Chemistry of natural compounds: educational manual 9786010446083

Table of contents :
Liquid chromatography
Gas chromatography
Picture 1.2. Thin layer chromatography and Rf calculations
Column chromatography
Flavanonols
Flavans
Anthocyanidins
Anthocyanidins are the aglycones of anthocyanins; they use the(2-phenylchromenylium) ion skeleton
Cyanidin, Delphinidin, Malvidin, Pelargonidin, Peonidin, Petunidin
/
3.2. Properties of alkaloids and Distribution in nature
Distribution in nature
Strychnine tree. Its seeds are rich in strychnine and brucine.Alkaloids are generated by various living organisms, especially by higher plants – about 10 to 25% of those contain alkaloids. Therefore, in the past the term «alkaloid» was associated with...
The alkaloids content in plants is usually within a few percent and is inhomogeneous over the plant tissues. Depending on the type of plants, the maximum concentration is observed in the leaves (black henbane), fruits or seeds (Strychnine tree),/ root...
Scheme 3.1. Isolation of alkaloid from plant material
3.4. Dimer alkaloids
In addition to the described above monomeric alkaloids, there are also dimeric, and even trimeric and tetrameric alkaloids formed upon condensation of two, three, and four monomeric alkaloids.
Dimeric alkaloids are usually formed from monomers of the same type through the following mechanisms:
3.5. Applications of alkaloids
In medicine
In agriculture
Laboratory work 6
Obtaining caffeine from tea in laboratory
105. Stein R.,Fahl K. Biomarker proxy shows potential for studying the entire Quaternary Arctic sea ice // Org Geochem.-2013.-№55,-p.98.
106. Tabatadze N, Bun S-S, Tabidze B, Mshvildadze V, Dekanosidze G, Ollivier E, Elias R. New triterpenoid saponins from Leontice smirnowii. Fitoterapia, 2010. – №81, – p.897-901.
121. Kleemann G .,Kellner R., Poralla K.Purification and properties of the squalene-hopene cyclase from Rhodopseudomonas palustris, a purple non-sulfur bacterium producing hopanoids and tetrahymanol//J Gen Microbiol. – 1990. – №136, – p. 2551.
122. Sinninghe Damste J.S.,Kenig F., Koopmans M.P., Koster J., Schouten S., Hayes J.M., de Leeuw J.W. Evidence for gammacerane as an indicator of water column stratification// Geochim Cosmochim Acta. –1995. – №59, – p. 1895.
123. Yang H.,Chen D., Cui Q.C., Yuan X., Dou Q.P.Celastrol, a triterpene extracted from the Chinese "Thunder of God Vine," is a potent proteasome inhibitor and suppresses human prostate cancer growth in nude mice//Cancer Res. – 2006. – №66, – p. 4758.
124. Chen M, Wu WW, Sticher O, Nanz D. Leonticins A−C, Three octasaccharide saponins from Leontice kiangnanensis // Journal of Natural Products, – 1996. – № 59, – p722-728.
125. Reif C., Arrigoni E., Schärer H., Nyström L., Hurrell R.F. Carotenoid database of commonly eaten Swiss vegetables and their estimated contribution to carotenoid intake // J. Food Compos. Anal. – 2013. – Vol 29, – p.64-72.
126. Hughes D. A. Effects of carotenoids on human immune function //Proc Nutr Soc. – 1999. – №58, – p.713-718.
127. Kavanagh C.J.,Trumbo P.R., Ellwood K.C. The U.S. Food and Drug Administration's evidence-based review for qualified health claims: tomatoes, lycopene, and cancer // J Natl Cancer Inst.-2007, – Vol 99, – p.1074.
128. Stanley J.C., Folch J., Lees M. A simple method for the isolation and purification of total lipids from animal tissues // Lipid technol. – 2008. – №20, – p.64.
129. Aust O.,Stahl W., Sies H., Tronnier H., Heinrich U.Supplementation with tomato-based products increases lycopene, phytofluene, and phy-toene levels in human serum and protects against UV-light-induced erythema / Int. J VitamNutr Res. – 2005, – Vo...
130. Handelman G.J. Nightingale Z.D., Lichtenstein A.H., Schaefer E.J., Blumberg J.B. Lutein and zeaxanthin concentrations in plasma after dietary supplementation with egg yolk // Am J ClinNutr. – 1999, – Vol 70, – p. 247.
131. Nimalaratne C., Lopes-Lutz D., Schieber A., Wu J. Effect of domestic cooking methods on egg yolk xanthophylls // J Agric Food Chem. – 2012. – №60, – p.12547.
132. Krinsky N.I.,Landrum J.T., Bone R.A.Biologic mechanisms of the protective role of lutein and zeaxanthin in the eye // Annu Rev Nutr. – 2003, – Vol 23, – p.171.
133. Olmedilla B., Granado F., Blanco I., Vaquero M.Lutein, but not alpha-tocopherol, supplementation improves visual function in patients with age-related cataracts: a 2-y double-blind, placebo-controlled pilot study // Nutrition. – 2003, – Vol 19, –...
134. Wagener S., Völker T., De Spirt S., Ernst H., Stahl W.3,3 – Dihydroxyi-sorenieratene and isorenieratene prevent UV-induced DNA damage in human skin fibroblasts // Free Rad Biol Med. – 2012. – № 53, – p.457.
135. Bennedsen M.,Wang X., Willén R., Wadström T., Andersen L.P. Treat-ment of H. pylori infected mice with antioxidant astaxanthin reduces gastric inflammation, bacterial load and modulates cytokine release by splenocytes // ImmunolLett.-1999, №70,-p...
136. Chew B.P.,Park J.S., Wong M.W., Wong T.S.A comparison of the anticancer activities of dietary beta-carotene, canthaxanthin and asta-xanthin in mice in vivo // Anticancer Res. – 1999, – Vol 19, – p.1849.
137. Park J.S., Chyun J.H., Kim Y.K., Line L.L., Chew B.P. Astaxanthin decreased oxidative stress and inflammation and enhanced immune response in humans // Nutr Metabol. – 2010, – Vol 7, – p.18.
138. Takaichi S., Mochimaru M. Carotenoids and carotenogenesis in cyano-bacteria: unique ketocarotenoids and carotenoid glycosides // Cell Mol Life Sci. – 2007. – №64, – p.2607.
139. Maoka T.,Akimoto N., Kuroda Y., Hashimoto K., Fujiwara Y. Pittospo-rumxanthins, cycloaddition products of carotenoids with alphatocophe-rol from seeds of Pittosporum tobira // J Nat Prod.-2008.-№71, – p. 622.
140. Maeda H.,Hosokawa M., Sashima T., Funayama K., Miyashita K. Fuco-xanthin from edible seaweed, Undaria pinnatifida, shows antiobesity effect through UCP1 expression in white adipose tissues // Biochem Biophys Res Comm. – 2005, – №332, – p.392.
141. Tsukui T.,Konno K., Hosokawa M., Maeda H., Sashima T., Miyashita K.Fucoxanthin and fucoxanthinol enhance the amount of doco-sahexaenoic acid in the liver of KKAy obese/diabetic mice // J Agric Food Chem. – 2007. – №55, – p.5025.
142. Tsuboi M.,Etoh H., Kato K., Nakatugawa H., Kato H., Maejima Y., Matsumoto G., Mori H., Hosokawa.M, Miyashita K., Tokuda H., Suzuki N., Maoka T
143. Dembitsky V.M.,Maoka T. Allenic and cumulenic lipids //Prog Lipid Res. – 2007. – №46, –p.328.
144. Pott I.,Breithaupt D.E., Carle R.Detection of unusual carotenoid esters in fresh mango (Mangifera indica L. cv. 'Kent') //Phytochemistry. – 2003. – №64, – p. 825.
145. Rogers E.W., Molinski T.F. A cytotoxic carotenoid from the marine sponge Prianos osiros // J Nat Prod. – 2005. – №68, – p. 450.
146. Hartgers W.A., Sinninghe Damsté J.S., Requejo A.G., Allan J., Hayes J.M., Ling Y., Xie T.M., Primack J., De Leeuw J.W. A molecular and carbon isotopic study towards the origin and diagenetic fate of diaromatic carotenoids // Organic Geochem.-1994...
147. Lutnaes B.F., New C(40)-carotenoid acyl glycoside as principal carotenoid in Salinibacter ruber, an extremely halophilic eubacterium // J Nat prod. – 2002. – №65, – p.1340.
148. Maoka T.,Akimoto N.Carotenoids and their fatty acid esters of spiny lobster Panulirus japonicas // J Oleo Sci.-2008,-Vol 57, – p.145.
149. FujiFlora Kazakhstan, Almaty, 1963. Т.3. – P. 204-213.
150. Xu X., Konirbay B., Jenis J., etal. The Kazakh Materia Medica / The Ethnic Press: Beijing, – 2009. – P.357.
151. Jenis, J., Burasheva G.Sh., Aisa H. Ba-hang; Abilov Zh.A. Study on Chemical Constitutes of the Volatiles from Atriplex tataricwara Y., Hashimoto K., Manabe K., Maoka T. Tetrahedron Lett. – 2002, –Vol 43, – p.4385.
123-133.pdf
121. Stein R., Fahl K. Biomarker proxy shows potential for studying the entire Quaternary Arctic sea ice // Org Geochem. – 2013. – N55. – P. 98.
122. Tabatadze N., Bun S-S., Tabidze B., Mshvildadze V., Dekanosidze G., Ollivier E., Elias R. New triterpenoid saponins from Leontice smirnowii. Fitoterapia. – 2010. – N 81. – P. 897-901.
137. Kleemann G., Kellner R., Poralla K. Purification and properties of the squalene-hopene cyclase from Rhodopseudomonas palustris, a purple non-sulfur bacterium producing hopanoids and tetrahymanol //J Gen Microbiol. – 1990. – N136. – P. 2551.
138. Sinninghe Damste J.S., Kenig F., Koopmans M.P., Koster J., Schouten S., Hayes J.M., de Leeuw J.W. Evidence for gammacerane as an indicator of water column stratification // Geochim Cosmochim Acta. –1995. – N 59. – P. 1895.
139. Yang H., Chen D., Cui Q.C., Yuan X., Dou Q.P. Celastrol, a triterpene extracted from the Chinese «Thunder of God Vine» is a potent proteasome inhibitor and suppresses human prostate cancer growth in nude mice // Cancer Res. – 2006. – N 66. – P. 4...
140. Chen M., Wu W.W., Sticher O., Nanz D. Leonticins A-C., Three octasaccharide saponins from Leontice kiangnanensis // Journal of Natural Products. – 1996. – N 59. – P. 722-728.
141. Reif C., Arrigoni E., Schärer H., Nyström L., Hurrell R.F. Carotenoid database of commonly eaten Swiss vegetables and their estimated contribution to carotenoid intake // J. Food Compos. Anal. – 2013. – Vol. 29. – P. 64-72.
142. Hughes D.A. Effects of carotenoids on human immune function // Proc Nutr Soc. – 1999. – N 58. – P. 713-718.
143. Kavanagh C.J., Trumbo P.R., Ellwood K.C. The U.S. Food and Drug Administration's evidence-based review for qualified health claims: tomatoes, lycopene, and cancer // J Natl Cancer Inst. – 2007. – Vol. 99. – P. 1074.
144. Stanley J.C., Folch J., Lees M. A simple method for the isolation and purification of total lipids from animal tissues // Lipid technol. – 2008. – N 20. – P. 64.
145. Aust O., Stahl W., Sies H., Tronnier H., Heinrich U. Supplementation with tomato-based products increases lycopene, phytofluene, and phy-toene levels in human serum and protects against UV-light-induced erythema / Int. J VitamNutr Res. – 2005. – ...
146. Handelman G.J. Nightingale Z.D., Lichtenstein A.H., Schaefer E.J., Blumberg J.B. Lutein and zeaxanthin concentrations in plasma after dietary supplementation with egg yolk // Am J ClinNutr. – 1999. – Vol. 70. – P. 247.
147. Nimalaratne C., Lopes-Lutz D., Schieber A., Wu J. Effect of domestic cooking methods on egg yolk xanthophylls // J Agric Food Chem. – 2012. – N 60. – P. 12547.
148. Krinsky N.I., Landrum J.T., Bone R.A.Biologic mechanisms of the protective role of lutein and zeaxanthin in the eye // Annu Rev Nutr. – 2003. – Vol. 23. – P.171.
149. Olmedilla B., Granado F., Blanco I., Vaquero M.Lutein, but not alpha-tocopherol, supplementation improves visual function in patients with age-related cataracts: a 2-y double-blind, placebo-controlled pilot study // Nutrition. – 2003. – Vol. 19. ...
150. Wagener S., Völker T., De Spirt S., Ernst H., Stahl W. 3,3 – Dihydroxyi-sorenieratene and isorenieratene prevent UV-induced DNA damage in human skin fibroblasts // Free Rad Biol Med. – 2012. – N 53. – P. 457.
151. Bennedsen M.,Wang X., Willén R., Wadström T., Andersen L.P. Treat-ment of H. pylori infected mice with antioxidant astaxanthin reduces gastric inflammation, bacterial load and modulates cytokine release by splenocytes // ImmunolLett. – 1999. – N ...
152. Chew B.P., Park J.S., Wong M.W., Wong T.S. A comparison of the anticancer activities of dietary beta-carotene, canthaxanthin and asta-xanthin in mice in vivo // Anticancer Res. – 1999. – Vol. 19. – P.1849.
153. Park J.S., Chyun J.H., Kim Y.K., Line L.L., Chew B.P. Astaxanthin decreased oxidative stress and inflammation and enhanced immune response in humans // Nutr Metabol. – 2010. – Vol. 7. – P.18.
154. Takaichi S., Mochimaru M. Carotenoids and carotenogenesis in cyano-bacteria: unique ketocarotenoids and carotenoid glycosides // Cell Mol Life Sci. – 2007. – N64. – P. 2607.
155. Maoka T., Akimoto N., Kuroda Y., Hashimoto K., Fujiwara Y. Pittospo-rumxanthins, cycloaddition products of carotenoids with alphatocophe-rol from seeds of Pittosporum tobira // J Nat Prod. – 2008. – N 71. – P. 622.
156. Maeda H., Hosokawa M., Sashima T., Funayama K., Miyashita K. Fuco-xanthin from edible seaweed, Undaria pinnatifida, shows antiobesity effect through UCP1 expression in white adipose tissues // Biochem Biophys Res Comm. – 2005. – N 332. – P. 392.
157. Tsukui T., Konno K., Hosokawa M., Maeda H., Sashima T., Miyashita K.Fucoxanthin and fucoxanthinol enhance the amount of doco-sahexaenoic acid in the liver of KKAy obese/diabetic mice // J Agric Food Chem. – 2007. – N 55. – P. 5025.
158. Tsuboi M., Etoh H., Kato K., Nakatugawa H., Kato H., Maejima Y., Matsumoto G., Mori H., Hosokawa M., Miyashita K., Tokuda H., Suzuki N., Maoka T
159. Dembitsky V.M., Maoka T. Allenic and cumulenic lipids // Prog Lipid Res. – 2007. – N 46. – P. 328.
160. Pott I., Breithaupt D.E., Carle R.Detection of unusual carotenoid esters in fresh mango (Mangifera indica L. cv. 'Kent') // Phytochemistry. – 2003. – N 64. – P. 825.
161. Rogers E.W., Molinski T.F. A cytotoxic carotenoid from the marine sponge Prianos osiros // J Nat Prod. – 2005. – N 68. – P. 450.
162. Hartgers W.A., Sinninghe Damsté J.S., Requejo A.G., Allan J., Hayes J.M., Ling Y., Xie T.M., Primack J., De Leeuw J.W. A molecular and carbon isotopic study towards the origin and diagenetic fate of diaromatic carotenoids // Organic Geochem. – 19...
163. Lutnaes B.F., New C(40)-carotenoid acyl glycoside as principal carotenoid in Salinibacter ruber, an extremely halophilic eubacterium // J Nat prod. – 2002. – N65. – P.1340.
164. Maoka T., Akimoto N. Carotenoids and their fatty acid esters of spiny lobster Panulirus japonicas // J Oleo Sci. – 2008. – Vol. 57. – P.145.
165. FujiFlora Kazakhstan Almaty. – 1963. – P. 204-213. – Т.3.
166. Xu X., Konirbay B., Jenis J., etal. The Kazakh Materia Medica / The Ethnic Press: Beijing. – 2009. – P. 357.
167. Jenis J., Burasheva G.Sh., Aisa H. Ba-hang; Abilov Zh.A. Study on Chemical Constitutes of the Volatiles from Atriplex tataricwara Y., Hashimoto K., Manabe K., Maoka T. Tetrahedron Lett. – 2002. –Vol. 43. – P. 4385.
123-133.pdf
121. Stein R., Fahl K. Biomarker proxy shows potential for studying the entire Quaternary Arctic sea ice // Org Geochem. – 2013. – N55. – P. 98.
122. Tabatadze N., Bun S-S., Tabidze B., Mshvildadze V., Dekanosidze G., Ollivier E., Elias R. New triterpenoid saponins from Leontice smirnowii. Fitoterapia. – 2010. – N 81. – P. 897-901.
137. Kleemann G., Kellner R., Poralla K. Purification and properties of the squalene-hopene cyclase from Rhodopseudomonas palustris, a purple non-sulfur bacterium producing hopanoids and tetrahymanol //J Gen Microbiol. – 1990. – N136. – P. 2551.
138. Sinninghe Damste J.S., Kenig F., Koopmans M.P., Koster J., Schouten S., Hayes J.M., de Leeuw J.W. Evidence for gammacerane as an indicator of water column stratification // Geochim Cosmochim Acta. –1995. – N 59. – P. 1895.
139. Yang H., Chen D., Cui Q.C., Yuan X., Dou Q.P. Celastrol, a triterpene extracted from the Chinese «Thunder of God Vine» is a potent proteasome inhibitor and suppresses human prostate cancer growth in nude mice // Cancer Res. – 2006. – N 66. – P. 4...
140. Chen M., Wu W.W., Sticher O., Nanz D. Leonticins A-C., Three octasaccharide saponins from Leontice kiangnanensis // Journal of Natural Products. – 1996. – N 59. – P. 722-728.
141. Reif C., Arrigoni E., Schärer H., Nyström L., Hurrell R.F. Carotenoid database of commonly eaten Swiss vegetables and their estimated contribution to carotenoid intake // J. Food Compos. Anal. – 2013. – Vol. 29. – P. 64-72.
142. Hughes D.A. Effects of carotenoids on human immune function // Proc Nutr Soc. – 1999. – N 58. – P. 713-718.
143. Kavanagh C.J., Trumbo P.R., Ellwood K.C. The U.S. Food and Drug Administration's evidence-based review for qualified health claims: tomatoes, lycopene, and cancer // J Natl Cancer Inst. – 2007. – Vol. 99. – P. 1074.
144. Stanley J.C., Folch J., Lees M. A simple method for the isolation and purification of total lipids from animal tissues // Lipid technol. – 2008. – N 20. – P. 64.
145. Aust O., Stahl W., Sies H., Tronnier H., Heinrich U. Supplementation with tomato-based products increases lycopene, phytofluene, and phy-toene levels in human serum and protects against UV-light-induced erythema / Int. J VitamNutr Res. – 2005. – ...
146. Handelman G.J. Nightingale Z.D., Lichtenstein A.H., Schaefer E.J., Blumberg J.B. Lutein and zeaxanthin concentrations in plasma after dietary supplementation with egg yolk // Am J ClinNutr. – 1999. – Vol. 70. – P. 247.
147. Nimalaratne C., Lopes-Lutz D., Schieber A., Wu J. Effect of domestic cooking methods on egg yolk xanthophylls // J Agric Food Chem. – 2012. – N 60. – P. 12547.
148. Krinsky N.I., Landrum J.T., Bone R.A.Biologic mechanisms of the protective role of lutein and zeaxanthin in the eye // Annu Rev Nutr. – 2003. – Vol. 23. – P.171.
149. Olmedilla B., Granado F., Blanco I., Vaquero M.Lutein, but not alpha-tocopherol, supplementation improves visual function in patients with age-related cataracts: a 2-y double-blind, placebo-controlled pilot study // Nutrition. – 2003. – Vol. 19. ...
150. Wagener S., Völker T., De Spirt S., Ernst H., Stahl W. 3,3 – Dihydroxyi-sorenieratene and isorenieratene prevent UV-induced DNA damage in human skin fibroblasts // Free Rad Biol Med. – 2012. – N 53. – P. 457.
151. Bennedsen M.,Wang X., Willén R., Wadström T., Andersen L.P. Treat-ment of H. pylori infected mice with antioxidant astaxanthin reduces gastric inflammation, bacterial load and modulates cytokine release by splenocytes // ImmunolLett. – 1999. – N ...
152. Chew B.P., Park J.S., Wong M.W., Wong T.S. A comparison of the anticancer activities of dietary beta-carotene, canthaxanthin and asta-xanthin in mice in vivo // Anticancer Res. – 1999. – Vol. 19. – P.1849.
153. Park J.S., Chyun J.H., Kim Y.K., Line L.L., Chew B.P. Astaxanthin decreased oxidative stress and inflammation and enhanced immune response in humans // Nutr Metabol. – 2010. – Vol. 7. – P.18.
154. Takaichi S., Mochimaru M. Carotenoids and carotenogenesis in cyano-bacteria: unique ketocarotenoids and carotenoid glycosides // Cell Mol Life Sci. – 2007. – N64. – P. 2607.
155. Maoka T., Akimoto N., Kuroda Y., Hashimoto K., Fujiwara Y. Pittospo-rumxanthins, cycloaddition products of carotenoids with alphatocophe-rol from seeds of Pittosporum tobira // J Nat Prod. – 2008. – N 71. – P. 622.
156. Maeda H., Hosokawa M., Sashima T., Funayama K., Miyashita K. Fuco-xanthin from edible seaweed, Undaria pinnatifida, shows antiobesity effect through UCP1 expression in white adipose tissues // Biochem Biophys Res Comm. – 2005. – N 332. – P. 392.
157. Tsukui T., Konno K., Hosokawa M., Maeda H., Sashima T., Miyashita K.Fucoxanthin and fucoxanthinol enhance the amount of doco-sahexaenoic acid in the liver of KKAy obese/diabetic mice // J Agric Food Chem. – 2007. – N 55. – P. 5025.
158. Tsuboi M., Etoh H., Kato K., Nakatugawa H., Kato H., Maejima Y., Matsumoto G., Mori H., Hosokawa M., Miyashita K., Tokuda H., Suzuki N., Maoka T
159. Dembitsky V.M., Maoka T. Allenic and cumulenic lipids // Prog Lipid Res. – 2007. – N 46. – P. 328.
160. Pott I., Breithaupt D.E., Carle R.Detection of unusual carotenoid esters in fresh mango (Mangifera indica L. cv. 'Kent') // Phytochemistry. – 2003. – N 64. – P. 825.
161. Rogers E.W., Molinski T.F. A cytotoxic carotenoid from the marine sponge Prianos osiros // J Nat Prod. – 2005. – N 68. – P. 450.
162. Hartgers W.A., Sinninghe Damsté J.S., Requejo A.G., Allan J., Hayes J.M., Ling Y., Xie T.M., Primack J., De Leeuw J.W. A molecular and carbon isotopic study towards the origin and diagenetic fate of diaromatic carotenoids // Organic Geochem. – 19...
163. Lutnaes B.F., New C(40)-carotenoid acyl glycoside as principal carotenoid in Salinibacter ruber, an extremely halophilic eubacterium // J Nat prod. – 2002. – N65. – P.1340.
164. Maoka T., Akimoto N. Carotenoids and their fatty acid esters of spiny lobster Panulirus japonicas // J Oleo Sci. – 2008. – Vol. 57. – P.145.
165. FujiFlora Kazakhstan Almaty. – 1963. – P. 204-213. – Т.3.
166. Xu X., Konirbay B., Jenis J., etal. The Kazakh Materia Medica / The Ethnic Press: Beijing. – 2009. – P. 357.
167. Jenis J., Burasheva G.Sh., Aisa H. Ba-hang; Abilov Zh.A. Study on Chemical Constitutes of the Volatiles from Atriplex tataricwara Y., Hashimoto K., Manabe K., Maoka T. Tetrahedron Lett. – 2002. –Vol. 43. – P. 4385.

