“PHYTOCHEMICAL SCREENING ON THE CONSTITUENTS OF RUMEX OBTUSIFOLIUS” Ph.D Thesis By ABDUL KHABIR KHAN Institute of Chemical Sciences, Gomal University, Dera Ismail Khan, Pakistan. 2017 “PHYTOCHEMICAL SCREENING ON THE CONSTITUENTS OF RUMEX OBTUSIFOLIUS” Thesis submitted for the fulfillment of the degree of DOCTOR OF PHILOSOPHY IN CHEMISTRY BY ABDUL KHABIR KHAN Institute of Chemical Sciences, Gomal University, Dera Ismail Khan, Pakistan. 2017 CONTENTS DEDICATION i ACKNOWLEDGMENTS ii SUMMRY iii S.No DESCRIPTIONS Page 1 Chapter No 1: Introduction 1 1.1 Importance of medicinal plants 2 1.2 Family Polygonaceae 5 1.2.1 Botany of the family Polygonaceae 6 1.2.2 Chemistry of family Polygonaceae 6 1.2.3 Pharmacology of the family Polygonaceae 7 1.3 Genus Rumex 8 1.3.1 Distribution of genus Rumex 8 1.3.2 Morphology of genus Rumex 14 1.4 Rumex Obtusifolius 16 1.4.1 Habitat 17 1.4.2 Morphology 17 1.4.2.1 Roots 17 1.4.2.2 Stems 17 1.4.2.3 Leaves 17 1.4.2.4 Flower and Inflorescence 17 1.4.2.5 Taxonomic position 18 2 Chapter 2: Literature Review 19 2.1 Traditional uses of Rumex species 20 2.2 Traditional medicinal uses of Rumex species 21 2.3 Pharmacological and Biological screening of rumex species 23 2.4 Compound isolated from Rumex species. 29 4.2.1 Structures of the isolated compounds 33 2.5 R. Obtusifolius 46 2.6 Biosynthesis of anthraquinones 47 3 Chapter No 3: Result and Discussion 50 Preliminary phytochemical screeninig (qualitative) of crude extracts of 3.1 R.Obtusifolius 51 Biological Screening of Dichloromethane Sub-fractions of Rumex 3.2 Obtusifolius 53 3.2.1 Antibacterial screening 53 3.2.2. Antifungal screening 55 3.2.3. Cytotoxicity screening 58 3.3. Secondary metabolites from Rumex Obtusifolius 62 3.4. DCM (Dichloromethane) soluble fraction 62 3.5. Structure elucidation of compounds. 63 3.5.1. Obtusifolate A (102) 63 3.5.1.1. UV-Visible and IR of Obtusifolate A (102) 63 3.5.1.2. Mass spectrometry of Obtusifolate A (102) 64 3.5.1.3. 1H-NMR spectrum of Obtusifolate A (102) 64 3.5.1.4. 13C NMR spectrum of Obtusifolate A (102) 65 3.5.1.5. Other chemical tests for identification Obtusifolate A (102) 65 3.5.2. Obtusifolate B (103) 67 3.5.2.1 UV-Visible and IR of Obtusifolate B (103) 67 3.5.2.2 Mass spectrometry of Obtusifolate B (103) 68 3.5.2.3 1H-NMR spectrum of Obtusifolate B (103) 68 3.5.2.4 13C NMR spectrum of Obtusifolate B (103) 69 3.5.2.5 Other chemical tests for identification Obtusifolate B (103) 69 3.5.3. Obtusifolate C (104) 71 3.5.3.1 UV-Visible and IR of Obtusifolate C (104) 71 3.5.3.2 Mass spectrometry of Obtusifolate C (104) 72 3.5.3.3 1H-NMR spectrum of Obtusifolate C (104) 72 3.5.3.4 13C NMR spectrum of Obtusifolate C (104) 72 3.5.3.5 Other chemical tests for identification Obtusifolate C (104) 73 3.5.4. Obtusifolate D (105) 75 3.5.4.1 UV-Visible and IR of Obtusifolate C (104) 75 3.5.4.2 Mass spectrometry of Obtusifolate C (104) 75 3.5.4.3. 1H-NMR and 13C-NMR spectra of compound D (105) 76 3.5.4.4. Other chemical tests for identification Obtusifolate C (104) 76 3.6. Free radical scavenging activity of the isolated compounds 78 4 Chapter No 4: Experimental 80 4.1. Plant collection, Identification and Grinding 81 4.2. Extraction and Fractionations 81 4.3. Preliminary qualitative phytochemical analysis 81 4.3.1. Alkaloids 81 4.3.2. Flavonoids 81 4.3.3. Tannins 82 4.3.3. Tannins 82 4.3.4. Cardiac glycosides 82 4.3.5. Anthraquinones 82 4.3.6. Steroids 82 4.3.7. Saponins 83 4.4. Sub-fractionation of Dichloromethane soluble fraction 83 4.5. Biological Screening of Dichloromethane Sub-fractions of Rumex 85 Obtusifolius 4.5.1. Antibacterial screening 85 4.5.2. Antifungal screening 85 4.5.3. Cytotoxic screening 86 4.6. Secondary metabolites from R. Obtusifolius 87 4.6.1. Instrumentation 87 4.6.2. Chromatography 87 4.6.3. Isolation, Purification and Characterization of compounds 88 4.6.4. Obtusifolate A (102) 91 4.6.5 Obtusifolate A (103) 93 4.6.6 Obtusifolate C (104) 95 4.6.7 Obtusifolate D (105) 96 4.7. Free radical scavenging activity (RSA) of the isolated compounds 97 5 Chapter No 5: References 99 TABLES Table No Table Name Page 1.1 Rumex species 9 2.1 Compound isolated from Rumex species. 29 Preliminary qualitative phytochemical analysis of crude extracts of 3.1. 52 R.Obtusifolius Antibacterial screening of the sub- fractions of DCM fraction of Rumex 3.2 54 obtusifolius (in mm) 3.3 % Inhibition of fungi of sub-fractions of DCM fraction of Rumex Obtusifolius 56 Illustration of percentage mortality of brine shrimps at different 3.4. 