Phytochemical Studies on the Polar Fractions of Plants Belonging to The
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Department of Environmental Biology PhD in Environmental and Evolutionary Biology XXX cycle - Curriculum Botany Phytochemical studies on the polar fractions of plants belonging to the Lamiaceae family with aspects of chemotaxonomy, ethno-pharmacology, nutraceutics and phytochemical evolution PhD student: Dr. Claudio Frezza Student identification number: 1172494 Supervisor: Prof. Mauro Serafini Co-Supervisor: Prof. Armandodoriano Bianco Academic year 2016/2017 1 “Nature is not a place to visit. It is home.” Gary Snyder 2 Index Front page 1 Literature citation 2 Index 3 1. Introduction 6 1.1. Lamiaceae 6 1.1.1. General 6 1.1.2. Botany 6 1.1.3. Phytochemistry 9 1.1.4. Chemotaxonomy 13 1.1.5. Ethno-pharmacology 14 1.1.6. Nutraceutics 15 1.1.7. Phylogeny 16 1.2. Studied sub-families and species 17 1.2.1. General 17 1.2.2. Ajugoideae and Lamioideae 17 1.2.3. List of studied species 19 1.2.4. Botanical characteristics of the studied species 20 1.2.4.1. Ajuga chamaepitys (L.) Schreb. 20 1.2.4.2. Ajuga genevensis L. 20 1.2.4.3. Ajuga reptans L. 21 1.2.4.4. Ajuga tenorei C. Presl - leaves 22 1.2.4.5. Galeopsis ladanum subsp. angustifolia (Ehrh. ex Hoffm.) Gaudin 23 1.2.4.6. Melittis melissophyllum subsp. albida (Guss.) P.W. Ball 24 1.2.4.7. Melittis melissophyllum L. subsp. melissophyllum 24 1.2.4.8. Sideritis montana L. subsp. montana 25 1.2.4.9. Sideritis romana L. 25 1.2.4.10. Stachys affinis Bunge 26 1.2.4.11. Stachys alopecuros (L.) Benth. subsp. divulsa (Ten.) Grande 27 1.2.4.12. Stachys annua L. 27 1.2.4.13. Stachys germanica subsp. salviifolia L. (Ten.) Gams. 28 1.2.4.14. Stachys palustris L. 29 1.2.4.15. Teucrium chamaedrys L. 30 1.2.4.16. Teucrium polium L. 30 1.2.5. Phytochemistry of Ajugoideae and Lamioideae and related studied 31 genera and species 1.2.6. Chemotaxonomy of Ajugoideae and Lamioideae and related studied 36 genera and species 1.2.7. Ethno-pharmacology of Ajugoideae and Lamioideae and related 37 studied genera and species 1.2.8. Nutraceutics of Ajugoideae and Lamioideae and related studied 40 genera and species 1.2.9. Phylogeny of Ajugoideae and Lamioideae and related studied 41 genera and species 2. Aims and objectives 43 3. Results and discussion 44 3.1. General 44 3.2. Phytochemistry 44 3.2.1. Ajuga chamaepitys (L.) Schreb. - aerial parts 44 3 3.2.2. Ajuga chamaepitys (L.) Schreb. - roots 46 3.2.3. Ajuga genevensis L. 47 3.2.4. Ajuga reptans L. 48 3.2.5. Ajuga tenorei C. Presl 49 3.2.6. Galeopsis ladanum subsp. angustifolia (Ehrh. ex Hoffm.) Gaudin 50 3.2.7. Melittis melissophyllum subsp. albida (Guss.) P.W. Ball 51 3.2.8. Melittis melissophyllum L. subsp. melissophyllum 52 3.2.9. Sideritis montana L. subsp. montana 52 3.2.10. Sideritis romana L. 53 3.2.11. Stachys affinis Bunge - rhizomes 55 3.2.12. Stachys alopecuros (L.) Benth. subsp. divulsa (Ten.) Grande 56 3.2.13. Stachys annua L. 56 3.2.14. Stachys germanica subsp. salviifolia L. 57 3.2.15. Stachys palustris L. - Hungary 58 3.2.16. Stachys palustris L. - France 59 3.2.17. Teucrium chamaedrys L. 60 3.2.18. Teucrium polium L. - Northern Iran 61 3.2.19. Teucrium polium L. - Southern Iran 64 3.3. Chemotaxonomy 65 3.4. Ethno-pharmacology 72 3.5. Nutraceutics 79 3.6. Phytochemical evolution 82 4. Conclusions 97 5. Experimental 100 5.1. Plant materials 100 5.2. Chemicals 102 5.3. Instrumentation 102 5.4. Methodology 103 5.4.1. Extraction of metabolites 103 5.4.2. Separation of metabolites 104 5.4.2.1. Column Chromatography 104 5.4.2.2. Preparation of acidic silica gel 105 5.4.2.3. HPLC 106 5.4.3. Active charcoal treatment 107 5.4.4. Identification of metabolites 109 5.4.4.1. NMR Spectroscopy 109 5.4.4.2. Mass Spectrometry 111 5.4.5. Preparation of the samples 115 5.4.5.1. For Column Chromatography 115 5.4.5.2. For HPLC analysis 116 5.4.5.3. For NMR analysis 117 5.4.5.4. For MS analysis 118 5.5. Supplementary Material 118 5.5.1. Ajuga chamaepitys (L.) Schreb. - aerial parts 119 5.5.2. Ajuga chamaepitys (L.) Schreb. - roots 124 5.5.3. Ajuga genevensis L. 128 5.5.4. Ajuga reptans L. 134 5.5.5. Ajuga tenorei C. Presl - leaves 145 5.5.6. Galeopsis ladanum subsp. angustifolia (Ehrh. ex Hoffm.) Gaudin 148 5.5.7. Melittis melissophyllum subsp. albida (Guss.) P.W. Ball 152 4 5.5.8. Melittis melissophyllum L. subsp. melissophyllum 157 5.5.9. Sideritis montana L. subsp. montana 163 5.5.10. Sideritis romana L. 169 5.5.11. Stachys affinis Bunge - rhizomes 177 5.5.12. Stachys alopecuros (L.) Benth. subsp. divulsa (Ten.) Grande 180 