Citation preview

AL-FARABI KAZAKH NATIONAL UNIVERSITY

J. Jenis

CHEMISTRY OF NATURAL COMPOUNDS Educational manual Stereotypical publication

Almaty «Qazaq universitety» 2020

UDС 54 (075.8) LBC 24 я 73 J 41 Recommended for publication by the Academic Council of the Faculty of Chemistry and Chemical Technology , Editorial-Publishing Council, and Educational and methodical association of the Republic educational- methodical council of higher and postgraduate education of Ministry of education and science of the Republic of Kazakhstan on the basis of Al-Farabi of Kazakh National University (Protocol №1 from 07.10.2015) Reviewer Doctor of chemistry, Professor B. Zh. Dzhiembaev Doctor of chemistry, Professor K. Yu Valentina Doctor of chemistry, Professor Zh. A. Abilov Doctor of chemistry, Professor D.U. Korulkin

Jenis J. J 41 Chemistry of natural compounds: educational manual / J. Jenis – Ster. pub. –Almaty: Qazaq universitety, 2020. – 134 р. ISBN 978-601-04-4608-3 The educational manual performed a series of theory on chemistry of natural compounds which in-cluding explanation some important terms, classification, structure, biological properties, and the extraction, separation and identification of biologically active substances, together with practical comprehensive experiments and analytical studies such as qualitative and quantitative analyzing biological active compounds by chromatographic methods, and obtaining natural products from plant material in laboratory. The educational manual on the discipline «Chemistry of natural compounds» is qualified standards in higher education and recommended for 3rd course students of faculty of che-mistry and chemical technology.

UDС 54 (075.8) LBC 24 я 73 © Jenis J., 2020 © Al-Farabi KazNU, 2020

ISBN 978-601-04-4608-3

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CONTENTS

List of abbreviations.................................................................................... 5 Introduction ................................................................................................. 7 1. Historically important natural products............................................... 9 1.1. Natural products chemistry, natural products, classes, and primary or secondary metabolism ................................................ 11 1.2 Isolation and purification and structure elucidation of natural products ............................................................................. 14 1.2.1. Extraction of plant material ....................................................... 15 1.2.2. Isolation and purification of natural products by using chromatography ....................................................................... 21 2. 2.1. 2.2. 2.3.

Phenols and flavones........................................................................... 26 Classification, structures, and properties phenolic compounds ........... 26 Flavanoids, classification, and biological activities ............................ 29 Galloylbergenin from Kazakh traditional medicinal plant of Bergenia crassifolia with anti- lipid droplet accumulation activity .......................................................................... 34

3. 3.1. 3.2. 3.3. 3.4. 3.5.

Alkaloids ............................................................................................. 40 Introduction, occurrence , nomenclature and classification ................ 40 Properties of alkaloids and distribution in nature ............................... 53 Extraction and isolation method for alkaloids ..................................... 54 Dimer alkaloids ................................................................................... 56 Applications of alkaloids .................................................................... 56

4. Terpens ............................................................................................... 59 4.1. Introduction and classification ............................................................ 59 4.2. Monoterpens, essential oils and the methods of extracting essential oils ........................................................................................ 64 4.3. Sesquiterpenes .................................................................................... 68 4.4. Diterpens and sesterterpens ................................................................. 74 4.5. Triterpenoid and steroids..................................................................... 80 4.6. Carotenoids ......................................................................................... 88

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4.7. Essential oils from Kazakh traditional medicinal plant of Thymus altaica............................................................................... 97 5. Laboratory works for course of chemistry of natural compounds ....... 103 6.

The examination questions for the course of chemistry of natural compounds .......................................................................... 118

References ................................................................................................... 123

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LIST OF ABBREVIATIONS

AMD APCI-MS ASE CCD CLC C-RPC DMSO EPC FFSLE FFPC GC GC-MS GLC GSC HPLC HPTLC IR LC LLC LSC LC-MS MPLC MPSLE MS NMR NP N-RPC OPLC PC PEC PHWE PLE PS Rf RPC

automated multiple development atmospheric pressure chemical ionization mass spectrometry accelerated solvent extraction charge-coupled device column liquid chromatography column rotation planar chromatography dimethylsulfoxide electro-planar chromatography forced-flow solid-liquid extraction forced-flow planar chromatography gas chromatography gas chromatograph coupled to mass spectrometer gas-liquid chromatography gas-solid chromatography high performance liquid chromatography high performance thin-layer chromatography infrared liquid chromatography liquid-liquid chromatography liquid-solid chromatography liquid chromatography-mass spectrometry medium pressure liquid chromatography medium pressure solid-liquid extraction mass spectrometry nuclear magnetic resonance spectroscopy normal phase normal chamber rotation planar chromatography over-pressured layer chromatography paper chromatography planar electrochromatography pressurized hot water extraction pressurized liquid extraction selectivity point retention factor rotation planar chromatography –5–

RPE RP RPLC SFE SLE S-RPC ST SWE TLC TLE U-RPC UV UV/VIS

rotation planar extraction reversed-phase reversed phase liquid chromatography supercritical fluid extraction solid-liquid extraction sequential rotation planar chromatography solvent strength subcritical/superheated water extraction thin-layer chromatography thin-layer electrochromatography ultra-microchamber rotation planar chromatography ultraviolet ultraviolet/visible

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INTRODUCTION

The traditionally natural products have played an important role in developing of natural product chemistry which continues to expand to exciting new frontiers of great importance in medicine. Natural Products Chemistry is a co urse in the chemistry discipline area. We will explore the historical and contemporary role of secondary natural products in health care and commerce. We will learn how natural products are normally classified according to their biosynthetic origins and chemical properties. A special emphasis will be placed on how chemical structure affects the physiological function of various natural products. These "structure activity relationships" help us learn about the interaction of small molecules in living systems and pharmacology of drugs. At present the own production of medical drugs in the Republic of Kazakhstan is 11% (including vaccines – 1.1%), veterinary drugs 78% (mainly the manufacture of drugs from imported substances), while other drugs are being imported into the country. The main problem is the lack of original domestic biotechnological drugs in the presence of perspective experimental developments. To date, virtually the production of genetically engineered drugs is not adjusted, while there are original domestic or joint developments that passed or passing phase of preclinical and clinical studies. In Kazakh traditional medicine,the plant resources have been efficiently used in the treatments of different kinds of diseases such as bronchitis, bronchial asthma, brohepatitis, urethritis, chronic rheumatoid arthritis, nephritis, urolithiasis, pharyngitis, periodontitis, stomach pain, hyperacidity, diarrhea, hemostasia, metrorrhagia, snakebite, cancer and so on. In Kazakhstan grow over six thousand –7–

kinds of plants in which more than 6000 species of highest vascular plants, about 5000 species of mushrooms, 4851 s pecies of lichen, more than 2000 species of seaweed are registered. The study revealed that investigated plants belonging to familyChenopodiaceae is good sources for biologically active substances which include: saponins, triterpenes, organic acids, amino acids flavonoids, chromones and carbohydrates. The choice of plant species which belong to family of Chenopodiaceaedue to the fact that on the territory of the Republic of Kazakhstan salt tolerant plants have a huge reserves and in depth studies not performed before which promotes the study of their chemical composition and biological activity for the purpose of development of new drugs needed for the domestic pharmaceutical industry which is one of the main priorities of socio-economic policy of the Government of Kazakhstan. The plant kingdom offers a rich source of structural biodiversity in the form of a variety of natural products. By using the rich resources of medicinal plants from Kazakhstan to find new bioactive natural products which as new drug leads, new anticancer or antitumor drugs and to solve in our opinion with a success a problem of creation and introduction in the industry (medicine and agriculture) of Kazakhstan of new highly effective medical products.

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1. HISTORICALLY IMPORTANT NATURAL PRODUCTS

For centuries, drugs were entirely of natural origin and composed of herbs, animal products, and inorganic materials. Early remedies may have combined these ingredients with witchcraft, mysticism, astrology, or religion, but it is certain that those treatments that were effective were subsequently recorded and documented, thus leading to the early Herbals. Traditional medicinal practices have formed the basis of most of the early medicines followed by subsequent clinical, pharmacological and chemical studies [1-2]. Probably the most famous and well known example to date would be the synthesis of the antiinflammatory agent, acetylsalicyclic acid (1) (aspirin) derived from the natural product, salicin (2) isolated from the bark of the willow tree Salix alba L. I nvestigation of Papaver somniferum L. (opium poppy) resulted in the isolation ofseveral alkaloids including morphine (3), a commercially important drug, first reported in 1803 (Figure 1). It was in the 1870s that crude morphine derived from the plant P. somniferum, was boiled in acetic anhydride to yield diacetylmorphine (heroin) and found to be readily converted to codeine (painkiller). Historically, it is documented that the Sumerians and Ancient Greeks used poppy extracts medicinally, whilst the Arabs described opium to be addictive. Digitalis purpurea L. (foxglove) had beentraced back to Europe in the 10th century but it was not until the 1700s that the active constituent digitoxin (4), a cardiotonic glycoside was found to enhance cardiac conduction, thereby improving the strength of cardiac contractibility. Digitoxin (4) and its analogues have long been used in the management of congestive heart failure and have possible long term detrimental –9–

effects and are being replaced by other medicines in the treatment of «heart deficiency». The anti-malarial drug quinine(5) approved by the United States FDA in 2004, isolated from the bark of Cinchona succirubra Pav. exKlotsch, had been used for centuries for the treatment of malaria, fever, indigestion, mouth and throat diseases and cancer. Formal use of the bark to treat malaria was established in the mid-1800s when the British began the worldwide cultivation of the plant [30]. Pilocarpine (6) found in Pilocarpus jaborandi (Rutaceae) is an L-histidine-derived alkaloid, which has been used as a clinical drug in the treatment of chronic open-angle glaucoma and acute angle-closure glaucoma for over 100 years. In 1994, an oral formulation of pilocarpine was approved by the FDA to treat dry mouth (xerostomia) which is a side effect of radiation therapy for head and neck cancers and also used to stimulate sweat glands to measure the concentrations of sodium and chloride (Figure 1.1) [3]. In 1998, the oral preparation was approved for the management of Sjogren's syndrome, an autoimmune disease that damages the salivary and lacrimal glands.

Figure 1.1. Acetylsalicyclic acid (1), Salicin (2), Morphine (3), Digitoxin (4), Quinine (5)and Pilocarpine (6). – 10 –

1.1. Natural products chemistry, natural products, classes, and primary or secondary metabolism Natural products chemistry is a chemistry branch that examines chemical compounds produced by living organisms. Natural products are the chemical compounds found in nature that usually has a pharmacological or biological activity for use in pharmaceutical drug discovery and drug design. Natural Products Chemistry & Research emphasizes the study of chemistry and biochemistry of naturally occurring compounds or the biology of living systems from which they are obtained. A natural product is a chemical compound or substance produced by a living organism – found in nature. In the broadest sense, natural products include any substance produced by life. The term natural product has also been extended for commercial purposes to refer to cosmetics, dietary supplements, and foods produced from natural sources without added artificial ingredients. Within the field of organic chemistry, the definition of natural products is usually restricted to mean purified organic compounds isolated from natural sources that are produced by the pathways of primary or secondary metabolism. Within the field of medicinal chemistry, the definition is often further restricted to secondary metabolites. Secondary metabolites are not essential for survival, but nevertheless provide organisms that produce them an evolutionary advantage.Many secondary metabolites are cytotoxic and have been selected and optimized through evolution for use as «chemical warfare» agents against prey, predators, and competing organisms[4]. Natural products sometimes have pharmacological or biological activity that can be of therapeutic benefit in treating diseases. As such, natural products are the active components of many traditional medicines. Furthermore synthetic analogs of natural products with improved potency and safety can be prepared and therefore natural products are often used as starting points for drug discovery. In fact, natural products are the inspiration for approximately one half of U.S. Food and Drug Administration-approved drugs. The broadest definition of natural product is anything that is produced by life. – 11 –

A more restrictive definition of a natural product is an organic compound that is synthesized by a living organism. The remainder of this article restricts itself to this more narrow definition. Natural products may be classified according to their biological function or the chemical structure/biosynthetic pathway as described below. Following Albrecht Kossel's original proposal in 1891,natural products are often divided into two major classes, the primary and secondary metabolites. Primary metabolites have an intrinsic function that is essential to the survival of the organism that produces them. Secondary metabolites in contrast have an extrinsic function that mainly affects other organisms. Secondary metabolites are not essential to survival but do increase the competitiveness of the organism within its environment. A more restrictive definition limiting natural products to secondary metabolites is commonly used within the fields of medicinal chemistry and pharmacognosy [5]. Medicinal chemistry is the chemistry discipline concerned with the design, development and synthesis of pharmaceutical drugs. The discipline combines expertise from chemistry and pharmacology to identify, develop and synthesize chemical agents that have a therapeutic use and to evaluate the properties of existing drugs. Pharmacognosy is the study of medicines derived from natural sources. The American Society of Pharmacognosy defines pharmacognosy as «the study of the physical, chemical, biochemical and biological properties of drugs, drug substances or potential drugs or drug substances of natural origin as well as t he search for new drugs from natural sources». It is also defined as the study of crude drugs. Chemistry of Natural Products deals with the chemistry of metabolites. Metabolites arenaturally-occurring organic compounds synthesised by plants, through metabolic actitivities inplants, aided by enzymes.There are two types of metabolites, viz: (a) Primary metabolites such as c arbohydrates, proteins, fatty acids and glycerol,mevalonicacids, etc. (b) Secondary metabolites such as steroids, alkaloids, triterpenes, tannins, saponnins,flavonoids, etc. Chemistry of Natural Products is dated far back to early century. An aspect of Natural Products that deals with plants is known as – 12 –

Phytochemistry. Natural Products is define as organic compounds and other chemicals synthesised by plants through metabolic processes aided by sunlight, involving CO2, H2O vapour and chlorophyll. Generally, natural products are characterised by specific functions they form in plants and animals. Categories of natural Products are called metabolites. Primary metabolites are usually found in all living organisms such as plants and animals. They form the fundamental building block of living material e.g. mevalonic acids and nucleotides. Primary metabolites have wide distribution in living systems and are usually involved in essential life processes. However, secondary metabolites are chemicals synthesized by plants but are not directly used by them, but are used indirectly by man as a s ource of pharmaceutical preparations. Secondary metabolites are generally built from primary metabolites. Secondary metabolites have restricted distribution and are characteristics of individual genera or species. Secondary metabolites are essential to the existence of the organism but play an important role to the survival of the plant. Reports have shown presence of secondary metabolites in yeast and microbes. All living organisms from the simplest protozoan to the most developed animals contain a wide range of organic compounds. Millions of secondary metabolites have been isolated from plants and animals, some of which are known to have medicinal features. Examples are: the popular Quinine-an antimalarial drug and Chloroquuine isolated from medicinal plants. It is good to note that Natural Products is restricted specifically to major organic compounds obtained from natural origin, especially from marine plants and plants growing on land. Plants are generally classified on the basis of the family they belong. Each family of plant contains a GENUS and SPECIES. Leguminoseae is a family of a p ant of the genus Berlinia and several species of Berlinia exists; such as B. confusa, B. grandiflora, B. auriculata,etc. In the present days, millions of secondary metabolites are in existence; and majorities are isolated from natural sources such as plants. Examples are steroids, terpenoids, alkaloids, glycosides, flavonoids, etc. However, some secondary metabolites have varying structures within a group or a specific specie e.g. monoterpenoids, diterpenoids, triterpenoids, sesquiterpenoids, etc. Some are acyclic – 13 –

while others are cyclic in their structures. Some secondary metabolites are synthesised via biosynthesis and or biogenesis. Higher plants synthesised chemical compounds in vivo and degrade them by means of series of chemical reactions, each aided by enzymes, by a process known as metabolism. The products of metabolic pathways are called metabolites.

