University of Bath PHD Phytochemical and biological activity studies on Salvia viridis L Rungsimakan, Supattra Award date: 2011 Awarding institution: University of Bath Link to publication Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 09. Oct. 2021 Phytochemical and biological activity studies on Salvia viridis L. Supattra Rungsimakan A thesis submitted for the degree of Doctor of Philosophy University of Bath Department of Pharmacy and Pharmacology November 2011 Copyright Attention is drawn to the fact that copyright of this thesis rests with the author. A copy of this thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that they must not copy it or use material from it except as permitted by law or with the consent of the author. Restrictions on use This thesis may be made available for consultation within the University Library and may be photocopied or lent to other libraries for the purposes of consultation. Signature……………………………………………….. Table of contents Title i Abstract vi Acknowledgements vii List of abbreviations viii Chapter 1 Introduction 1 1.1 Terpenoids in the genus Salvia 2 1.2 Polyphenols in the genus Salvia 15 1.3. Biological activities of compounds in the genus Salvia 23 1.4 Investigated plant: Salvia viridis L. 48 Aims of this study 51 Chapter 2 Experimental 52 2.1 Plant materials 52 2.2 Materials 52 2.3 Thin layer chromatography (TLC) 52 2.3.1 TLC chemoautographic method: 54 DPPH radical scavenging activity 2.3.2 Antibacterial activity 54 2.3.3 Acetylcholinesterase inhibitory activity 56 2.4 High performance liquid chromatography (HPLC) 57 2.5 Spectroscopic examinations 57 2.6 Extraction procedures of aerial part 58 2.7 Chromatography of aerial part 60 2.8 Extraction procedures of root part 64 2.9 Chromatography of root part 64 2.10 Spectroscopic data of isolated compounds from aerial part 68 2.11 Spectroscopic data of isolated compounds from root part 78 Chapter 3 Results and discussions 83 3.1 TLC patterns of crude fractions and their detection 83 ii 3.2 TLC-chemoautographic method: 88 DPPH radical scavenging activity 3.3 Antibacterial activity 90 3.3.1 TLC-bioautographic method: agar overlay technique 90 3.3.2 Minimal inhibitory concentration 93 3.4 Acetylcholinesterase inhibitory activity 97 3.5 Separation compounds from aerial part 99 3.5.1 HPLC of aerial part crude fraction 99 3.5.2 Open column chromatography of aerial part crude fraction 101 3.6 Structure elucidations of compounds from aerial part 102 3.6.1 Caffeic acid derivatives 102 3.6.1.1 Caffeic acid 102 3..6.1.2 Trans-verbascoside 105 3.6.1.3 Cis-verbascoside 116 3.6.1.4 Leucosceptoside A 120 3.6.1.5 Martynoside 123 3.6.1.6 Rosmarinic acid 127 3.6.1.7 6-O-Caffeoyl-glucose 130 3.6.2 Flavonoid glycosides 133 3.6.2.1 Luteolin-7-O-β-glucopyranoside 133 3.6.2.2 Luteolin-7-O-β-galactopyranoside 136 3.6.2.3 Luteolin-7-O-rutinoside 139 3.6.2.4 Compound 6 (luteolin-7-O-α-rhamnopyranosyl- 143 (1→6)-β-galactopyranoside) 3.6.2.5 Apigenin-7-O-glucopyranoside 148 3.6.3 Triterpenoids 151 3.6.3.1 Ursolic acid 151 3.6.3.2 Oleanolic acid 158 3.6.3.3 Lup-20(29)-ene-2α-3β-diol 163 3.6.3.4 Compound 1 (lup-20(29)-ene-2α-acetate-3β-ol) 171 3.6.3.5 Compound 2 (lup-20(29)-ene-2α-ol-3β-acetate) 177 3.6.3.6 β-Sitosterol 186 3.6.3.7 β-Sitosterol glucoside 191 3.6.4 Miscellaneous 196 iii 3.6.4.1 Salidroside 196 3.7 Separation compounds from root part 199 3.7.1 Open column chromatography and HPLC of root part crude 199 fractions 3.7.2 Open column chromatography of root part crude fractions 201 3.8 Structure elucidations of compounds from root part 201 3.8.1 Caffeic acid derivatives 201 3.8.1.1 2'',3''-Di-O-acetyl-martynoside 201 3.8.1.2 1-Docosyl ferulate 205 3.8.2 Diterpenoids 208 3.8.2.1 Ferruginol 208 3.8.2.2 Salvinolonyl-12-methyl ether 214 3.8.2.3 7α-Acetoxy-14-hydroxy-8,13-abietadiene-11,12-dione 219 3.8.2.4 7α,14-Dihydroxy-8,13-abietadiene-11,12-dione 224 3.8.2.5 Microstegiol 229 3.8.2.6 Compound 3 (1-oxomicrostegiol) 234 3.8.2.7 Compound 4 (viroxocane) 240 