58 concentrations of sub-fractions and respective LD50 values 3.5 1HNMR and 13CNMR data of Obtusifolate A(102) (δ=chemical shift in ppm) 66 3.6 1HNMR and 13CNMR data of Obtusifolate B(103), (δ=chemical shift in ppm) 70 3.7 1HNMR and 13CNMR data of Obtusifolate C(104), (δ=chemical shift in ppm) 74 3.8 1HNMR and 13CNMR data of Obtusifolate D(105), (δ=chemical shift in ppm) 77 Percentage of RSA of compounds 102, 103, 104 and 105 from Rumex 3.9 78 Obtusifolius 4.1 Sub-fractionation of Dichloromethane soluble fraction 84 FIGURES AND SCHEMES Figure and scheme Page Scheme 2.1:Polyketide pathway for anthraquinone biosynthesis 48 Scheme 2.2:Shikimate pathway for anthraquinone biosynthesis 49 Fig 3.1. % Inhibition of fungi of sub-fractions of DCM fraction of 57 Rumex obtusifolius Fig No: 3.2. Illustration of percentage mortality of brine shrimps at 60 different concentrations of sub-fractions. Fig 3.3. Illustration of respective LD values of brine shrimps at 50 61 different concentrations of sub-fractions Fig 3.4 % DPPH inhibibition zone of compound the four isolated 79 compounds from Rumex Obtusifolius Scheme 4.1 Extraction and Fraction of R. obtusifolius 89 Scheme 4.2 Chromatographic resolution on Si-gel column eluted 90 with n-hexane, DCM and EtOAs as solvent system. Dedication Dedicated to my parents (Late) and brothers, their foresight, unconditional support and values paved the way for a privileged education. Acknowledgements First of all I bow down my head to the Omnipotent, the most Merciful, the Compassionate, and the Omniscient Al-Mighty ALLAH, whose clemency resulted into my success. I wish to pay homage to the most perfect personality of the world Hazrat Muhammad (PBUH), who enlightened our minds to recognize our Creator. Firstly I would like to express my feelings of gratitude for the kind support and persistent encouragement of my ever smiling supervisor Dr. Shafiullah Khan. I am very thankful to him for his kind, cool calm and nice behaviour. I am pleased to acknowledge Dr. Shafiullah Khan, Director of the Institute of Chemical Sciences for providing me with all the facilities to complete this task. Due to his tremendous efforts and dynamic effort for research facilities the Institute is now progressing day by day and the institute which was formerly Department of Chemistry got the name of institute of chemical sciences. I would also like to acknowledge the contributions of Dr. Kamran khan, Dr. Syed Badsha, Dr. Saed Ahmad and Dr. Hidayat Ullah for their encouraging and supporting behavior. I am extremely thankful to Dr. Afzal Shah (Chairman Chemistry Department UST Bannu) for providing lab space and Dr. Mushtaq Ahmad (Assistant Prof. UST, Bannu) for helping me in carrying out the biological activities. I am extremely thankful to Beijing University of Chemical Technology China for helping in characterization of the compounds. I like to express my gratitude to friends especially, Dr. Farman ullah khan and Mr. Fada Khan for their nice company and kind support during the study. Thanks to all technical and non-technical staff of the Department of Chemistry especially to Hafiz Aziz and Dr. Naqeeb Ullah for their assistance. Also, thank you to all the academic staff. ABDUL KHABIR KHAN Summary The present Ph.D. thesis deals with the phytochemical screening on the constituents of Rumex Obtusifolius. Rumex obtusifolius is commonly known as ‘broad-leaf dock’. Itis perennial Herb that grows to a height of 50 to 130 cm. R. obtusifolius occurs along with its very close relative’s R. longifolius and R. crispus. It is widely distributed in ditches, wetlands, riparian areas, roadsides, meadows, waste grounds, disturbed damp areas and pasture fields. This plant is very important in research point of view because of its traditional uses in medicine in several countries of South America. According to folk medicine this plant’s root has a prominent detoxifying result on the liver and is used against fever, jaundice, and as an anti- anemic tonic. The roots are also laxative. In addition, the leaves of this Rumex are used against hepatic, dermatological and eye problems. They are functional in the relief of furuncles, bruises and are also used as antiseptic and as scar healer. It is used as an antidote to nettle, astringent, depurative, tonic and laxative. It is also used for treatment of tumors, blisters, sores, burns and cancer. The thesis is presented in following two parts. Part-A: Biological screeing The present study has been carried out to investigate the antibacterial, antifungal and cytotoxic screening of sub-fractions of dichloromethane.