1.2. Isolation and purification and structure elucidation of natural products Penicillin G, the first of its class fungal antibiotic, first studied by Scottish microbiologist Alexander Fleming in the late 1920s, and made practical as a therapeutic via natural product isolation in the late 1930s by Ernst Boris Chain, Howard Florey, and others, these three named sharing the 1945 Nobel Prize in Medicine for the work. Fleming recognized the antibiotic properties and remarkable lack of toxicity of «pen G» in humans, but was unable to capture its clinical utility because he could only prepare aqueous extracts; his removal of water led to decomposition of the drug. Developments in chromatographic separations and freeze drying helped move progress forward in the production of commercial quantities of penicillin and other natural products [6-7]. All natural products of interest begin as mixtures with other compounds from the natural source, often very complex mixtures, from which the particular product of interest must be isolated and purified. The isolation of a natural product refers, depending on context, either to the isolation of the considerable quantities of pure chemical matter required for chemical structure elucidation, derivitzation/degradation chemistry, biological testing, and other research needs (generally milligrams to grams, but historically, often more),or to the isolation of «analytical quantities» of the substance of interest, where the focus is on identification and quantitation of the substance (e.g., in biological tissue or fluid), and where the amount isolated depends on the analytical method applied (but is – 14 –

generally always sub-microgram in scale). The ease with which the active agent can be isolated and purified depends on the structure, stability, and quantity of the natural product. The methods of isolation applied toward achieving these two distinct scales of product are likewise distinct, but generally involve extraction, precipitation, adsorptions, chromatography, and sometimes crystallizations. In both cases, the isolated substance is purified to chemical homogeneity, i.e., specific combined separation and analytical methods such as LC-MS methods are chosen to be «orthogonal» – achieving their separations based on distinct modes of interaction between substance and isolating matrix – with the goal being repeated detection of only a single species present in the putative pure sample. Early isolation is almost inevitably followed by structure determination, especially if an important pharmacologic activity is associated with the purified activity. Structure determination refers to methods applied to determine the chemical structure of an isolated, pure natural product, a process that involves an array of chemical and physical methods that have changed markedly over the history of natural products research; in earliest days, these focused on chemical transformation of unknown substances into known substances, and measurement of physical properties such as melting point and boiling point, and related methods for determining molecular weight.In the modern era, methods focus on mass spectrometry and nuclear magnetic resonance methods (NMR), often multidimensional, and when feasible, small molecule crystallography. For instance, the chemical structure of penicillin was determined by Dorothy Crowfoot Hodgkin in 1945, work for which she later received a Nobel Prize in Chemistry (1964) [8]. 1.2.1. Extraction of plant material Most of the bulk of the biomass, irrespective of whether it is plants or microbes, exists asfairly inert, insoluble, and often polymeric material, such as cellulose of plants or fungi and the microbial cell wall [9]. The first step of the extraction is therefore to release and solubilize the smaller secondary metabolites in the matrix, resulting in the initial extract. – 15 –

In liquid extractions the choice of extraction solvent or solvents provides the first and most obvious means of sample preparation [10]. Initial extraction with low-polarity solvents yields the more lipophilic components, while alcohols isolate a broader spectrum of apolar and polar compounds from the material. In addition to the choice of extraction solvent, there are also different approaches to the actual extraction procedure. While stirring or mechanical agitation are the most common methods, percolation or even pressurized solid-liquid extraction are possible. The most commonly used extraction methods are reviewed in the following chapter. Selection of extraction method The most widely used extraction processes have traditionally been based either on different liquid extraction methods or on vaporphase extraction methods [11]. A more recent method whose application has steadily increased is supercritical fluidextraction (SFE), which is based on the properties of gases compressed and heated to a st ateabove their critical pressure and temperature, at which no d istinction between the gas andliquid phases can be discerned [12].At the present time, there are also a number of nonconventional extraction methods in usethat are all, in principle, solidliquid extractions (SLE) but which introduce some form of additional energy to the process in order to facilitate the transfer of analytes from sample to solvent. These methods include ultrasonic extraction, microwave-assisted extraction and pressurized liquid extraction [13], as well as vertical (turbo) extraction. Even extraction by electrical energy has been studied [14]. Forced-flow solid-liquid extraction (FFSLE) techniques, such as medium-pressure solid-liquid extraction (MPSLE) and rotation planar extraction (RPE), are methods in which the extraction solvent is forced through the sample bed either by means of pressure or by centrifugal force, respectively, thus increasing the efficiency of the extraction process [15]. The main advantage of these non-conventional methods compared to conventional SLE methods is the increased extraction efficiency, which leads to increased yields and/or shorter extraction times. The simplest method of extraction, however, needs no extraction medium. Mechanical pressing has been traditionally applied to the extraction of oils from oilseeds [16]. This process may be combined – 16 –

with some form of pretreatment such as cleaning, dehulling, crushing or flaking before the extraction but, in general, the only equipment needed is a hydraulic press. Despite the simple operating principle, there are several operating parameters that need to be controlled in order to obtain a sufficient extraction rate and yield. The most important parameters affecting the yield of the extraction procedure are the moisture content of the seeds and temperature . Traditional extraction processes may be classified as follows: extraction with organicsolvents: percolation, maceration, and extraction using a Soxhlet apparatus; and extraction with water: infusion, decoction, and steam distillation. An old method also worth mentioning is extraction with cold fat, called enfleurage, used mainly for the extraction of fragrances from flowers. Percolation is one of the most widespread methods employed in plant extraction since it does not require much sample manipulation or long pre-treatment times. The only equipment required is a conical glass container with a tap at the base used to set the rate of solvent elution. Percolation is a continuous process in which the saturated solvent is constantly displaced by fresh solvent, but normally the sample is steeped in solvent in the percolator for 24 hours for up to three times, and the extracts are then collected and pooled. In maceration the sample is placed in a stoppered container and is in contact with the solvent. This allows the solvent to penetrate into the cellular structure in order to dissolve the soluble compounds. Its efficiency may be increased by occasionally shaking the container or by using a mechanical or magnetic stirrer to homogenize the final solution andsaturate the solvent. As maceration is a discontinuous method, the solvent should be renewed until the plant material is exhausted; this requires filtration steps that may result in the loss of solvent, analytes, and/or plant material. Soxhlet extraction is a very old, clean-up method, but it is still relatively widely used in plant analysis. It is used mainly with one solvent at a time due to the fact that individual solvents may distill off at different temperatures, with the result that the mixture in the chamber containing the drug becomes enriched in the solvent of lower boiling point. The main advantages of the Soxhlet technique are that it is an automatic and continuous method that does not require much manipulation. It has also been shown to be very effective in terms of extraction yield and – 17 –

therefore often used as areference method for newer extraction methods. One disadvantage is that the extractives are heated during extraction at the boiling point of the solvent employed and thermally labile compounds may hydrolyze, decompose, or produce artifacts. Infusion and decoction are simple methods for extraction with water. In the infusion technique, boiling or cold water is added to the milled sample; in decoction the sample is boiled for about 15 minutes in water. Extraction with pure water, however, is seldom used for plant material as hydrophilic compounds are usually extracted with methanol-water or ethanol-water mixtures. Steam distillation is an old extraction method that is primarily used to obtain essential oils from plant material. In this method, a packed bed of plant material is continuously flushedwith steam and the volatile organic compounds present in the material are taken up by the vapor phase due to their low partial vapor pressure. Compounds carried by the vapor stream are then separated after decreasing the temperature of the vapor by forced condensation. Ultrasonic extraction takes advantage of the very high effective temperatures (which increase solubility and diffusivity) and pressures (which favor penetration and transport) at the interphase between the solvent solution subjected to ultrasonic energy and a solid matrix, combined with the oxidative energy of radicals created during sonolysis, resulting in high extractive power [17]. Ultrasonically assisted extraction methods have been employed for a great number of different plant materials, e.g. Salvia officinalis L., Valeriana officinalis L., Calendula officinalis L., Gentiana lutea L., Hibiscus tiliaeus L., and chrysanthemum flowers to name a few [17-24]. Compound groups that have been obtained by ultrasonic extraction include polysaccharides, volatile oils, fatty acids and their esters, stigmasterol derivatives, and pyrethrins. Another way of increasing the efficiency of conventional extraction methods is to usemicrowave irradiation. Microwaveassisted extraction consists of heating the solvent in contact with the sample by means of microwave energy. The process involves disruption of hydrogen bonds, as a result of microwave-induced dipole rotation of molecules, and migration of the ions, which enhance penetration of the solvent into the matrix, allowing dissolution of the components to be extracted [23]. The main – 18 –

advantages of microwave assisted extraction over the conventional extraction techniques are reduced solventconsumption, shorter operational times, moderately high recoveries, good reproducibility andminimal sample manipulation for extraction process [24-29]. Microwave-assisted extraction methods have been applied to the extraction of oil from olive seeds, pigments from paprika powders, glycyrrhizic acid from liquorice root, lipids from several oleaginous seeds, cocaine and benzoylecgonine from coca leaves, and alkamides from Echinacea purpurea L. roots [30]. Pressurized liquid extraction (PLE, also commonly known as accelerated solvent extraction; ASE) works according to the principle of static extraction with superheated liquids. The method uses an organic solvent at high pressures and temperature above the boiling point. The main reasons for the enhanced performance of PLE are the higher solubility of analytes in solvent at higher temperatures, higher diffusion rate as a result of higher temperatures, and disruption of the strong solute matrix interaction caused by van der Waals forces, hydrogen bonding and dipole-dipole attractions between solute molecules and active sites on the matrix. The PLE technique is well suited for the extraction of various types of compound from different plant materials because parameters other than temperature can be varied and the polarity of the extraction solvent can be chosen from a wide range and adapted to the respective matrix. PLE has been reported to have been applied to e.g. the extraction of dianthrons from Hypericum perforatum L.,deacylsaponins from Aesculus hippocastanum L., silybin from Silybum marianum L., curcumin from Curcuma xanthorrhiza, thymol from Thymus vulgaris L., flavanones andxanthones from Maclura pomifera, aristolochic acids from Radix aristolochiae, berberinefrom Coptidis rhizoma and oxysterols from whole egg powder and egg-containing foods. Subcritical water extraction (SWE, also called pressurized hot water extraction, PHWE, orsuperheated water extraction) is based on the unique solvent properties of water, namely itsdisproportionately high boiling point for its mass, a high dielectric constant and high polarity. The method involves heating water above its boiling point but below itscritical point (i.e. 374°C) under elevated pressure so that the water remains in a liquid state. As the temperature rises there is a – 19 –

marked and systematic decrease in permittivity, an increasein the diffusion rate and a decrease in the viscosity and surface tension. SWE has been foundto be an efficient extraction method and a potential alternative to steam distillation andsolvent extraction in the extraction of essential oils from plant material. Satisfactory resultshave been reported for SWE of essential oils from marjoram (Thymus mastichina), clove(Syzygium aromaticum), fennel (Foeniculum vulgare) and sage (Salvia officinalis). Besides essential oils, the method has also beenapplied to the extraction of lactones from kava root (Piper methysticum) and iridoidglycosides from Veronica longifolia leaves. In recent years, the extraction method that has received increasing attention and manyindustrial applications in the isolation of natural products is supercritical fluid extraction(SFE). SFE has several advantages over the conventional liquid-liquid and solidliquidextraction techniques, e.g. the elimination of most of the organic solvents that may pose asafety risk during extraction, elimination of carry-over of the more or less toxic solvents in thefinal extracts, and the possibility of avoiding the detrimental effects of these solvents on theenvironment [31]. The disadvantages of SFE includethe low polarity of the most commonly used fluid, i.e. carbon dioxide, possible problems caused by the presence of water, unpredictability of the matrix effect and the need forspecialized/ expensive equipment. A number of compounds have been tested as supercritical fluids, including pentane, nitrousoxide, ammonia and Freon fluorocarbons but, on the grounds of cost and safety, carbondioxide either alone or modified with methanol or some other polar solvent is by far the most widely used supercritical extraction solvent. It has also the advantage of a l ow critical temperature and pressure, which enable pressurization of the gas into a su percritical fluid. Thepractical aspects of SFE and its applications have been recently reviewed [32]. Due to the low polarity of carbon dioxide it is best suited for the extraction of nonpolarcompounds. SFE has been successfully applied to e.g. the extraction of essential oilfrom Angelica archangelica L. roots, as well as from Matricaria chamomilla flowerheads,and the extraction of lycopene from tomato skins [33-34]. However, by varying the pressure and temperature of thesupercritical carbon dioxide and the amount of polar modifiers, – 20 –

the method has also beenfound suitable for the extraction of more polar compounds such as flavanones and xanthonesfrom Maclura pomifera, flavonoids from Scutellaria baicalensis roots and apigenin fromMatricaria chamomilla [35-37]. 1.2.2. Isolation and purification of natural products by using chromatography Chromatography is a physical method ofseparation in which the components to beseparated are distributed between twophases one of which is stationary(stationary phase) while the other (themobile phase) moves through it in a definite direction. The chromatographic process occurs due to differences in the distribution constant of the individual sample components [38]. Chromatographic methods: 1. Thin layer chromatography 2. Column chromatography 3. Gas liquid chromatography (GLC) 4. High performance liquid chromatography (HPLC). 5. Gel filtration chro-matography 6. Affinity chromatography 7. Paper chromatography. Classification of chromatography according to mobile phase (the stationary phase may be a solid or a liquid. And the mobile phase may be liquid or gas): 1. Liquid chromatography(LC): mobile phase is a liquid. (LLC, LSC). 2. Gas chromatography(GC): mobile phase is a gas. (GSC, GLC). Liquid chromatography Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. It can be carried out either in a column or a plane. Present day liquid chromatography that generally utilizes very small packing particles and a relatively high pressure is referred to as high performance liquid chromatographyhigh performance liquid chromatography (HPLC) [39]. In HPLC the sample is forced by a l iquid at high pressure (the mobile phase) through a column that is packed with a stationary phase composed of irregularly or spherically shaped particles, a porous monolithic layer, or a porous membrane. HPLC is historically divided into two different sub-classes based on the polarity of the – 21 –

mobile and stationary phases. Methods in which the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase, silica as the stationary phase) are termed normal phase liquid chromatography (NPLC) and the opposite (e.g., water-methanol mixture as the mobile phase and C18 = octadecylsilyl as the stationary phase) is termed reversed phase liquid chromatography (RPLC) [40]. Gas chromatography Gas chromatography (GC), also sometimes known as gas-liquid chromatography, (GLC), is a separation technique in which the mobile phase is a g as. Gas chromatographic separation is always carried out in a column, which is typically «packed» or «capillary». Packed columns are the routine work horses of gas chromatography, being cheaper and easier to use and often giving adequate performance. Capillary columns generally give far superior resolution and although more expensive are becoming widely used, especially for complex mixtures. Both types of column are made from nonadsorbent and chemically inert materials. Stainless steel and glass are the usual materials for packed columns and quartz or fused silica for capillary columns [41]. Gas chromatography is based on partition equilibrium of analyte between a solid or viscous liquid stationary phase (often a liquid silicone-based material) and a mobile gas (most often helium). The stationary phase is adhered to the inside of a s mall-diameter (commonly 0.53 - 0.18mm inside diameter) glass or fused-silica tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column). It is widely used in analytical chemistry; though the high temperatures used in GC make it unsuitable for high molecular weight biopolymers or proteins (heat denatures them), frequently encountered in biochemistry, it is well suited for use in the petrochemical, environmental monitoring and remediation, and industrial chemical fields. It is also used extensively in chemistry research. Classification according to the packing of the stationary phase: 1. Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2. Paper chromatography (PC): the stationary phase is a thin film of liquid – 22 –

supported on an inert support. 3. Column chromatography (CC): stationary phase is packed in a glass column. Classification according to the force of separation: 1. Adsorption chromatography. 2. Partition chromatography. 3. Ion exchange chromatography. 4. S ize exclusion chromatography. 5. A ffinity chromatography. Paper chromatography Paper chromatography is a technique that involves placing a small dot or line of sample solution onto a strip of chromatography paper. The paper is placed in a container with a shallow layer of solvent and sealed. As the solvent rises through the paper, it meets the sample mixture, which starts to travel up the paper with the solvent. This paper is made of cellulose, a polar substance, and the compounds within the mixture travel farther if they are non-polar. More polar substances bond with the cellulose paper more quickly, and therefore do not travel as far. Thin layer chromatography Thin layer chromatography (TLC) is a w idely employed laboratory technique and is similar to paper chromatography. Thin layer chromatography (TLC) is a method for identifying substances and testing the purity of compounds. TLC is a useful technique because it is relatively quick and requires small quantities of material. However, instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate. Compared to paper, it has the advantage of faster runs, better separations, and the choice between different adsorbents. For even better resolution and to allow for quantification, high-performance TLC can be used. An older popular use had been to differentiate chromosomes by observing distance in gel (separation of was a separate step). Retention Factor, Rf The retention factor, Rf, is a quantitative indication of how far a particular compound travels in a particular solvent. The Rfvalue is a good indicator of whether an unknown compound and a known compound are similar, if not identical. If the Rf value for the – 23 –

unknown compound is close or the same as the Rf value for the known compound then the two compounds are most likely similar or identical (Picture 1.1). The retention factor, Rf, is defined as Rf= distance the solute (D1) moves divided by the distance traveled by the solvent front (D2) Rf = D1 / D2 where D1 = distance that color traveled, measured from center of the band of color to the point where the food color was applied D2 = total distance that solvent traveled

