………………. 52 4.1. Euphorbia wallichii…………………………………………….. 52 4.1.1. Systematic position…………………………………………. 52 4.1.2. Morphology………………………………………………… 52 4.2. Pharmacognostic study…………………………………………… 52 4.2.1. Macroscopic features……………………………………….. 53 4.2.1.i. Leaf macroscopy……………………………………… 53 4.2.1.ii. Stem macroscopy……………………………………… 53 4.2.1.iii Rhizome macroscopy………………………………….. 53 4.2.2. Microscopic study…………………………………………… 55 4.2.2.i. Leaf surface microscopy………………………………. 55 4.2.2.ii. Powder drug study……………………………………... 57 4.2.3. Physicochemical analysis…………………………………… 58 4.2.3.i. Elemental composition………………………………… 58 4.2.3.ii. Proximate Analysis……………………………………. 62 4.3. Preliminary Phytochemical evaluation…………………………. 65 4.4. Nanoparticles characterization…………………………………… 65 4.4.1. Characterization of SE mediated AgNPs…………………….. 65 4.4.1.i. UV-Visible spectroscopy…………………………….. 65 4.4.1.ii. Scanning Electron Microscopy (SEM)………………… 66 4.4.1.iii Energy dispersive X-Ray Spectroscopy……………….. 67 4.4.1.iv Fourier transforms infra red (FTIR) spectroscopy…….. 67 4.4.1.v. X-ray diffraction (XRD) Crystallography……………... 68 4.4.2. Shoot Extract Mediated AuNPs………………………………. 69 4.4.2.i. UV-Visible spectroscopy……………………………. 69 4.4.2.ii. Scanning Electron Microscopy (SEM)………………… 70 4.4.2.iii Energy Dispersive X-ray Specroscopic analysis……… 71 4.4.2.iv X-Ray crystallography/ X-ray diffraction analysis…… 71 4.4.2.v. Fourier transformed infra red spectroscopy…………… 72 4.4.3. Characterization of RE Mediated AgNPs………………….. 74 4.4.3.i. UV-Visible spectroscopy……………………………… 74 4.4.3.ii. X-ray diffraction analysis……………………………… 74 4.4.3.iii Scanning Electron Microscopy (SEM)………………… 75 4.4.3.iv Energy Dispersive X-ray Specroscopy 76 4.4.3.v. Fourier transformed infra red spectroscopy…………… 77 4.4.4. Characterization of RE Mediated AuNPs…………………….. 77 4.4.4.i. UV-Visible spectroscopy……………………………… 77 4.4.4.ii. Scanning Electron Microscopy (SEM)……………….. 78 4.4.4.iii Energy Dispersive X-ray Specroscopy……………….. 79 4.4.4.iv X-ray diffraction analysis (XRD)…………………….. 80 4.4.4.v. Fourier transformed infra red spectroscopy (EDS)……. 81 4.5. Pharmacology……………………………………………………. 82 4.5.1. Acute toxicity………………………………………………. 82 4.5.2. Analgesic activity…………………………………………… 83 4.5.3. Antispasmodic activity……………………………………… 88 ix 4.5.4. Larvicidal activity…………………………………………….. 91 4.5.5. Cytotoxicity………………………………………………….. 94 4.5.6. Antioxidant (Free radical scavenging) activity………………. 98 4.5.7. Antibacterial activity……………………………………….. 103 4.5.8. Antifungal activity……………………………………………. 106 4.5.9. Antileishmanial activity…………………………………….. 107 4.5.10 Phytotoxicity………………………………………………….. 110 Conclusions…………………………………………………………… 114 References……………………………………………………………… 116 x LIST OF TABLES Table No. Table title Page No. 1.a. Macroscopic features of stem of E. wallichii. 54 1.b. Macroscopic features of leaf of E. wallichii. 54 1.c. Macroscopic features of rhizome of E. wallichii. 55 2. Leaf surface constants of E. wallichii leaf. 56 3. Elemental profile of different parts of E. wallichii 64 4. Nutraceutical profile of different parts of E. wallichii 64 5. Phytochemical screening of E. wallichii 65 6. Acute toxicity assay of E. wallichii shoot and rhizome. 83 Analgesic activity of E. wallichii shoot and its mediated AgNPs and 7.a. 85 AuNPs. Analgesic activity of E. wallichii rhizome and its mediated AgNPs and 7.b. 87 AuNPs. Antispasmodic activity of E. wallichii shoot and its mediated AgNPs 8.a. 90 and AuNPs. Antispasmodic activity of E. wallichii rhizome and its mediated 8.b. 91 AgNPs and AuNPs. 9.a. Larvicidal efficacy of AgNPs and AuNPs of E. wallichii shoot extract 93 Larvicidal efficacy of AgNPs and AuNPs of E. wallichii rhizome 9.b. 94 extract 10.a. Cytotoxic potential of AgNPs and AuNPs of E. wallichii shoot extract 96 Cytotoxic potential of AgNPs and AuNPs of E. wallichii rhizome 10.b. 97 extract Antioxidant activity of AuNPs and AgNPs of E. wallichii Shoot 11.a. 101 extract Antioxidant activity of AuNPs and AgNPs of E. wallichii rhizome 11.b. 102 extract 12. Antibacterial activity of E. wallichii mediated AuNPs and
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