3.8.2.8 Compound 5 (viridoquinone) 246 3.8.3 Miscellaneous 252 3.8.3.1 A mixture of 2-(4'-alkoxyphenyl) ethyl alkanoates 252 Chapter 4 In vitro testing 255 4.1 Introduction 255 4.2 Materials 258 4.3 Experimental 259 4.4 Results and discussions 263 4.4.1 MTT assay 265 4.4.2 Neutral red assay 269 4.4.3 Flow cytometric analysis 271 4.5 In vitro testing conclusions 275 Chapter 5 Conclusions 277 References 282 iv Appendices 310 Appendix 1 NMR spectra of caffeic acid 310 Appendix 2 NMR spectra of trans-verbascoside 313 Appendix 3 NMR spectra of a mixture of trans- and cis-verbascoside 316 Appendix 4 NMR spectra of leucosceptoside A 318 Appendix 5 NMR spectra of martynoside 321 Appendix 6 NMR spectra of rosmarinic acid 324 Appendix 7 NMR spectra of 6-O-caffeoyl-glucose 327 Appendix 8 NMR spectra of luteolin-7-O-glucopyranoside 330 Appendix 9 NMR spectra of luteolin-7-O-galactopyranoside 333 Appendix 10 NMR spectra of luteolin-7-O-rutinoside 336 Appendix 11 NMR spectra of compound 6 (luteolin-7-O-α-rhamnopyranosyl- 339 (1→6)-β-galactopyranoside) Appendix 12 NMR spectra of apigenin-7-O-glucopyranoside 342 Appendix 13 NMR spectra of ursolic acid 345 Appendix 14 NMR spectra of oleanolic acid 348 Appendix 15 NMR spectra of lup-20(29)-ene-2α-3β-diol 351 Appendix 16 NMR spectra of compound 1 (lup-20(29)-ene-2α-acetate-3β-ol) 354 Appendix 17 NMR spectra of compound 2 (lup-20(29)-ene-2α-ol-3β-acetate) 357 Appendix 18 NMR spectra of β-sitosterol 360 Appendix 19 NMR spectra of β-sitosterol glucoside 363 Appendix 20 NMR spectra of salidroside 366 Appendix 21 NMR spectra of 2'', 3''-di-O-acetyl-martynoside 369 Appendix 22 NMR spectra of 1-docosyl ferulate 372 Appendix 23 NMR spectra of ferruginol 375 Appendix 24 NMR spectra of salvinolonyl-12-methyl-ether 378 Appendix 25 NMR spectra of 7α-acetoxy-14-hydroxy-8,13-abietadiene-11,12-dione 381 Appendix 26 NMR spectra of 7α,14-dihydroxy-8,13-abietadiene-11,12-dione 384 Appendix 27 NMR spectra of microstegiol 387 Appendix 28 NMR spectra of compound 3 (1-oxomicrostegiol) 390 Appendix 29 NMR spectra of compound 4 (viroxocane) 393 Appendix 30 NMR spectra of compound 5 (viridoquinone) 396 Appendix 31 NMR spectra of a mixture of 2-(4'-alkoxyphenyl) ethyl alkanoates 399 Appendix 32 Abstracts and presentations from this work 402 v Abstract Six new compounds were isolated from the aerial and root parts of S. viridis L. cv. Blue Jeans. Two new triterpenoids, lup-20(29)-ene-2α-acetate-3β-ol, and lup-20(29)-ene-2α- ol-3β-acetate were found in the aerial part together with lup-20(29)-ene-2α-3β-diol, ursolic acid, oleanolic acid, β-sitosterol and β-sitosterol glucoside. Three new diterpenoids, 1- oxomicrostegiol, viroxocane, viridoquinone, together with five known diterpenoids, ferruginol, salvinolonyl 12-methyl ether, microstegiol, 7α-acetoxy-14-hydroxy-8,13- abietadiene-11,12-dione and 7α,14-dihydroxy-8,13-abietadiene-11,12-dione were found in roots. 1-Docosyl ferulate, 2'',3''-di-O-acetyl-martynoside and a mixture of 2-(4'-alkoxy- phenyl) ethyl alkanoates were also isolated from roots. Seven caffeic acid derivatives, five flavonoid glycosides, and salidroside were found in the crude aerial fraction. Four caffeic acid derivatives were known phenylpropanoids, i.e. trans-, cis-verbascoside, leucosceptoside A and martynoside, which are now reported in the genus Salvia for the first time. The others were caffeic acid, rosmarinic acid and 6-O-caffeoyl-glucose. A new flavonoid glycoside, luteolin-7-O-α-rhamnopyranosyl-(1→6)-β-galactopyranoside was also identified in the aerial part with four known flavone glycosides: luteolin-7-O-β-glucopyranoside, luteolin-7-O-β- galactopyranoside, luteolin-7-O-rutinoside and apigenin-7-O-β-glucopyranoside. Verbascoside (acteoside), which is a major component in this plant, showed a significant protective effect against UVA induced damage in a human skin fibroblast model in vitro. It exhibited 1.4 fold protective effect against UVA induced necrosis with 1.4 fold higher in cell survival.