Picture 1.2. Thin layer chromatography and Rf calculations

Column chromatography Column chromatography is a separation technique in which the stationary bed is within a tube. The particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube (packed column) or be concentrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube (open tubular column). Differences in rates of movement through the medium are calculated to different retention times of the sample. In 1978, W. Clark Still introduced a modified version of column chromatography called flash column chromatography. The technique is very similar to the traditional column chromatography, except for that the solvent is driven through the column by applying positive pressure. This allowed most separations to be performed in – 24 –

less than 20 minutes, with improved separations compared to the old method. Modern flash chromatography systems are sold as prepacked plastic cartridges, and the solvent is pumped through the cartridge. Systems may also be linked with detectors and fraction collectors providing automation. The introduction of gradient pumps resulted in quicker separations and less solvent usage. Problems 1. Explain: Historically important natural products, Distribution of plant resources in Kazakhstan. 2. Please explain: Kazakh traditional medicine. Development condition of pharmacology in Kazakhstan? 3. Please give definition for: Natural products chemistry, Natural products, and Phytochemistry. 4. Illustration: Major classes of natural products, Definition: Medicinal chemistry,Pharmacognosy. 5. Please making definition and explain: Metabolites, Primary metabolites and Secondary metabolites. 6. Please explain: Development of isolation and purification of natural products. 7. Chromatography -what does it mean? History of chromatography? 8. Principles and types of chromatography. Explain with applications. 9. Liquid chromatography, types, which kind of natural compounds and material could be analyzed by the method. 10. Making statements on: Adsorption chromatography,Partition chromatography, types, and applications. 11. Please explain: Paper chromatography, Thin layer chromatography, Column chromatography. 12. What is Rfvalue? What does it mean that difference of Rf value? Explain the affecting factors to Rf value.

– 25 –

2. PHENOLS AND FLAVONES

2.1. Classification, structures, and propertiesof phenolic compounds In organic chemistry, phenols, sometimes called phenolics, are a class of chemical compounds consisting of a hydroxylgroup (-OH) bonded directly to an aromatic hydrocarbon group. The simplest of the class is phenol, which is also called carbolic acid C6H5OH. Phenolic compounds are classified as simple phenols or polyphenols based on the number of phenol units in the molecule.

Phenol – the simplest of the phenols

Quercetin, a typical flavonoid, is a polyphenol

Phenolic compounds are synthesized industrially; they also are produced by plants and microorganisms, with variation between and within species.Although similar to alcohols, phenols have unique properties and are not classified as alcohols (since the hydroxyl group is not bonded to a saturated carbon atom). They have higher acidities due to the aromatic ring's tight coupling with the oxygen and a relatively loose bond between the oxygen and hydrogen. The acidity of the hydroxyl group in phenols is commonly intermediate – 26 –

between that of aliphatic alcohols and carboxylic acids (their pKa is usually between 10 and 12).Organisms that synthesize phenolic compounds do s o in response to ecological pressures such as pathogen and insect attack, UV radiation and wounding. As they are present in food consumed in human diets and in plants used in traditional medicine of several cultures, their role in human health and disease is a subject of research.Some phenols are germicidal and are used in formulating disinfectants. Others possess estrogenic or endocrine disrupting activity [42-43]. Classification There are various classification schemes. A commonly used scheme is based on the number of carbons and was devised by Jeffrey Harborne and Simmonds in 1964 and published in 1980 (Table 2.1): They can also be classified on the basis of their number of phenol groups. They can therefore be called simple phenols or monophenols, with only one phenolic group, or di- (bi-), tri- and oligophenols, with two, three or several phenolic groups respectively [44]. Table 2.1 Classification of phenolic compounds

Number of carbon atoms

Basic skelet on

1

2

Number of phenolic cycles

Class

3

Examples 4

5

6

C6

1

Simple phenols, Benzoquinones

Catechol, Hydroquinone, 2,6Dimethoxybenzoqui none

7

C6-C1

1

Phenolic acids, Phenolic aldehydes

Gallic, salicylic acids

Acetophenones, Tyrosine derivatives, Phenylacetic acids

3-Acetyl-6methoxybenzaldehy de, Tyrosol, pHydroxyphenylaceti c acid, Homogentisic acid

8

C6-C2

1

– 27 –

1

2

3

4

5

9

C6-C3

1

Hydroxycinnamic acids, Phenylpropenes, Coumarins, Isocoumarins, Chromones

10

C6-C4

1

Naphthoquinones

Juglone, Plumbagin

2

Xanthonoids

Mangiferin

2

Stilbenoids, Anthraquinones

Resveratrol, Emodin

15

C6-C3C6

2

Chalconoids, Flavonoids, Isoflavonoids, Neoflavonoids

Quercetin, cyanidin, Genistein

16

C6-C4C6

2

Halogenated algal phenolic compounds

Kaviol A, colpol

2

Lignans, Neolignans

Pinoresinol, Eusiderin

4

Biflavonoids

Amentoflavone

n > 12

Lignins, Catechol melanins, Flavolans (Condensed tannins), Polyphenolic proteins, Polyphenols

Raspberry ellagitannin, Tannic acid

C6-C1C6 C6-C2C6

13 14

(C6C3)2 (C6C3C6)2

18 30

Many

(C6C3)n, (C6)n, (C6C3C6)n

Caffeic, ferulic acids, Myristicin, Eugenol, Umbelliferone, aesculetin, Bergenon, Eugenin

Not in this Harborne classification are the C6-C7-C6 diarylheptanoids.

The largest and best studied natural phenols are the flavonoids, which include several thousand compounds, among them the flavonols, flavones, flavan-3ol (catechins), flavanones, anthocyanidins and isoflavonoids. – 28 –

2.2. Flavonoids, classification, and biological activities Flavonoids (from the Latin word flavus meaning yellow, their color in nature) are a class of plantsecondary metabolites. Flavonoids are a group of natural compounds with variable phenolic structures and are found in plants. In 1930 a new substance was isolated from oranges. At that time it was believed to be a member of a new class of vitamins and was designated as vitamin P. Later on it became clear that this substance was a flavonoid (rutin). Flavonoids consist of a large group of polyphenolic compounds having a benzo-γpyrone structure and are ubiquitously present in plants. They are synthesized by phenylpropanoid pathway. Available reports tend to show that secondary metabolites of phenolic nature including flavonoids are responsible for the variety of pharmacological activities. Chemically, they have the general structure of a 15-carbon skeleton, which consists of two phenyl rings (A and B) and heterocyclic ring (C). This carbon structure can be abbreviated C6-C3-C6. According to the IUPAC nomenclature, they can be classified into: 1. Flavonoids or Bioflavonoids, 2. Isoflavonoidsderived from 3-phenylchromen-4-one (3-phenyl1,4-benzopyrone) structure. 3. Neoflavonoids, derived from 4-phenylcoumarine (4-phenyl1,2-benzopyrone) structure.

Flavone

Isoflavone

Neoflavone

The three flavonoid classes above are all ketone-containing compounds, and as such, are anthoxanthins (flavones and flavonols). This class was the first to be termed bioflavonoids. The terms flavonoid and bioflavonoid have also been more loosely used to – 29 –

describe non-ketone polyhydroxy polyphenol compounds which are more specifically termed flavonoids. The three cycle or heterocycles in the flavonoid backbone are generally called ring A, B and C. Ring A usually shows a phloroglucinol substitution pattern. Over 5000 naturally occurring flavonoids have been characterized from various plants. They have been classified according to their chemical structure, and are usually subdivided into the following subgroups (Table 2.2): Table 2.2 Skeleton and examples of Flavonoids Flavones and flavonols Skeleton Group

Flavone

Flavonol or 3-hydroxyflavone

Structural formula

Description

Examples

2phenylchromen4-one

Luteolin, Apigenin, Tangeritin

3-hydroxy-2phenylchromen4-one

Quercetin, Kaempferol, Myricetin, Fisetin, Galangin, Isorhamnetin, Pachypodol, Rhamnazin, Pyranoflavonols, Furanoflavonols,

Flavanones Structural formula

Description

Flavanone

2,3-dihydro-2phenylchromen4-one

– 30 –

Hesperetin, Naringenin, Eriodictyol, Homoeriodictyol

Flavanonols Description

Flavanonol or 3 Hydroxyflavanone or 2,3 dihydroflavonol

Structural formula

3-hydroxy2,3-dihydro-2phenylchrome n-4-one

Taxifolin (or Dihydroquercetin), Dihydrokaempferol

Flavans

Flavan-3-ol (flavanol)

Flavan structure

Flavan-4-ol

Flavan-3,4-diol (leucoanthocyanidin)

Catechin (C), Gallocatechin (GC), Catechin 3-gallate (Cg), Gallocatechin 3gallate (GCg)), Epicatechins (Epicatechin (EC)), Epigallocatechin (EGC), Epicatechin 3gallate (ECg), Epigallocatechin 3-gallate (EGCg)

Anthocyanidins Anthocyanidins Anthocyanidins are the aglycones of anthocyanins; they use the(2phenylchromenylium) ion skeleton

Cyanidin, Delphinidin, Malvidin, Pelargonidin, Peonidin, Petunidin

– 31 –

Isoflavonoids Isoflavonoids Isoflavones use the 3-phenylchromen-4-one skeleton (with no hydroxyl group substitution on carbon at position 2

Genistein, Daidzein, Glycitein

Isoflavanes

Other isoflavonoids:Isoflavandiols, Coumestans, Pterocarpans.

Biological activities of flavonoids [44] Flavonoids have been shown to have a wide range of biological and pharmacological activities in in vitro studies: anti-allergic, antiinflammatory, antioxidant, anti-microbial (antibacterial, antifungal, and antiviral),anti-cancer, anti-diarrheal activities. Antioxidant Activity. Flavonoids possess many biochemical properties, but the best described property of almost every group of flavonoids is their capacity to act as antioxidants. The antioxidant activity of flavonoids depends upon the arrangement of functional groups about the nuclear structure. The configuration, substitution, and total number of hydroxyl groups substantially influence several mechanisms of antioxidant activity such as radical scavenging and metal ion chelation ability. The B ring hydroxyl configuration is the most significant determinant of scavenging of ROS and RNS because it donates hydrogen and an electron to hydroxyl, peroxyl, and peroxynitrite radicals, stabilizing them and giving rise to a relatively stable flavonoids radical. Hepatoprotective Activity. Flavonoids are known to be synthesized by plants in response to microbial infection; thus it should not be surprising that they have been found in vitro to be effective antimicrobial substances against a wide array of microorganisms. – 32 –

Flavonoid rich plant extracts from different species have been reported to possess antibacterial activity. Several flavonoids including apigenin, galangin, flavone and flavonol glycosides, isoflavones, flavanones, and chalcones have been shown to possess potent antibacterial activity. Antiviral Activity. Natural compounds are an important source for the discovery and the development of novel antiviral drugs because of their availability and expected low side effects. Naturally occurring flavonoids with antiviral activity have been recognized since the 1940s and many reports on the antiviral activity of various flavonoids are available. Search of effective drug against human immunodeficiency virus (HIV) is the need of hour. Most of the work related with antiviral compounds revolves around inhibition of various enzymes associated with the life cycle of viruses. Structure function relationship between flavonoids and their enzyme inhibitory activity has been observed. Anticancer Activity. Dietary factors play an important role in the prevention of cancers. Fruits and vegetables having flavonoids have been reported as cancer chemopreventive agents. Consumption of onions and/or apples, two major sources of the flavonol quercetin, is inversely associated with the incidence of cancer of the prostate, lung, stomach, and breast. In addition, moderate wine drinkers also seem to have a lower risk to develop cancer of the lung, endometrium, esophagus, stomach, and colon. The critical relationship of fruit and vegetable intake and cancer prevention has been thoroughly documented. It has been suggested that major public health benefits could be achieved by substantially increasing consumption of these foods. Quercetin is known to produce cell cycle arrest in proliferating lymphoid cells. In addition to its antineoplastic activity, quercetin exerted growth-inhibitory effects on several malignant tumor cell lines in vitro. These included P-388 leukemia cells, gastric cancer cells (HGC-27, NUGC-2, NKN-7, and MKN-28), colon cancer cells (COLON 320 DM), human breast cancer cells, human squamous and gliosarcoma cells, and ovarian cancer cells.It has been experimentally proved that increased signal transduction in human breast cancer cells is markedly reduced by quercetin acting as an antiproliferative agent. – 33 –

2.3. Galloylbergenin from Kazakh traditional medicinal plant of Bergenia crassifolia with anti- lipid droplet accumulation activity[45] Bergenia crassifolia (Saxifragaceae) has been used for treatments of bronchitis, gastro-enteritis, diarrhea, hemostasia, and metrorrha-gia in Kazakh traditional medicine [46]. The phytochemical constituents including bergenin, tannins, flavonoids, phenols, polysaccharaide, and cumarines with some pharmacological actions such as antioxidant, antimicrobial, antiviral, anti-inflammatory, diuretic, immunostimulating, and lipase inhibiting activities have also been reported [47-51]. Adipogenesis is a process from fibroblast-like preadipocytes to mature adipocytes. Increases in the mass and accumulation of lipid droplet of adipocytes are observed in cases of severe human obesity which presents a risk to health and may lead to the development of hypertension, hyperlipidemia, diabetes, and cardiovascular disease under the number of pathological disorders[52-53]. Our research for novel lead natural products from Kazakh medicinal plants led to isolation of a new galloylbergenin, 3,11-di-Ogalloylbergenin (1) to show anti-lipid accumulation active along with 4,11-di-O-galloylbergenin (2) [54], 11-O-p-hydroxybezoyl bergenin (3) [55], (+)-catechin 3-O-gallate (4) [51], and (+)-catechin 3,5-di-Ogallate (5) [56] from the roots of B. crassifolia. In this paper, we describe the isolation and structure elucidation of 1, and anti-lipid accumulation activities of isolated compounds (Figure 2.1). 3,11-Di-O-galloylbergenin (1),amorphous powder, [α]D32 – 49.6 (c 0.53, MeOH), showed a molecular formula, C28H24O17, which was determined by HRESIMS [m/z 655.0921 (M+Na)+]. The presence of a carbonyl and a hydroxy group were inferred by the absorptions observed in the IR spectrum of 1 appearing at 1740 and 3380 cm-1. – 34 –

The 1H NMR spectrum of 1 (Table 2.3) suggested the presence of a methoxy group at δH 3.91(s), five oxymethine groups at δH 4.24 (m), 5.26 ( t), 4.22 ( m), 4.26 ( m), and 5.14 ( d), one aromatic proton at δH 7.12 (1H, s), and two 3,4,5-trisubstituted benzoyl groupscharacteristic signals at 7.11 (2H, s) and 7.15 (2H, s).

Figure 2.1. Isolated phenolic compounds from Bergenia crassifolia

The gross structure of 1was deduced from extensive analyses oftwo-dimensional NMR data, including 1H–1H COSY, HSQC, and HMBC spectra in CD3OD (Fig. 2.2). The 1H–1H COSY and HSQC spectra revealed the presence of a partial structure (C-11, C-2 – C-4a, C-10b) as shown in Figure 2.2. The connectivity between aromatic rings and this partial structure was revealed by the HMBC correlations of H-10b to C-2 and C-6a, and H-4a to C-6 and C-10a. The locations of methoxy group and aromatic proton at δH 7.12 (1H, s) were proved to be C-9 and C-7, respectively, by HMBC correlations of 9-OMe to C-9 and H-7 to C-6 (δC 165.5), C-9, and C-10a. Further analysis of the 1H, 13C and 2D-NMR data indicated the structure of 1 was virtually identical to those of a bergenin derivative, 4,11-di-O-galloylbergenin (2) except for difference in downfield chemical shift of H-3 at δH 5.26 (t) and up field shift of H4 at δH 4.19 (m) in 1. The location of two galloyl groups at C-3 and C-11 of bergenin were confirmed by 1H NMR and HMBC spectra as shown in Figure 3. The typical downfield shift of H-3 (δH 5.26) and H2-11 (δH 4.64) due – 35 –

to esterification indicated the position of C-3 and C-11 linked with the galloyl groups(Table 1).11 Moreover, the HMBC correlations of H-11 and H-2' to 1'-CO (δC 168.0), and H-3 and H-2'' to 1''-CO (δC 167.4) also served to conclude positions of the galloyl groups to be at C-3 and C-11.

Figure 2.2. Selected 2D NMR Correlations for 3,11-Di-O-galloylbergenin (1).