5.5.13. Stachys annua L. 185 5.5.14. Stachys germanica subsp. salviifolia L. (Ten.) Gams. 188 5.5.15. Stachys palustris L. - Hungary 191 5.5.16. Stachys palustris L. - France 193 5.5.17. Teucrium chamaedrys L. 193 5.5.18. Teucrium polium L. - Northern Iran 198 5.5.19. Teucrium polium L. - Southern Iran 207 6. References 217 Acknowledgements and thanks 242 5 1. Introduction 1.1. Lamiaceae 1.1.1. General Lamiaceae or Labiatae is a family of plants comprised in the Lamiales order, in the Euasterids I clade of the Asterids class, in the Eudicots type of the Angiosperms group, in the Magnoliophyta division of the Spermatophyta superdivision (Pignatti, 1982). The first citation of this family is dated back to the Latin writer and naturalist Gaius Pliny Secundus who also indicated the etymology of this word deriving from its most typical genus Lamium and descending from the greek term “λάιμος” (Laimos) meaning “jaws” and referring to the fact that the flowers of this plants are able to swallow small insects after they enter the corolla searching for its nectar. Actually, the scientific name was coined in 1789 by Antoine Laurent de Jussieu in one of his works entitled "Genera Plantarum - 110" where he used for the first time the word Labiatae (De Jussieu, 1789). In 1820, this name was changed into Lamiaceae by the Russian botanist Ivan Ivanovic Martinov in his work "Tekhno-Botanicheskii Slovar': na latinskom i rossiiskom i-azykakh. Sanktpeterburgie - 355" and nowadays both names are generally accepted even if Lamiaceae is considered to be more correct (Martinov, 1820). 1.1.2. Botany From the botanical standpoint, species belonging to this family are characterized by an annual or perennial carriage (Figure 1). The main biological forms are emicryptophyte and therophyte even though chamaephyte, nanofanerophyte, bushy, arboreal and shrubby species are also present. The indumentum has a simple pubescence with glandular and non-glandular trichomes. The stem is principally with a quadrangular section due to the presence of big bundles of collenchyma at its four vertices. No spines are present (Conti et al., 2005; Pignatti, 1982). 6 Figure 1: Example of the family carriage: Scutellaria altissima L. The leaves are opposite and decussate or verticillated along the stem. They can be sessile or petiolate with a very variable shape ranging from lanceolate to ovate with entire or dentate margins (Figure 2) (Conti et al., 2005; Pignatti, 1982). Figure 2: Example of the family leaves: Collinsonia canadensis L. The flowers are hermaphrodite and zygomorphous or actinomorphic. They are tetramers or pentamers and generally collected in verticillasters. Each of them is constituted by many flowers (from 2 to 20) and is located circularly on two big leafy bracts which present an alternate disposition. These bracts are subulate or spinose and are densely colored. Bracteoles may be present, too. The floreal formula is X, K (5), [C (2+3), A 2+2] G (2), (superior), drupe, four nuculae. The calyx is gamosepalous and more or less actinomorphic. It is constituted by five equal straight lobes which can be lanceolate or triangular. The tube of the calex has about 10 superficial venations. 7 The corolla is gamopetalous and zygomorphous with the shape of a dilated pipe in the distal part ending with two lips having five lobes. The upper one is short and presents a hood shape and generally an entire margin. The lower one is flattened and is formed by three lobes with the central one bigger then the other two. Sometimes, it is fully pubescent. Its colors are very variable. The androecium is formed by four fertile didinamous stamens included or protruding form the corolla and located under the lower lip. The anthers are close, paired and pubescent. The ovary is superior and formed by two sealed carpels. The placentation is axyle. There are four ovules with tegument and kernel. The stylus is inserted into the base of the ovary. The stigma is bifid with equal lobes (Figure 3) (Conti et al., 2005; Pignatti, 1982). Figure 3: Example of the family inflorescence: Amasonia campestris (Aubl.) Moldenke The fruit is a schizocarp composed by four dry and fleshy nuculae. These are also rounding at the apex. The surface is smooth, glabrous or pubescent or tuberculate. The fruit is able to release semen without endosperm with extreme readiness (Figure 4) (Conti et al., 2005; Pignatti, 1982). Figure 4: Example of the family fruit: Callicarpa japonica Thunb. 8 In general, Lamiaceae can be found everywhere all over the planet even if some species are endemic (Figure 5).