The absolute configuration of 1 was determined by the hydrolysis of gallate group. On enzymatic hydrolysis with tannase, 1 gave gallic acid and bergenin to confirm the deduced structure. UV and CD spectral data of bergenin as a h ydrolyte of 1 was in good agreement with authentic sample. Anti-lipid droplet accumulation activityfor 1 - 5 was evaluated. 3,11-Di-O-galloylbergenin (1) and 4,11-di-O-galloylbergenin (2) exhibited moderate anti-lipid accumulation activities with IC50 values of 38.40 µM and 60.50 µM, respectively. As a comparison, berberine used as a positive control showed anti-lipid accumulation activity with IC50 values of 14.18 µM. On the other hand, (+)-catechin 3-Ogallate (4) and (+)-catechin 3,5-di-O-gallate (5), which also possessed one or two galloyl groups, showed practically no anti-lipid accumulation activity. It is interesting to note that the presence of galloyl group in bergenin may play an important role to show antilipid accumulation activity. – 36 –

Table 2.3 1

H&13C NMR Data of 3,11-Di-O-galloylbergenin (1) in CD3OD at 300Ka

Position

δH (J,Hz)]

δC

HMBC

2 3 4

4.24, m 5.26, t, J=8.8 4.22, m

81.2 72.0 73.4

1'-CO

4a 6 6a 7 8 9 10 10a 10b 11 9-OMe 1' 2', 6' 3', 5' 4' 1'-CO 1'' 2'', 6'' 3'', 5'' 4'' 1''-CO

4.26, m

78.7 165.5 119.4 111.4 152.4 142.4 149.3 116.8 74.4 63.7 61.0 120.6 110.4 146.5 140.1 168.0 120.8 110.5 146.6 140.3 167.4

7.12, s

5.14, d, J=10, 4.25, m, 4.67, dd, J=10, 6 3.91, s 7.11, s

7.15, s

6, 10a 6, 9, 10a

2, 6a 3, 1'-CO 9 1'-CO

1''-CO

a

δ in ppm

Extraction and Isolation.The roots of B. crassifolia were extracted with 70% EtOH, and a part (35 g) of the extract was partitioned with n-hexane, CHCI3, n-BuOH, and H2O. The n-BuOH fraction was subjected to an HP-20 column (H2O/MeOH, 0:1 to 1:0), and the 80% MeOH fraction (3.2 g) was further separated by using an ODS column (H2O/MeOH, 8:2 to 0:1) to give 5 fractions. Fractions 3-5 were separated by an ODS HPLC (25% CH3CN/0.1% formic acid) and a Sephadex LH-20 column (CHCl3/MeOH, 1:1) to afford 3,11-di-O-galloylbergenin (1, 7.2 mg), 4, 11-di-O-galloyl– 37 –

bergenin (2,2.1 mg), 11-O-p-hydroxybezoyl bergenin (3,1.7 mg), (+)catechin 3-O-gallate (4, 2.0 mg) and (+)-catechin 3,5-di-O-gallate (5, 9.5 mg).

Scheme 2.1. The extraction and isolation of B. crassifolia

3,11-Di-O-galloylbergenin1, colorless amorphous powder; [α]D32 - 49.6 (c 0.53, MeOH), IR νmax (KBr) 3380 and 1740 cm−1; UV (MeOH) λmax219 (ε69000), and 278 (25400) nm; CD (MeOH) λmax 213 (Δ ε -10.6), 243 (Δ 0), 264 (Δ -2.25), and 288 (Δ 4.96) nm; 1H and 13C NMR data, see Table 1; EIMS m/z 655 M+Na+; HRESIMS [m/z 655.0921 M+Na; calcd for C28H24O17Na, 655.0911]. Enzymatic hydrolysis of 1:A solution of 3,11-Di-Ogalloylbergenin (1, 2.00 mg)in H2O (3 mL) was incubated with tannase (0.5 mg) at 40°C for 8 h. After evaporation, the residue was suspended in MeOH. To identify gallic acid and bergenin, the methanol-soluble portion was subjected to an ODS HPLC (25% CH3CN/0.1% formic acid). The spectral data (UV and CD) of bergenin were compared with authentic sample. – 38 –

Problems 1. Explain: Historically important natural products, Distribution of plant resources in Kazakhstan. Please explain: Kazakh traditional medicine. Development condition of pharmacology in Kazakhstan? 2. Please give definition for: Natural products chemistry, Natural products, and Phytochemistry. 3. Illustration: Major classes of natural products, Definition: Medicinal chemistry,Pharmacognosy. 4. Please making definition and explain: Metabolites, Primary metabolites and Secondary metabolites. 5. Please explain: Development of isolation and purification of natural products. 6. Please make illustration: Phenols, structures, classification and their biological properties. 7. Explain detail: Flavonoids, and their classification, please show structures of each class. 8. Illustration: Extraction and isolation of flavonoids from plant resources, and draw the isolation block scheme, indicate the applied solvents and methods. 9. Please discuss: Chemical and biochemical properties of flavonoids, identification method. 10. Make statement on Galloylbergenin, structure, distribution in plant material, isolation and biological activites.

– 39 –

3. AlKALOIDS

3.1. Introduction, occurrence ,nomenclature and classification Introduction Alkaloids are a group of naturally occurring chemical compounds (natural products) that contain mostly basicnitrogen atoms. This group also includes some related compounds with neutral and even weakly acidic properties. Some synthetic compounds of similar structure are also termed alkaloids. In addition to carbon, hydrogen and nitrogen, alkaloids may also contain oxygen, sulfur and more rarely other elements such as chlorine, bromine, and phosphorus [57]. Alkaloids are produced by a large variety of organisms including bacteria, fungi, plants, and animals. They can be purified from crude extracts of these organisms by acid-base extraction. Many alkaloids are toxic to other organisms. They often have pharmacological effects and are used as medications, as recreational drugs, or in entheogenic rituals. Examples are the local anesthetic and stimulantcocaine, the psychedelic psilocin, the stimulant caffeine, nicotine,the analgesicmorphine, the antibacterial berberine, the anticancer compound vincristine, the antihypertension agent reserpine, the cholinomimeticgalantamine, the anticholinergic agent atropine, the vasodilator vincamine, the antiarrhythmia compound quinidine, the antiasthma therapeutic ephedrine, and the antimalarial drugquinine. Although alkaloids act on a diversity of metabolic systems in humans and other animals, they almost uniformly invoke a bitter taste. The boundary between alkaloids and other nitrogen-containing natural compounds is not clear-cut.Compounds like amino – 40 –

acidpeptides, proteins, nucleotides, nucleic acid, amines, and antibiotics are usually not called alkaloids. Natural compounds containing nitrogen in the exocyclic position (mescaline, serotonin, dopamine, etc.) are usually attributed to amines rather than alkaloids. Some authors, however, consider alkaloids a special case of amines [57]. Characteristics of alkaloids: 1. They are basic in nature due to the presence of nitrogen in their ring. 2. They have complex structures. 3. They have bitter principles. 4. They are mostly obtained from plant materials. 5. They have high pharmacological and physiological activities. Examples of alkaloids are: Quinine – an antimalarial drug isolated from a plant called Cinchonia officialis.Quinine is an antipyretic alkaloid. Its molecular formular is C20H24N2O2. Functional groups present in quinine are: methoxyl -OCH3, hydroxyl -OH, tertiary amine group, etc. Quinineis a natural white crystallinealkaloid having antipyretic (fever-reducing), antimalarial, analgesic (painkilling), and antiinflammatory properties and a bitter taste. It is a stereoisomer of quinidine, which, unlike quinine, is an antiarrhythmic. Quinine contains two major fused-ring systems: the aromaticquinoline and the bicyclicquinuclidine. Quinine occurs naturally in the bark of the cinchona tree, though it has also been synthesized in the laboratory. The medicinal properties of the cinchona tree were originally discovered by the Quechua, who are indigenous to Peru and Bolivia; later, the Jesuits were the first to bring cinchona to Europe. Quinine was the first effective Western treatment for malaria caused by Plasmodium falciparum, appearing in therapeutics in the 17th century. It is pre-dated as a m alarial treatment by the Chinese herbalist's use of Artemisia annua, described in a 4th-century text, a plant from which the antimalarial drug artemisinin was derived. It remained the antimalarial drug of choice until the 1940s, when other – 41 –

drugs such as chloroquine that have fewer unpleasant side effects replaced it. Since then, many effective antimalarials have been introduced, although quinine is still used to treat the disease in certain critical circumstances, such as severe malaria, and in impoverished regions due to its low cost. Quinine is available with a prescription in the United States and «over-the-counter» (in minute quantities) in tonic water. Quinine is also used to treat lupus and arthritis. Quinine was also frequently prescribed in the US as an off-label treatment for nocturnal leg cramps, but this has become less prevalent due to a Food and Drug Administration statement warning against the practice. Quinine is highly fluorescent (quantum yield ~0.58) in 0.1 Msulfuric acid solution and it is widely used as a standard for fluorescence quantum yield measurement.It is on the World Health Organization's List of Essential Medicines, a list of the most important medication needed in a basic health system. Other examples of alkaloids are: morphine, cocaine, heroine, etc. Morphine is highly narcotic and analgesic. Morphine is an opium alkaloid which isolated from the plant Papavera omniferous Occurrence of Alkaloids Like other natural products, alkaloids are found in tissues of plants at point of intense cell activities, found majorly in stems, leaves and roots, seeds and barks of plants. Alkaloids are common in some higher plants such as Rubiaceae, Rotaceae, Papaveraceae, etc. They perform certain functions in plants and animals as a result of their pharmacological activities. Pharmacologically, they acts as chelating agents, in which case they select one metal in preference to another from the soil while rejecting others. They are usually solid, though some exists in liquid form. Nomenclature of Alkaloids There is no systemic nomenclature for alkaloids due to the complexity in their structures; hence trivial names are often employed in the nomenclature of alkaloids. However, the names often end with -ine and this indicates the basic nature of the compound. Sometimes, names of alkaloid depict the source of the alkaloid in question. An example is Nicotine isolated from the plant Nicotina tobaccullum. At times, names of alkaloids indicates the discoverer of such alkaloid or even the society or tradition where – 42 –

such plants originated; e.g. morphine alkaloid came from the name Morphens (the ancient god of Greek) [58-60]. Classification: Alkaloids are classified into two broad classes: Classification based on the chemical structure of alkaloids: Heterocyclic alkaloids-pyrollidine nucleus, pyridine nucleus, piperidine nucleus, pyridine-piperidine nucleus, etc. Classification based on plant source: The classifications based on plant’s source: in this case alkaloids are classified on the basis of the plant source such as the family and the genus. For instance, morphine alkaloid is from Apocynaceae family. Opium alkaloids such as morphine, codeine, nicotine and papaverine are derived from opium plant. Rauwolfia alkaloids is reserpine, derived from Rauwolfia family. Reserpine is an antihypertensive alkaloid. It equally act as tranquilizer. Chintonia alkaloids: quinine, from Cinchonine, etc. Alkaloids are often divided into the following major groups[61]: 1. «True alkaloids», which contain nitrogen in the heterocycle and originate from amino acids. Their characteristic examples are atropine, nicotine, and morphine. This group also includes some alkaloids that besides nitrogen heterocycle contain terpene (e.g., evonine) or peptide fragments (e.g. ergotamine). This group also includes piperidine alkaloids coniine and coniceine although they do not originate from amino acids. 2. «Protoalkaloids», which contain nitrogen and also originate from amino acids. Examples include mescaline, adrenaline and ephedrine. 3. Polyamine alkaloids – derivatives of putrescine, spermidine, and spermine. 4. Peptide and cyclopeptide alkaloids. Pseudalkaloids – alkaloid-like compounds that do not originate from amino acids.This group includes, terpene-like and steroid-like alkaloids, as well as purine-like alkaloids such as caffeine, theobromine, theacrine and theophylline. Some authors classify as pseudoalkaloids such compounds such as ephedrine and cathinone. Those originate from the amino acid phenylalanine, but acquire their nitrogen atom not from the amino acid but through transamination. – 43 –

– 44 –

Tropane derivatives

Pyrrolidine derivatives

1

Class

Cocaine group Substitution in positions 2 and 3

Atropine group Substitution in positions 3, 6 or 7

2

Main synthesis steps

Ornithine or arginine → putrescine → Nmethylputrescine → N-methylΔ1-pyrroline

Ornithine or arginine → putrescine → Nmethylputrescine → N-methylΔ1-pyrroline

3

4

Examples

Table 3.1

Cocaine, ecgonine

Atropine, scopolamine, hyoscyamine

Cuscohygrine, hygrine, hygroline, stachydrine

Alkaloids with nitrogen heterocycles (true alkaloids)

Major groups

Classification and main groups of alkaloids [57]

Some alkaloids do not have the carbon skeleton characteristic of their group. So, galantamine and homoaporphines do n ot contain isoquinoline fragment, but are, in general, attributed to isoquinoline alkaloids. Classifications and major groups of alkaloids, their main synthesis procedures with related examples showing at Table 3.1 below:

– 45 –

Quinolizidine derivatives

Piperidine derivatives

Pyrrolizidine derivatives

1

Ormosanine, piptantine

Ormosanine group

Sparteine, lupanine, anahygrine Matrine, oxymatrine, allomatridine

Lysine → cadaverine → Δ1piperideine

Matrine group

Sparteine group

Cytisine

Coniine, coniceine

Octanoic acid → coniceine → coniine

Cytisine group

Sedamine, lobeline, anaferine, piperine

Lysine → cadaverine → Δ1piperideine

Lupinine, nupharidin

Loline, N-formylloline, N-acetylloline

In fungi: L-proline + Lhomoserine →N-(3-amino-3carboxypropyl)proline → norloline

Platyphylline, trichodesmine

Lupinine group

1-aminopyrrolizidines (lolines)

Macrocyclicdiesters

Indicine, lindelophin, sarracine

In plants: ornithine or arginine → putrescine → homospermidine → retronecine

Complex esters of monocarboxylic acids

4 Retronecine, heliotridine, laburnine

3

Non-esters

2

– 46 –

Isoquinolinederi vatives and related alkaloids [63-64]

Pyridine derivatives

Indolizidine derivatives

1

Protoberberines

Simple derivatives of pyridine Polycyclic noncondensing pyridine derivatives Polycyclic condensed pyridine derivatives Sesquiterpene pyridine derivatives Simple derivatives of isoquinoline Derivatives of 1- and 3isoquinolines Derivatives of 1- and 4-phenyltetrahydroisoquinolines Derivatives of 5-naftilisoquinoline Derivatives of 1- and 2benzyl-izoquinolines Cularine group Pavines and isopavines Benzopyrrocolines

2

Tyrosine or phenylalanine → dopamine or tyramine (for alkaloids Amarillis)

Nicotinic acid, isoleucine

Nicotinic acid → dihydronicotinic acid → 1,2dihydropyridine

Lysine → δ-semialdehyde of αaminoadipic acid → pipecolic acid → 1 indolizidinone

3

Cularine, yagonine Argemonine, amurensine Cryptaustoline Berberine, canadine, ophiocarpine, mecambridine, corydaline

Papaverine, laudanosine, sendaverine

Ancistrocladine

Cryptostilin

N-methylcoridaldine, noroxyhydrastinine

Salsoline, lophocerine

Evonine, hippocrateine, triptonine

Actinidine, gentianine, pediculinine

Nicotine, nornicotine, anabasine, anatabine

Trigonelline, ricinine, arecoline

Swainsonine, castanospermine

4

– 47 –

Isoxazole derivatives

Oxazole derivatives

Erythrina alkaloids Phenanthrene derivatives Protopins Aristolactam

Amaryllis alkaloids

Phthalidisoquinolines Spirobenzylisoquinolines Ipecacuanha alkaloids Benzophenanthridines Aporphines Proaporphines Homoaporphines Homoproaporphines Morphines Homomorphines Tropoloisoquinolines Azofluoranthenes

Ibotenic acid → Muscimol

Tyrosine → tyramine

Ibotenic acid, Muscimol

Annuloline, halfordinol, texaline, texamine

Hydrastine, narcotine (Noscapine) Fumaricine Emetine, protoemetine, ipecoside Sanguinarine, oxynitidine, corynoloxine Glaucine, coridine, liriodenine Pronuciferine, glaziovine Kreysiginine, multifloramine Bulbocodine Morphine, codeine, thebaine, sinomenine Kreysiginine, androcymbine Imerubrine Rufescine, imeluteine Lycorine, ambelline, tazettine, galantamine, montanine Erysodine, erythroidine Atherosperminine Protopine, oxomuramine, corycavidine Doriflavin

– 48 –

Quinoline derivatives

Acridine derivatives

Quinazoline derivatives

1 Thiazole derivatives

Quinines

Furanoquinoline derivatives

Tricyclic terpenoids

Simple derivatives of quinoline derivatives of 2 – quinolones and 4-quinolone

3,4-Dihydro-4-quinazolone derivatives 1,4-Dihydro-4-quinazolone derivatives Pyrrolidine and piperidinequinazoline derivatives

2

Tryptophan → tryptamine → strictosidine (with secologanin) → korinanteal → cinhoninon

Anthranilic acid → 3carboxyquinoline

Anthranilic acid

Anthranilic acid or phenylalanine or ornithine

1-Deoxy-D-xylulose 5-phosphate (DOXP), tyrosine, cysteine

3

Quinine, quinidine, cinchonine, cinhonidine

Dictamnine, fagarine, skimmianine

Flindersine

Cusparine, echinopsine, evocarpine

Rutacridone, acronicine

Vazicine (peganine)

Glycorine, arborine, glycosminine

Febrifugine

Nostocyclamide, thiostreptone

4

– 49 –

Imidazole derivatives

See also: indole alkaloids

Indole derivatives

1

Iboga-type alkaloids Aspidosperma-type alkaloids

Corynanthe type alkaloids

Ergot alkaloids

Pyrroloindole alkaloids

Simple derivatives of βcarboline

Simple indole derivatives

2

Harman, harmine, harmaline, eleagnine

Ergotamine, ergobasine, ergosine

Directly from histidine

Histamine, pilocarpine, pilosine, stevensine

Monoterpenoidindole alkaloids Ajmalicine, sarpagine, vobasine, ajmaline, yohimbine, reserpine, mitragynine, group strychnine and (Strychninebrucine, Tryptophan → tryptamine → aquamicine, vomicine) strictosidine (with secologanin) Ibogamine, ibogaine, voacangine Vincamine, vinca alkaloids, vincotine, aspidospermine

Tryptophan → chanoclavine → agroclavine → elimoclavine → paspalic acid → lysergic acid

Physostigmine (eserine), etheramine, physovenine, eptastigmine Semiterpenoidindole alkaloids

Tryptophan → tryptamine or 5hydroxitriptofan

4 Serotonin, psilocybin, dimethyltryptamine (DMT), bufotenin

Non-isoprene indole alkaloids

3

– 50 –

Muscarine

Colchicine alkaloids

βPhenylethylamin e derivatives

1 Purine derivatives

2

Glutamic acid → 3-ketoglutamic acid → muscarine (with pyruvic acid)

Tyrosine or phenylalanine → dopamine → autumnaline → colchicine

Tyrosine or phenylalanine → dioxyphenilalanine → dopamine → adrenaline and mescalinetyrosine → tyramine phenylalanine → 1phenylpropane-1,2-dione → cathinone → ephedrine and pseudoephedrine

4 Caffeine, theobromine, theophylline, saxitoxin

Muscarine, allomuscarine, epimuscarine, epiallomuscarine

Colchicine, colchamine

Tyramine, ephedrine, pseudoephedrine, mescaline, cathinone, catecholamines (adrenaline, noradrenaline, dopamine)

Alkaloids with nitrogen in the side chain (protoalkaloids)

Xanthosine (formed in purine biosynthesis) → 7 methylxantosine → 7-methyl xanthine → theobromine → caffeine

3

– 51 –

Peptide alkaloids with a 14membered cycle

Peptide alkaloids with a 13membered cycle

Spermine derivatives

Frangulanine, scutianine J Scutianine A

Frangulanine type Scutianine A type

Integerrine, discarine D

Ziziphine A, sativanine H

Ziziphine type

Integerrine type

Nummularine C, Nummularine S

Nummularine C type

From different amino acids

Peptide (cyclopeptide) alkaloids

Verbascenine, aphelandrine

Lunarine, codonocarpine

ornithine → putrescine → spermidine → spermine

Capsaicin, dihydrocapsaicin, nordihydrocapsaicin, vanillylamine

4

Spermidine derivatives

Polyamines alkaloids

Phenylalanine with valine, leucine or isoleucine

3

Paucine

2

Putrescine derivatives

1 Benzylamine

– 52 –

Steroids

Diterpenes

Peptide alkaloids with a 15membered cycle

1

Lycoctonine type

Mucronine A type

Cholesterol, arginine

Mevalonic acid → izopentenilpyrophosfate → geranyl pyrophosphate

Pseudoalkaloids (terpenes and steroids)

Solasodine, solanidine, veralkamine, batrachotoxin

Aconitine, delphinine

Mucronine A

Amphibine B, lotusine C

Amfibine B type

4 Amphibine F, spinanine A

3

Amphibine F type

2

3.2. Properties of alkaloids and Distribution in nature Most alkaloids contain oxygen in their molecular structure; those compounds are usually colorless crystals at ambient conditions. Oxygenfree alkaloids, such as nicotine or coniine, are typically volatile, colorless, oily liquids. Some alkaloids are colored, like berberine (yellow) and sanguinarine (orange). Most alkaloids are weak bases, but some, such as theobromine and theophylline, are amphoteric. Many alkaloids dissolve poorly in water but readily dissolve in organic solvents, such as diethyl ether, chloroform or 1,2-dichloroethane. Caffeine,cocaine, codeine and nicotineare water soluble (with a solubility of ≥1g/L), whereas others, including morphineand yohimbine arehighly water soluble (0.1–1 g/L). Crystals of piperine extracted from black pepper. These salts are usually soluble in water and alcohol and poorly soluble in most organic solvents. Exceptions include scopolamine hydrobromide, which is soluble in organic solvents, and the watersoluble quinine sulfate. Most alkaloids have a bitter taste or are poisonous when ingested. Alkaloid production in plants appeared to have evolved in response to feeding by herbivorous animals; however, some animals have evolved the ability to detoxify alkaloids. Some alkaloids can produce developmental defects in the offspring of animals that consume but cannot detoxify the alkaloids. Distribution in nature Strychnine tree. Its seeds are rich in strychnine and brucine.Alkaloids are generated by various living organisms, especially by higher plants – about 10 to 25% of those contain alkaloids. Therefore, in the past the term «alkaloid» was associated with plants. The alkaloids content in plants is usually within a few percent and is inhomogeneous over the plant tissues. Depending on the type – 53 –

of plants, the maximum concentration is observed in the leaves (black henbane), fruits or seeds (Strychnine tree), root (Rauwolfia serpentina) or bark (cinchona). Furthermore, different tissues of the same plants may contain different alkaloids. Beside plants, alkaloids are found in certain types of fungi, such as psilocybin in the fungus of the genus Psilocybe, and in animals, such as bufotenin in the skin of some toads. Many marine organisms also contain alkaloids. Some amines, such as adrenaline and serotonin, which play an important role in higher animals, are similar to alkaloids in their structure and biosynthesis and are sometimes called alkaloids [59]. 3.3. Extraction and isolation method for alkaloids Isolation of Alkaloids: In order to isolate pure alkaloid from plant source, the procedures to be followed are as follows: Step 1: Collection of plant material, air-drying the plant material to remove water, pulverization of the air-dried plant material and solvent extraction protocol of the powdered plant materials. Step 2: Purification of the alkaloid-This is done to separate alkaloid from the solution. This is done by running chromatography on the syrupy form of the extract. Step 3: involves the crystallization and further purification of the isolates. Crystals of piperine extracted from black pepper, because of the structural diversity of alkaloids, there is no single method of their extraction from natural raw materials.Most methods exploit the property of most alkaloids to be soluble in organic solvents but not in water, and the opposite tendency of their salts. Most plants contain several alkaloids. Their mixture is extracted first and then individual alkaloids are separated. Plants are thoroughly ground before extraction. Most alkaloids are present in the raw plants in the form of salts of organic acids. The extracted – 54 –

alkaloids may remain salts or change into bases. Base extraction is achieved by processing the raw material with alkaline solutions and extracting the alkaloid bases with organic solvents, such as 1, 2-dichloroethane, chloroform, diethyl ether or benzene. Then, the impurities are dissolved by weak acids; this converts alkaloid bases into salts that are washed away with water. If necessary, an aqueous solution of alkaloid salts is again made alkaline and treated with an organic solvent. The process is repeated until the desired purity is achieved [65-66]. In the acidic extraction, the raw plant material is processed by a weak acidic solution (e.g., acetic acid in water, ethanol, or methanol). A base is then added to convert alkaloids to basic forms that are extracted with organic solvent (if the extraction was performed with alcohol, it is removed first, and the remainder is dissolved in water). The solution is purified as described above. Alkaloids are separated from their mixture using their different solubility in certain solvents and different reactivity with certain reagents or by distillation.

Scheme 3.1. Isolation of alkaloid from plant material – 55 –

The extraction procedure often used in the isolation of alkaloid is summarized in the scheme 1 below. 3.4. Dimer alkaloids In addition to the described above monomeric alkaloids, there are also dimeric, and even trimeric and tetrameric alkaloids formed upon condensation of two, three, and four monomeric alkaloids. Dimeric alkaloids are usually formed from monomers of the same type through the following mechanisms: – Mannich reaction, resulting in, e.g., voacamine – Michael reaction (villalstonine) – Condensation of aldehydes with amines (toxiferine) – Oxidative addition of phenols (dauricine, tubocurarine) – Lactonization (carpaine).

Voacamine

Dauricine

3.5. Applications of alkaloids In medicine Medical use of alkaloid-containing plants has a long history, and, thus, when the first alkaloids were isolated in the 19th century, they immediately found application in clinical practice. Many synthetic and semisynthetic drugs are structural modifications of the alkaloids, which were designed to enhance or change the primary effect of the drug and reduce unwanted sideeffects. For example, naloxone, an opioid receptorantagonist, is a derivative of thebaine that is present in opium [67-68]. – 56 –

Many alkaloids are still used in medicine, usually in the form of salts, including the following (Table3.2 ): Table 3.2 Important alkaloids with biological actions Alkaloid Ajmaline Atropine, scopolamine, hyoscyamine Caffeine Codeine Colchicine Emetine Ergot alkaloids

Action antiarrhythmic anticholinergic Stimulant, adenosine receptor antagonist cough medicine, analgesic remedy for gout antiprotozoal agent sympathomimetic, vasodilator, antihypertensive

Morphine Nicotine

analgesic Stimulant, Nicotinic acetylcholine receptor agonist inhibitor of acetylcholinesterase antiarrhythmic antipyretics, antimalarial antihypertensive Muscle relaxant antitumor vasodilating, antihypertensive Stimulant, Aphrodisiac

Physostigmine Quinidine Quinine Reserpine Tubocurarine Vinblastine, vincristine Vincamine Yohimbine

In agriculture Prior to the development of a wide range of relatively low-toxic synthetic pesticides, some alkaloids, such as salts of nicotine and anabasine, were used as insecticides. Their use was limited by their high toxicity to humans.

Thebaine

Naloxone – 57 –

Use as psychoactive drugs Preparations of plants containing alkaloids and their extracts, and later pure alkaloids, have long been used as psychoactive substances. Cocaine, caffeine, and cathinone are stimulants of the central nervous system. Mescaline and many of indole alkaloids (such as psilocybin, dimethyltryptamine and ibogaine) have hallucinogenic effect. Morphine and codeine are strong narcotic pain killers. There are alkaloids that do not have strong psychoactive effect themselves, but are precursors for semi-synthetic psychoactive drugs. For example, ephedrine and pseudoephedrine are used to produce methcathinone and methamphetamine. Thebaine is used in the synthesis of many painkillers such as oxycodone. Problems 1. Definition: What is Alkaloid, show structures, and make short statements on historical important alkaloids. 2. Please make detail discuss on: Classification of alkaloids, show related structures and make examples. 3. What are the chemical and biochemical properties of Alkaloids, and identification methods? 4. Extraction and isolation of alkaloids from plant resources, draw block scheme, please indicated the applying solvents and methods. 5. Explain Protoalkaloids, together with Pyrrolidine, Tropane, and Pyrrolizidine types alkaloids, discuss detail with examples. 6. Explain Piperidine, Quinolizidine, Isoquinoline, and Indolizidine types alkaloids, discuss detail with examples. 7. Explain Pyridine, Oxazole, Isoxazole and Indole alkaloids types alkaloids, discuss detail with examples. 8. Explain: Distribution of alkaloids in plant materials. And applications of alkaloids.

– 58 –

4. TERPENS

4.1. Introduction and classification Terpenoids (or isoprenoids), a su bclass of the prenyllipids (terpenes, prenylqunones, and sterols), represent the oldest group of small molecular products synthesized by plants and are probably the most widespread group of natural products. Terpenoids can be described as modified terpenes, where methyl groups are moved or removed, or oxygen atoms added. Inversely, some authors use the term «terpenes» more broadly, to include the terpenoids. During the 19th century, chemical works on t urpentine led to name «terpene» the hydrocarbons with the general formula C10H16 found in that complex plant product. These terpenes are frequently found in plant essential oils which contain the «Quinta essentia», the plant fragrance [69]. They are universally present in small amounts in living organisms, where they play numerous vital roles in plant physiology as well as important functions in all cellular membranes. The various functions of terpene natural products in the natural world have been reviewed [70]. On the other hand, they are also accumulated in many cases, and it is shown that the extraordinary variety they then display can be due to ecological factors playing an evolutionary role.

Isoprene – 59 –

They may be defined as a group of molecules whose structure is based on a various but definite number of isoprene units (methylbuta-1,3-diene, named hemiterpene, with 5 carbon atoms). Terpenoids are extraordinarily diverse but they all originate through the condensation of the universal phosphorylated derivative of hemiterpene, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) giving geranyl pyrophosphate (GPP) Fig.4.1.

Figure 4.1. Biosynthesis pathways of the terpenoids

In higher plants, IPP is derived from the classic mevalonic acid pathway in the cytosol but from the methylerythritol phosphate pathway in plastids. It is generally accepted that the cytosolic pool of IPP serves as a precursor of sesquiterpenes, triterpenes, sterols and polyterpenes whereas the plastid pool of IPP provides the precursors of mono-, di- and tetraterpenes. Some exceptions have been described showing that interactions between the two biosynthetic pathways may exist. A rational classification of the terpenes has been established based upon the number of isoprene (or isopentane) units incorporated in the basic molecular skeleton (Table 4.1): Table 4.1 Classification of terpens № 1 1 2 3 4 5

Terpenes 2 Monoterpenes Sesquiterpenes Diterpenes Sesterterpenes Triterpenes

Isoprene units 3 2 3 4 5 6 – 60 –

Carbon atoms 4 10 15 20 25 30

6 7

1

Carotenoids Rubber

2

8 > 100

3

40 > 500

4

Mono-, sesqui-, di-, and sesterterpenes contain the isoprene units linked in a head to tail fashion. The triterpenes and carotenoids (tetraterpenes) contain two C15 and C20 units respectively linked head to head. Many terpenes are hydrocarbons, but oxygencontaining compounds such as alcohols, aldehydes or ketones are also found. These derivatives are frequently named terpenoids. Mono- and sesquiterpenes are the chief constituents of the essential oils while the other terpenes are constituents of balsams, resins, waxes, and rubber. Oleoresin is a roughly equal mixture of turpentine (85% C10monoterpenes and 15% C15- sesquiterpenes) and rosin (C20-diterpene) that acts in many conifer species to seal wounds and is toxic to both invading insects and their pathogenic fungi. A number of inducible terpenoid defensive compounds (phytoalexins) from angiosperm species are well known. These include both sesquiterpenoid and diterpenoid types. Isoprenoid units are also found within the framework of other natural molecules. Thus, indole alkaloids, several quinones (vitamin K), alcohols (vitamin E, vitamin A formed from b-carotene), phenols, isoprenoid alcohols (also known as terpenols or polyprenols) also contain terpenoid fragments. The origin of the ubiquitous isoprene unit and its conversion into various compound has been extensively studied.The biogenesis, molecular regulation and function of plant terpenoids has been extensively reviewed by Bouvier F et al.(Prog Lipid Res 2005, 44, 357). According to Bohlmann J., there are in excess of 1000 monoterpenes, more than 7000 sesquiterpenes and more than 3000 diterpenes [71]. 4.2. Monoterpens, essential oils and the methods of extracting essential oils They are the terpenes that have been known for several centuries as components of the fragrant oils obtained from leaves, flowers and fruits. Monoterpenes, with sesquiterpenes, are the main constituents – 61 –

of essential oils. While a few, such as camphor, occur in a near pure form, most occur as complex mixtures, often of isomers difficult to separate. These essential oils have numerous actions, such as allelochemical functions between plants and between plants and predators. A role in wound healing was also observed [72]. Essential oils The answer is terpenes. Terpenes (TUR-peen) are a large class of organic hydrocarbons produced by a wide variety of plants, and are referred to as terpenoids when denatured by oxidation (drying and curing the flowers). They are the main building block of any plant resin or «essential oils» and contribute to the scent, flavor, and colors. Some are even known to have medicinal value. Terpenes are the main class of aromatic compounds found in cannabis and have even been proven to interact synergistically with cannabinoids to provide for a range of different effects. While many people believe that it is the sticky glands of THC (delta9-tetrahydrocannabinol) that provide cannabis with its peculiar aroma, it is in fact the more unstable monoterpenes and sesquiterpenes that are responsible. In fact, it is the smell of the specific sesquiterpene, Caryophyllene oxide that drug dogs are able to detect when probing for cannabis [73]. The methods of extracting essential oils There are many different ways to extract essential oils and they are listed below: – Enfleurage – Expressed Oils – Steam Distillation – Solvent Extraction – Fractional Distillation and Percolation – Carbon Dioxide Extraction – Phytonic Process Enfleurage Enfleurage is one of the oldest methods of extracting essential oils and is rarely used these days because of its high cost. It involves placing the flower petals on a layer of glass that is first spread with a thin layer of fat called «chassis». The volatile oil diffuses into the fat, then the fat is collected and the oil is extracted from the fat using alcohol. – 62 –

Expression of oils When oils are taken from the rind of fruits they are called «expressed oils». This method is cold and does not involve any solvents or heat of any kind. Most of the citrus oils are extracted in this manner including grapefruit, lime, orange and lemon essential oils. These are not technically considered essential oils for this reason, but they are still definitely therapeutic oils. The most important thing to consider with cold pressed oils is the source of the crop since citrus fruits are constantly sprayed with pesticides and it can be very concentrated in the oil [74-75]. Steam distillation method essential oils Steam distillation has been used for hundreds of years and today remains one of the most favorably methods of extracting essential oils. Technically speaking if it not extracted using steam distillation or cold expression it is not a therapeutic grade essential oil. There are actually three different steam processes that are described further in the distilling section. But in all of the methods, steam is used to rupture the oil membranes in the plant and release the essential oil. The steam carries the essential oil to a condenser and then as it re-liquefies the lighter essential oil floats on top. The water and oil is then separated and the oil portion, of course, is the essential oil [73]. Solvent extraction method Solvent extraction is a method of extracting essential oils that is dominated by the perfume industry. And technically does not produce therapeutic grade oils because chemicals such as h exane, acetone, di-methylene-chloride and others are used in the process. Percolation and fractional distillation Fractional distillation separates the volatile oil in different fractions or portions at various boiling points. This is used in oil refineries for distillation of petroleum products and is not suited for therapeutic grade essential oils. Percolation is one of the newer methods of extraction essential oils. It is similar to normal distillation but the equipment is literally upside down. It has been used successfully in France; however sometimes an emulsion is produced that can not be separated out, so until it can be further developed you will not see it on a large scale. – 63 –

Super critical fluid extraction (Carbon Dioxide Extraction) Super critical fluid extraction or carbon dioxide extraction is also a fairly new solvent extraction process that uses carbon dioxide at very high pressure. The carbon dioxide is injected into the tank where the plant material is contained and acts as a solvent to extract the oil. The carbon dioxide is colorless and odorless liquid and can be completely removed which is a good thing; however, there is no research at this point to verify if there are any toxic effects of using this procedure. Additionally, the distilling equipment is extremely expensive. So until more is known about this process, it is advised to use only steam distilled and expressed oils. The Phytonic Process The Phytonic process is a one of the newest methods of extracting essential oils using non-CFCs (non-chlorofluorocarbons). It is also called Florasol Extraction and the oils are referred to as phytols. Acyclic monoterpens They can be considered as derivatives of 2,6-dimethyloctane. Among natural molecules, the followings are well known and have several structural isomers.

2,6-dimethyloctane α-myrcene cis-α-osimene 4-trans-6-alloosimene

Citronellol

Linalool

Nerol

– 64 –

Geraniol

Defensive role of simple terpenes have been demonstrated as for more complex compounds. Ocimene and linalool (with farnesene) were shown to be produced by de novo biosynthesis in plants damaged by insect herbivories [76]. These compounds likely mediate the interaction between herbivores and their natural enemies, attracted by terpenes. Experimentally, a study of Arabidopsis thaliana engineered to overexpress a terpene synthase leading to the emition of large amounts of linalool, which is normally produced only in trace levels. Compared with wild-type A. thaliana, the transgenic plants significantly repelled Myzus persicae aphids [77]. That approach may be a solution to the protection of cultivated plants against insect attacks. Geraniol, produced by geranium, rose and lemon, has been determined to be also an alarm pheromone of the insect Corythucha ciliata which attacks the sycamore tree. A great number of orchid species in the New World tropics have coevolved with the insects Euglossini by producing floral scents, highly attractive to insect males over great distances, for efficient pollination. Ipsdienol has been shown to be the main attractive component of the orchid scent .

Ipsdienol

Monocyclic monoterpens They are derived from cyclohexane with an isopropyl substituent. The most typical are:

limonene γ-terpinene α-phellandrene p-cymene ascaridole pulegone – 65 –

Limonene is an important volatile emitted by the holm oak (Quercus ilex), and acts as allelochemical in inhibiting seed germination of other plant species [78]. About 30,000 tons/year of limonene are extracted from natural sources (turpentine oil) and are used for the syntheses of other optically active products. Bicyclic monoterpens Thuyone is best known for being a toxic chemical (the a form is the most active) in absinthe, a product extract from Artemisia absinthium. Its psychedelic effects consecutive to absinthe consumption is disputed.

car-3-ens sabinene α-thujene

camphor

umbellulol

α-pinene

α-Thujone

Pharmacologically, thujone acts mainly on the GABA receptors in the brain and exhibits psychoactive response. In many countries the amount of thujone allowed in food or drink products is regulated (in Europe, the maximum level tolerated is 25 m g/l). Other plants containing thujone, such as the coniferous Thuja occidentalis, are used in herbal medicine, mainly for their immune-system stimulating effects. Pinene is, as limonene, an allelochemichal emitted by the roots of Quercus ilex. Camphor and pinene are also allelochemicals emitted – 66 –

by Salvia leucophylla [79]. Pinene is among the more readily available optically active substance (about 30,000 tons/year) and is used for the syntheses of other chemical products. Iridoids are a cl ass of bicyclic monoterpenes found in a wide variety of plants and in some animals. They are often intermediates in the biosynthesis of alkaloids. Chemically, iridoids usually consist of a cyclopentane ring fused to a six-membered oxygen heterocycle, as exemplified by nepetalactone, the active ingredient in catnip (Nepeta spp), plant known for the behavioral effects they have on cats.

Nepetalactone

Iridoids are typically found in plants as glycosides, most often bound to glucose. Cleavage of a bond in the cyclopentane ring gives rise to a subclass known as seco-iridoids. Iridoids are found in many medicinal plants and may be responsible for the some of their pharmaceutical activities. Isolated and purified, iridoids exhibit a wide range of bioactivities including cardiovascular, hypoglycemic, anti-inflammatory, antispasmodic, antitumor, antiviral, immunomodulator and purgative activities [80]. They are produced by plants primarily as a d efense against herbivores or against infection by microorganisms. To humans and other mammals, they have also a deterrent bitter taste. It can also be used as a m osquito repellent. As other terpenes, iridoids may function as protective substances in the animal kingdom, especially for insects. These compounds are obtained from the diet or, as t he iridoids of leaf beetles, they are made by the insect itself. Many monoterpenes possess antitumor activity in animal and cell models. They have also antioxidant properties, g-terpene and hydroxytyrosol being among the most effective. – 67 –

4.3. Sesquiterpenes Sesquiterpenoids are defined as the group of 15 carbon compounds derived by the assembly of 3 i soprenoid units and they are found mainly in higher plants but also in invertebrates. Sesquiterpenes, with monoterpenes, are an important constituent of essential oils in plants. They are the most diverse group of isoprenoids. In plants, they function as pheromones and juvenile hormones. Sesquiterpene structures present several acyclic, mono-, bi-, tri-, and tetracyclic systems. Some of natural sesquiterpenoids are shown below. Acyclic compounds The acyclic representative are also called farnesans, term derived from the basic structure, farnesol. Farnesol and nerolidol are very common and are isolated from essential oils of various sources.

Farnesol

Nerolidol

Farnesol is widely distributed in many essential oils such as citronella, neroli, cyclamen, lemon grass, tuberose, rose, musk, and balsam. It is used in perfumery to emphasize the odors of perfumes. Moreover, it is a natural pesticide for mites and is also a pheromone for several insects and mammals, including elephants (teritorial marking, individual recognition, mate attraction). Farnesyl acetate is found in the web of some spiders (Pholcidae) to attract females [81]. Farnesol was also show to be the «quorum-sensing molecule» identified in fungi [82]. The presence of farnesol prevents the yeastto-mycelium conversion, resulting in actively budding yeasts without influencing cellular growth rates. This study is the first to identify an extracellular molecule mediating an eukaryotic quorum-sensing system. Farnesol is active against a v ariety of Candida albicans at concentrations between 1 and 50 m M [83].Farnesol is frequently – 68 –

esterified with one fatty acid having 8 to 12 carbon atoms.Farnesene, an analogue of farnesol, is known to act as an alarm pheromone in aphids. Released during predator attack, it causes aphids to stop feeding, disperse, and give birth to winged (rather than wingless) forms, which leave their host plants. Farnesene is produced by de novo biosynthesis by cotton plants when damaged by insect herbivories [84]. These compounds likely mediate the interaction between herbivores and their natural enemies, attracted by terpenes.

Farnesene

Nerolidol is present in neroli, ginger, jasmine, lavender, tea tree and lemon grass. The aroma of nerolidol is woody and reminiscent of fresh bark. It is used as a flavoring agent and in perfumery. It was also shown to be produced by the leaves of a large number of plant species in response to herbivory insects and then to be transformed into a C11-homoterpene (4,8-dimethyl-1,3,7-nonatriene) which attracts predatory insects [85].

4,8-Dimethyl-1,3,7-nonatriene

Among the acyclic species, two compounds are well known for their importance in invertebrate endocrinology. One, methyl farnesoate is now considered as the crustacean juvenile hormone [86] and is synthesized by the mandibular organs and is present in the hemolymph. Its structure is nearly identical to the other one, the insect juvenile hormone III which regulates metamorphosis and reproduction. Both are synthesized from farnesoic acid in the corpora allata. Farnesoic acid has been determined to be the autoregulatory substance involved in the regulation of the morphological transition – 69 –

of the yeast Candida albicans between a budding form and a multicellular invasive filamentous form[87]. This transformation has been postulated to contribute to the virulence of this organism. These findings might have medicinal value in the development of antifungal therapies.

Cyclic compounds Abscisic acid plays a key role in plants in the regulation of stomatal closure by regulating ion channel activities and water exchanges across the plasma membrane of guard cells.

Abscisic acid

Cyclic ADP-ribose (cADPR) has been shown to mediate signaling of abscisic acid in the drought-stress response leading to – 70 –

activation of gene transcription and to stomatal closure. It was shown that diacylglycerol pyrophosphate plays a role as phospholipid second messenger in abscisic signaling. A review of the signaling network may be found in a paper by Giraudat J and in the «Plant hormones» textbook. Abscisic acid is an end product of neoxanthin or violaxanthine peroxidation and reduction giving an apocarotenal (apocarotenoid) with a short side chain (5 carbons), followed by a final oxidation into an acid form [88-90]. Abscisic acid has also a variety of roles in plant development, bud and seed dormancy, germination, cell division and movement. It induces also storage protein synthesis in seeds and may be involved in defense against insect attack. Abscisic acid is biosynthesized via carotenoids (zeaxanthin, neoxanthin, violaxanthin) in roots and mature leaves. Its direct precursor is xanthoxin which is a natural inhibitor of plant growth. Abscisic acid is ubiquitous in lower and higher plants, it is present also in algae. Only the C2-cis, C4-trans isomer is biologically active. A mechanism of abscisic signaling in connection with cyclic ADP-ribose and calcium movement has been demonstrated to mediate temperature signaling in spongess well as tissue regeneration in Cnidaria. Abscisic acid is not restricted to the plant kingdom and primitive invertebrates since it has been shown to be present in the central nervous system of pigs and rats. Evidence was provided that it is also involved in the stimulation of human granulocytes with cyclic ADPribose as second messenger. This lipid may be considered as a new pro-inflammatory cytokine in humans. Human granulocytes and smooth muscle cells are also functionally activated by abscissic acid. That messenger can be considered as a new signal molecule involved in the development of atherosclerosis [91-93]. Abscisic acid was shown to be a endogenous stimulator of insulin release from human pancreatic b cells with cyclic ADP-ribose as second messenger. This observation suggests that this lipid phytohormone may be involved in the physiology of insulin release, mainly in its dysregulation under conditions of inflammation. Together with jasmonates, abscisic acid was shown to control excitability and closure of the insect trap of Dionaea muscipula (Venus flytrap) [94-95]. Cadalene has the cadinane skeleton and is present in essential oils and in many plants. It is used as a b iomarker in paleobo– 71 –

tanic studies. In connection with retene (1-methyl-7-isopropyl phenanthrene), it enables the estimation in sediments of the importance of Pinaceae in ancient forests. Some important sesquiterpenes

The function of curcumene as insecticide, repellent, and insect feeding deterrent has been previously reported. A parent compound, bisabolol, isolated from essential oils of a variety of plants, showed at low concentration a differentiating effect in endothelial cells but an apoptotic effect at higher concentration. Gossypol, a sesquiterpene dimer found in cotton that is formed from two cadinane units. All the cotton plant contains gossypol. That terpene occurs as a mixture of two enantiomers but each has different biological activities. For nonruminant animals (rodents, chickens, – 72 –

humans), (–)-gossypol is significantly more toxic than the (+) enantiomer. It has anti-cancer properties and inhibits male fertility in humans. In contrast, cotton plants containing high levels of (+)-gossypol are resistant to insect damage. These terpenes must be removed from the plant parts and oil before use as animal foods. Bicyclic sesquiterpenes with a driman unit are widespread in plants, liveworts, fungi and certain marine organisms (sponges). Some drimanes have been identified in petroleum and are probably derived from a microbial source. They have generally potent antibacterial and antifungal activities, and they are toxic to several invertebrates and also in fish. In addition, they deter feeding by i nsects on p lants and by fish on sponges. Capsidiol is a sesquiterpenoid compound that accumulates in tobacco Nicotiana tabacum and chili pepper Capsicum annuum in response to fungal infection. It is considered as a phytoalexin.

Driman squeleton

Capsidiol

Avarol, and its quinone derivative avarone, are biologically active sesquiterpenoids which exhibited antimicrobial and antifungal activities and also active against AIDS virus. They were isolated from a Med iterranean (Dysidia avara) and an Australian sponge (Dysidia spp). They have also potent antileukemic activity both in vitro and in vivo in mice. The 1,4-benzoquinone moietylinked to the sesquiterpene is a common structural feature in a large number of terpenoid compounds which have a large spectrum of biological activities. Besides avarol, several other natural quinones and hydroquinones, such as illimaquinone, nakijiquinone and bolinaquinone, are present in sponges of the order Dictyoceratida. All have cytotoxic and – 73 –

antiproliferative properties which offer promising opportunities for the development of new antitumor agents.

Avarol

(E)-b-Caryophyllene

A review of the cytotoxic termene quinones from marine sponges has been released by Gordaliza M [96]. Rasmann E et al. discovered that insect-induced (E)-b-caryophyllene emission from maize roots attracts nematodes that prey on the attacking insect larvae. 4.4. Diterpenes and Sesterterpens They have 20 carbon atoms and are derived fromgeranylgeraniol pyrophosphate. They are of fungal or plant origin and are found in resins, gummy exudates, and in the resinous high-boiling fractions remaining after distillation of essential oils. The rosin remaining after distilling pine turpentine, for instance, is rich in diterpenoids. In ancient times, conifer exudates were used for caulking boats and waterproofing ropes. Resin secretion is also recognized to be part of the resistance mechanism conifers employ against bark beetles and their associated pathogenic fungi. Diterpenoid groups that are physiologically active include: vitamin A activity (retinol), phytohormones that regulate plant growth and germination, e.g. gibberellin, fungal hormones that stimulate the switch from asexual to sexual reproduction, e.g. tris-poric acid; disease resistance agents (phytoalexins), e.g. casbene and podocarpic acid, the anticancer drug, taxol, from the bark of the yew tree, the cancer promoter, phorbol, and natural cannabinoids [97]. – 74 –

The diterpenes have exceptionally open chain, as found in geranylgeraniol or phytol which forms a part of chlorophyll and the side chain of vitamin E and K, and crocetinwhich is a diacid diterpenoid and the lipid part of the crocins, glycosylated derivatives present in saffron. Examples of diterpene substances are given below:

all trans retinol

– 75 –

Abietic acid is an irritant compound present in pine wood and resin. It is the most abundant compound present in rosin, the solid fraction of the oleoresin of coniferous trees. It is mainly used to make lacquers and varnishes and metal resinates. These resinates are produced in reacting abietic acid (or a similar compound) with a metal salt (gold, indium, nickel, palladium, platinum, silver) and used in a wide variety of applications where high purity metals in organic solution form is needed (gravure printing inks, vitrifiable colors, antifouling agent, dryers for paints and varnishes). Tanshinones are abietane diterpenes, isolated mainly from Salvia miltiorrhiza (Lamiaceae), a plant largely used in traditional Chinese medicine for the treatment of cardiovascular and inflammatory diseases. Among them, tanshinone I is an apoptose inductor and displays several anticancerous biological properties [98]. Several analogues have been synthesized for clinical trials.

Tanshionone I

Steviol

Steviol is the aglycone of stevia's sweet glycosides, one of them being formed by replacing one hydrogen atom (bottom) with glucose via an ester link, and another hydrogen atom (top) with a disaccharide (glucose and rhamnose). The steviol glycosides are responsible for the sweet taste of the leaves of the stevia plant (Stevia rebaudiana, Asteraceae). These compounds are 40 to 300 times sweeter than sucrose. They are developed to be used in sweet drinks. Retene is present in tars obtained by distillation of resinous wood, it is an important pollutant eliminated by the paper factories. This diterpene is present in geological sediment where it is formed by diagenesis from abietic acid, several intermediates having been. – 76 –

Thus, with cadalene (sesquiterpene), retene, a diterpenoid dehydrogenation product, is used in paleobotanic to estimate the importance of ancient pine forests.

Retene

Dehydroleucodine

Gibberellins are a family of compounds, over 130 members exist. The most important in plants is gibberellin A1 which is responsible for stem elongation. The most widely available compound is gibberellic acid (one double bond in the right cycle). Among the physiological properties, gibberellins are involved in stem growth, seed germination and fruit setting and growth. Dehydroleucodine was isolated from Artemisia douglasiana, a popular medicinein Argentina and was shown to have several physiological and therapeutic properties: anti-proliferative activity in G2 phase [99], cytoprotective agent for gastric ulcers and a general antioxidant. Cafestol and kahweol are present in high concentrations (up to 18% diterpene esters) in the oil derived from coffee beans. The only difference between cafestol and kahweol is an extra double bond present in the second cycle of kahweol. These diterpenes are esterified with one fatty acid (C14 to C24), palmitic and linoleic acids being the major esterified fatty acids. Terpenes were also shown to induce an elevated plasma cholesterol content in human.

Cafestol

kahweol – 77 –

Phorbol is a diterpene isolated in 1934 from croton oil (seeds of Croton tiglium). Various fatty acid esters of phorbol have important biological properties, the most notable of which is the capacity to act as tumor promoters through activation of protein kinase C as they mimic diacylglycerols. The most common phorbol ester is phorbol12-myristate-13-acetate (PMA), which is used as a research tool in models of carcinogenesis.

Phorbol

Δ9-Tetrahydrocannabinol (THC)

Cannabinoids are a group of diterpenes present in Cannabis (Cannabis sativa L). All these substances are structurally related to tetrahydrocannabinol (THC) and are able to bind to specific cannabinoid receptors. Tetrahydrocannabinol, also known as Δ9-THC or Δ9-tetrahydrocannabinol, is the main psychoactive substance found in the plant. It was isolated by Mechoulam R et al. in 1964 [100]. Sesterterpens They are derived from geranylfarnesol pyrophosphate and have 25 carbon atoms (the sester- prefix means half to three, i.e. two and a half.). These relatively rare lipids were first isolated from insect protective waxes and from fungal sources. Now, they are known to be widespread, they have been isolated from terrestrial fungi, lichens, higher plants, insects, and various marine organisms, especially sponges.

Geranyl farnesol – 78 –

They exhibit diverse biological properties such as antiinflammatory, cytotoxic, anticancer, antimicrobial, and anti-biofilm activities. The structure of the most recent sesterterpenoids and their activities have been reviewed [101]. Three examples of sesterterpenes are shown below.

Ophiobolin A, a fungal metabolite and a phytotoxin stimulates the net leakage of electrolytes and glucose from maize seedling roots and was found to be a potent inhibitor of calmodulin-activated cyclic nucleotide phosphodiesterase [102]. Gascardic acid is the first sesterterpenes that was isolated in 1965 from the secretion of an insect, Gascardia madagascariensis. Variously unsaturated and branched sesterterpenes, known as Haslenes, were found in species of diatomaceous algae [103]. They have 25 c arbons and 3, 4 and 5 double bonds. They are widely distributed and abundant in marine sediments. Several haslenes were found to be produced by a pennate diatom Haslea ostrearia according to the culture temperature and were shown to have cytostatic properties. One of them, with only one double bond and known as IP25, is shown below. Haslene-C25 monounsaturated isoprenoid hydrocarbon (IP25).This compound has been detected in sea ice in Canadian Arctic and was shown to be a specific biomarker for studying the Quaternary Arctic ice history. The monitoring of this lipid appears – 79 –

valuable for reconstruct paleo-sea ice distributions and accurate calibration of climate prediction models [104-105].

Haslene

4.5. Triterpenoids and Steroids They form a large group of natural products which includes steroids and consequently sterols, they derived from C30 precursors. Nearly 200 different triterpene skeletons are known from natural sources and represent structurally cyclization products of Squalene which is the immediate biological precursor of all triterpenoids. In 1934, Robinson proposed a direct cyclization of squalene to the steroid molecule. In 1936, Nobel laureate researcher Paul Karrer described the biochemical structure of squalene for the first time. It has major specificities, which are related to anticancer properties, the maintenance of the oxidation/antioxidation balance, and its antiaging capabilities. Shark liver oil is considered the richest source of squalene, accounting for at least 40% of its weight. It is also widely distributed in nature, in lesser proportions in amaranth oil (6-9%), in wheat germ oil, and in olive oil (usually from 0.4% up to 1% in extra virgin olive oil). It has been shown that the human body synthesizes an average amount of 1.5 g/day [106]. Biochemistry, molecular biology, and applications of squalene, and the origin of their skeletal diversity have been reviewed [107].

Squalene – 80 –

Squalene epoxide (2,3-oxidosqualene) is produced by the enzyme squalene epoxidase which use NADPH and oxygen to oxidize squalene. This metabolic step is the first in sterol biosynthesis leading to the formation of lanosterol or cycloartenol.

Squalene epoxide

Squalane is a completely saturated derivative of squalene. Present in sebum, it is largely used as a component in many cosmetic products. It is obtained by hydrogenation of squalene extracted from olive oil. The large group of steroids, including sterolsare present in very small amounts in bacteria but at larger amounts in plants and animals while the hopanoids are very abundant in prokaryotes where they replace cholesterol. Triterpenoids are widely distributed in edible and medicinal plants and are an integral part of the human diet. They are being evaluated for use in new functional foods, drugs, cosmetics and healthcare products. Screening plant material has identified fruit peel and especially fruit cuticular waxes as promising and highly available sources [108]. Amyrin isomers are the predominant compounds in tomato fruit wax, with quantities that vary considerably in different cultivars and during the subsequent stages of fruit development. A methyl ether derivative of an amyrin isomer, miliacin, has been proposed as a sedimentary molecular tracer of the history of Panicum (broomcorn millet) cultivation. Among the large number of triterpenoid structures, some of them are shown below. Serratane-type compounds constitute an important group of pentacyclic triterpenes with an unusual seven carbons ring. They – 81 –

are synthesized through the cyclisation of bis-epoxy-squalene and not from epoxy-squalene, the precursor of more common pentacyclic triterpene. More than 100 different serratenoids (serratenes and derivatives have been reported, most having been identified in club mosses, conifers, and in few angiosperms (Primulaceae and Leguminosae).

Cycloartenol

Lanesterol

β-Amyrin

Friedelin

Serratene

As opposed to other serratenoids, methoxy-serratenes appear constrained to conifers, and more specifically to Pinaceae (mainly Pinus, Picea) [109]. In addition to diterpenoids, they potentially constitute a new powerful tool for palaeoenvironmental reconstruction. Steroids are modified triterpenes which derived also from squalene by cyclization, unsaturation and substitution. The nucleus of all steroids is the tetracyclic C17 hydrocarbon 1,2-cyclopentanoperhydrophenanthrene (gonane or sterane) substituted by methyl groups at C10 and C13, as well as an alkyl side-chain at C17. – 82 –

Steroids may possess a nucleus derived from the former one by one or more C-C bond scissions or ring expansions or contractions. Gonane and three examples of basic unsubstituted steroids are shown below:

Unsaturated steroids with most of the skeleton of cholestane containing a 3β -hydroxyl group and an aliphatic side chain of 8 or more carbon atoms attached to position 17 forms the group of sterols. The hopanoids are pentacyclic triterpenoids based on the hopane skeleton (with a f ive-membered ring E) are widely distributed in prokaryotes but were not detected in Archaea[110]. In most cases, the hopanoid content of the cell is comparable with the cholesterol content of eukaryotic cells. They are considered as membrane rigidifiers. Furthermore, they are the precursors of several derived compounds (homohopanoids) in sediments and oils and thus could be considered as the most abundant natural products on earth [111]. Although oxygen is not required for hopanoid biosynthesis, the vast majority of known hopanoid producers are aerobic or microaerophilic bacteria [112]. Thus, hopanoids are found predominantly in aerobic methanotrophs, heterotrophs and cyanobacteria, but have also been found in some anaerobic bacteria. The simplest C30 hopanoid is diploptene.

Hopane

Diploptene – 83 –

Cyanobacteria are presently the only known bacteria to synthesize abundant 2-methylhopanoids with an extended polyhydroxylated side chain. The most abundant hopanoids are C35 bacteriohopanepolyols in which the side-chain of the parent structure contains a variable number of vicinal hydroxyl groups

The hopanoids are widely distributed in bacteria and blue-green algae where they are important cell membrane constituents. It is often said that hopanoids are the «most abundant natural products on Earth». As hopanoids are very stable and are not easily degraded, they are receiving an intense attention as biological markers with applications for geochemical studies of petroleum source rocks and oils. These biomarkers are mainly derived from bacterial bacteriohopanepolyols (biohopanoids). The 2-methylhopane derivatives are abundant in organic-rich sediments as old as 2,500Myr. These lipids may help constrain the age of the oldest cyanobacteria and the advent of oxygenic photosynthesis. They could also be used to quantify the ecological importance of cyanobacteria through geological time. More recent investigations have shown that the production of substantial quantities of polyhydroxylated 2-methylhopanoids may occur in phototrophs other than cyanobacteria and that their biosynthesis does not require molecular oxygen [113]. Aminohopanoids not very different from the previous one have been identified in several methylotrophic bacteria [114]. One of the most abundant is a aminohopane pentol, mixed with other parent compounds (triol and tetrol). This C30 aminopentol is a possible precursor of the widespread C29 hopanoid chemical fossils. – 84 –

Different bacterial groups possess recognizable biohopanoid distributions, giving hopanoids marker potential for specific bacterial populations and environmental conditions. More than 150 individual hopane derivatives have been isolated from various types of sedimentary organic matter. Other pentacyclic triterpenoids based on the lupane skeleton include a v ery large number of naturally occurring members with different functional groups which are found in vegetables and fruit. Among them, lupeol is one of the most ubiquitous compounds.

Aminobacteriohopane

Lupeol was shown to have various pharmacological properties, inducing anti-inflammatory and anti-arthritic responses and inhibition of tumor growthin animal cells. Long-chain fatty acid esters (C20, C22 and C26) of lupeol extracted from an African plant, Holarrhena floribunda (Apocynaceae), were shown to have strong antimalarial activity, especially against drug-resistant strains of Plasmodium falciparum [115-116]. Fatty acid esters (palmitic or stearic acid) of lupeol have been isolated from green propolis produced by honeybees from vegetative apices of the Asteraceae Baccharis dracunculifolia from Brazil [117]. These compounds were named procrim a and b.

Lupeol – 85 –

Palmitic acid ester of lupeol (Procrim a)

Ursolic acid is present in numerous plants and is the best known triterpene from the ursane group. It amounts at more than 1 g per 100 g dry weight in Thymus, Rosmarinus, Salvia, Lavandula, and Eucalyptus. It is found at high concentration in coffee seeds and in apple fruit (mainly in apple peels). It is used in cosmetics as an antiinflammatory, antibacterial and antifungal drug. Ursolic acid is also able to inhibit some form of cancer, particularly multiple myeloma [118].

Ursolic acid

Oleanolic acid

A compound very similar to ursolic acid, oleanolic acid, is widely distributed in food and medicinal plants. It is hepatoprotective and exhibits antitumor and antiviral properties. It is the main constituent of grape berry cuticular waxes [119]. Its presence could be at the origin of the numerous health benefits, including protection against heart disease, ascribed to the moderate consumption of wine. Oleanolic acid and another parent compounds, maslinic acid, are concentrated in olive cuticular waxes, representing 31–44 % and 55–68 % of total wax extract, respectively. These compounds could be related to the numerous health-promoting properties, such as – 86 –

anticancer, antihyperglycemic and antiparasitic activities, reported for olive consumption. As ursolic acid but in contrast with the hopane series, which possesses a f ive-membered ring E, the gammacerane skeleton is characterized by a six-membered ring (see below).

Tetrahymanol

Tetrahymanol (gammaceran-21a-ol), a typical representative of the series, was first isolated from the ciliate protozoan Tetrahymena pyriformis. Later, it was detected in a number of other eukaryotes, e.g. in ferns, fungi and some other ciliates. Its occurrence was long thought to be restricted to eukaryotes but its presence in sediments pointed out a much more widespread distribution in living organisms. The finding of tetrahymanol in the purple nonsulfur bacterium Rhodopseudomonas palustris opened new insights into the biochemistry of these molecules in bacteria. Tetrahymanol and novel methylated homologues were discovered in nitrogen-fixing bacterium Bradyrhizobium japonicum [120-121]. Gammacerane structures were shown to be reliable geochemical indicators for water column stratification in marine or in lacustrine deposits [122].

Tetrahymanol

Gammacerane

– 87 –

Celastrol is a hopanoid triterpene extracted from a plant used in the Chinese medicine, Tripterygium wilfordii. That plant is useful to treat inflammatory and autoimmune diseases. New research has demonstrated that it is a potent proteasome inhibitor and can help in the treatment of prostate cancer [123]. Several biochemical properties bring about several investigations in the field of cancer treatment or prevention. Its antioxidant and anti-inflammatory properties has been the basis of research on diseases induced by monocytes and macrophages activation, including neurodegenerative process [124]. 4.6. Carotenoids The nature of these compounds was discovered during the 19th century. In 1831, Wachen roder H. proposed the term «carotene» for the hydrocarbon pigment he had cristallized from carrot roots. Berzelius J. called the more polar yellow pigments extracted from autumn leaves «xanthophylls» and Tswett M., who separated many pigments by column chromatography, called the whole group «carotenoids». Among this important group, the numerous compounds consist of C40 chains (tetraterpenes) with conjugated double bonds, they show strong light absorption and often are brightly colored (red, orange). They occur as pigments in bacteria, algae and higher plants. Carotenoids perform three major functions in plants: accessory pigments for light harvesting, prevention of photooxidative damage and pigmentation attracting insects. The hydrocarbon carotenoids are known as carotenes, while oxygenated derivatives of these hydrocarbons are known as xanthophylls. To date, 700 carotenoids have been identified, of which only 50 are regularly consumed in the human diet and 24 have so far been detected in human plasma. In Western diets, the most abundant carotenoids are the three oxygenated xanthophylls lutein, zeaxanthin and b-cryptoxanthin and the three major carotenes, acarotene b-carotene and lycopene. The human intake of carotenoids may be appreciated using databases such as that establlished for Swiss vegetables [125]. – 88 –

Carotenoids are important components of the light harvesting in plants, expanding the absorption spectra of photosynthesis. The major carotenoids in this context are lutein, violaxanthin and neoxanthin. Additionally, there is considerable evidence which indicates a photoprotective role of xanthophylls preventing damage by dissipating excess light. In mammals, carotenoids exhibit immunomodulatory actions, likely related to their anticarcinogenic effects. b-Carotene was thus shown to enhance cell-mediated immune responses. The decrease in prostate cancer risk has been linked to the consumption of tomatoes, vegetable rich in lycopene, as p rostatic tissues. While there is yet limited direct evidence linking lycopene and prostate cancer, several observations, including the ability of the prostate to concentrate lycopene, suggest a special protection of lycopene against that pathology [126-128]. Carotenoids consist of eight isoprenoid units joined in such a manner that the arrangement of isoprenoid units is reversed at the center of the molecule so that the two central methyl groups are in a 1,6-position relationship and the remaining nonterminal methyl groups are in a 1,5-position relationship. They are, by far the predominant class of tetraterpenes. They may be also classified in the terpenoids. Carotenoids can be considered derivatives of lycopene, found in tomatoes, fruits and flowers. Its long straight chain is highly unsaturated and composed of two identical units joined by a double bond between carbon 15 and 15'. Each of these 20 carbon units may be considered to be derived from 4 isoprene units. Lycopene is a bioactive red colored pigment naturally occurring in plants. Interest in lycopene is increasing due to increasing evidence proving its antioxidant activities and its preventive properties toward numerous diseases. In vitro, in vivo and ex vivo studies have demonstrated that lycopene-rich foods are inversely associated to diseases such as cancers, cardiovascular diseases, diabetes, and others. A review of all these aspects may be consulted. Carotenoids may be acyclic (seco-carotenoids) or cyclic (mono- or bi-, alicyclic or aryl). Oxyfunctionalization of various carotenoids leads to a large number of xanthophylls in which the function may be a hydroxyl, methoxyl, carbonyl, oxo, f ormyl or epoxy group. – 89 –

Only some of the most common carotenes and xanthophylls are given below: Phytoene is one of the first intermediates in the biosynthesis of carotenoids. It is formed by coupling of two molecules of geranylgeranyl pyrophosphate by the action of the phytoene synthase. The diphosphate is removed and proton shift leads to the formation of phytoene. It is a colorless product and absorbs light in the UV range only. Dietary phytoene is accumulated in human skin where it can potentially protect the skin (UV absorber, antioxidant, anti inflammatory). Among the represented molecules, – 90 –

b-carotene is probably the most important as a precursor of vitamin A by central cleavage into retinol and some retinoic acid (b-carotene provides about 40% of human dietary retinol equivalents). The majority of carotenoids are still manufactured chemically, but, currently, the microbial production of b-carotene is of commercial importance. Blakeslea trispora is one of the best fungal strain employed for the production of b-carotene, commercially utilized by DSM. B. trispora produces up to 3% carotene per cell dry weight. B. trispora is also employed for the production of natural lycopene, commercialized by Vitatene. The marine algae Dunaliella salina is also employed for manufacturing of b-carotene. Carotene and lycopene are known to improve skin properties when ingested as supplements or in vegetal products.They protect against sunburn, increasing the basal defense against UV lightmediated damage to the skin, although their efficacy is not comparable to the use of a sunscreen [129]. In human serum several carotenes and xanthophylls have been detected. If a- , b-carotene and lycopene are frequently quoted in specialized papers, some others are now determined with precise HPLC methods (lutein, zeaxanthin, cantaxanthin and b-cryptoxanthin).

Xanthophylls – 91 –

These compounds originate from ingested fruit, green leaves, berries and yellow corn. Egg yolk usually contains about 175400 mg of lutein and about 200-300 mg of zeaxanthin, but this could be affected by hen's feed composition. Up to 18% losses of xanthophylls have been observed after various cooking methods [130-131]. Lutein has its maximum absorption at 450 nm, cryptoxanthin at 453 nm and zeaxanthin at 454 nm. Lutein and zeaxanthin are the only xanthophylls found in human serum that are present in retina, macula (the central region of the eye (they are frequently referred to as macular pigment), and lenses. Thus, they are at the origin of the name of the central part of the retina, macula lutea (yellow spot). By absorbing blue-light, the macular pigments protect the underlying photoreceptor cell layer from light damage, possibly initiated by the formation of reactive oxygen species. Increasing the intake of lutein or zeaxanthin may prove to be protective against the development of age-related macular degeneration [132]. Several nutritional investigations have suggested that lutein supplements may improve visual function and optical density in aged people [133]. Natural aromatic derivative of lutein, 3,3'-dihydroxyisorenieratene and its non-hydroxylated parent compound, isorenieratene, are produced by green sulfur bacteria and by Brevibacterium linens, a bacteria used in the dairy industry for production of smear cheese.

3,3'-Dihydroxyisorenieratene (R1, R2 = OH) Isorenieratene (R1, R2 = H)

Their high antioxidant activity against free radicals and their specific ability to prevent oxidatively generated damage to DNA make them interesting compounds for the development of endogenous photoprotective systems [134].

– 92 –

Astaxanthin, present in yeast, microalgae, crustaceans and fish, is a natural nutritional component, used as a food supplement for human and animal consumption. Its commercial production comes from both natural and synthetic sources.

Astaxanthine

In vitro, astaxanthin is several fold more active as a free radical antioxidant than b-carotene and a-tocopherol. In animal models, its modulates immune response, inhibits cancer cell growth, and reduces bacterial load and gastric inflammation in vitro and rodent models. In humans, dietary astaxanthin decreases a DNA damage biomarker and acute phase protein, and enhances immune response [135-137]. Ketocarotenoids belong to the xanthophyll group and are quite unique in nature. Among these compounds, echinenone and canthaxantin are abundant in cyanobacteria are characteristic of cyanobacteria [138]. Violaxanthin is an epoxidized derivative of antheraxanthin (hydroxylated cryptoxanthin) and forms with zeaxanthin the xanthophyll cycle that is said to protect the photosynthetic system of plants against damage by excess light.

Violaxanthin

Two cycloaddition products of trans-violaxanthin with atocopherol have been isolated from seeds of Pittosporum tobiraand their structures elucidated. These C69 carotenoids were named pittosporumxanthins. Their global structure is given below. – 93 –

Pittosporumxanthin

Further investigations have revealed the existence of six other forms based on addition of a-tocopherol with antheraxanthin, neoxanthin or violaxanthin [139]. Fucoxanthin, as an allenic carotenoid with a 5 ,6-monoepoxide group, is one of the most abundant carotenoid in brown algae and diatoms. This compound contributes more than 10% of the estimated total production of carotenoids in nature.

Fucoxanthin

This carotenoid has shown interesting biological properties, such as anticarcinogenetic effects, anti-inflammatory effects and radical scavenging activity. Furthermore, it has been shown that fucoxanthin has an anti-obesity effect in connection with the expression of the uncoupling protein UCP1 in white adipose tissue. Its ability to enhance the docosahexaenoic acid concentration in the liver of treated obese mice remains to be examined more thoroughly. A great part of the biological properties of fucoxanthin may be related to its ability to take up pe roxynitrite through the formation of nitrofucoxanthin and the inhibition of the nitration of tyrosine by peroxynitrite. Therefore, fucoxanthin may have the potential to reduce the risk of cancer induced by reactive nitrogen species [141-142]. Neoxanthin is another common allenic carotenoid, widely – 94 –

distributed in higher plants and algae, which was first isolated from the green leaves of barley in 1938 by Strain HH. Neoxanthin is thought to be a part of the light harvesting complexes in thylakoids and as a precursor to the plant growth hormone abscisic acid.

Neoxanthin

More than 40 allenic carotenoids have been described in vegetals and accumulated in animals. An unusual carotenoid ester was identified in fresh mango lipids as violaxanthin dibutyrate. Another unusual acetylenic carotenoid was discovered and identified in a marine sponge (Prianos osiros) [143-145].

That new molecule was shown to be strongly cytotoxic toward cultured human colon tumor cells. Specific carotenoids are found in mineral sediments or crude oils and are used as biomarkers. Among them, isorenieratane, found in oils of Devonian age, is derived from the carotenoid isorenieratene (an homologue of the previous one but with an unsaturated isoprenoid chain), which is synthesized by photosynthetic green sulfur bacteria (Chlorobiaceae). – 95 –

Isorenieratane

Another C40 carotenoid, paleorenieratane, also thought to be derived from the same bacteria has been identified in Devonian aged sediments and crude oils[146].

Paleorenieratane

These green sulfur bacteria are strict anaerobes that require light and hydrogen sulfide in stratified water columns to carry out photosynthesis and are thus markers for these photic zones (euxinic) in depositional environments. C50-carotenoids, including bacterioruberin which is present in halophilic Archaea, characterize the colored saltern ponds [147].

Bacterioruberin

Although green leaves contain unesterified hydroxy carotenoids, most carotenoids in ripe fruit are esterified with fatty acids. However, those of a few fruits, particularly those that remain green when ripe, such as kiwi, undergo limited or no esterification. Carotenoid mono- and diesters were identified in mandarin essential oil, in Haematococcus pluvialisand in the Antarctic krill Euphausia superba. – 96 –

Carotenoids fatty acid esters were also described in the carapace of the spiny lobster Panulirus japonicus. Astaxanthin, adonixanthin, and pectenolone were esterified with fatty acids with a large range of chain length and unsaturation.Several esterified seco-carotenoids, the tobiraxanthins, have been isolated from the seeds of Pittosporum tobira. Two fatty acid molecules (lauric or myristic acid) acylated the carotenoid part. One of these compounds is shown below (Tobiraxanthin A1) [148-149].

Tobiraxanthin A1

4.7. Essential oils from Kazakh traditional medicinal plant of Thymus altaicus Introduction The traditionally natural products have played an important role in developing of natural product chemistry which continues to expand to exciting new frontiers of great importance in medicine. In Kazakhstan 49 genuses, 247 species of plants are growing that belong to family Lamiaceae and genus Thymus have around 27 species [150]. Thymus altaicus has efficiently been used in the treatments of cold and cough with the properties of immunostimulant, expectorant, diuretic and detumescence in Kazakh traditional medicine [151]. Essential oils (EO) are also known as volatile oils, which is a concentrated hydrophobic liquid containing volatile aroma compounds and internationally defined as the product obtained by hydrodistillation, steam distillation or dry distillation or by a suitable mechanical process without heating (for Citrus fruits) of a plant or of some parts of it. Essential oils of aromatic plants and spices are used – 97 –

in industries for production of bath products, cosmetics, soap, perfumes and toiletries. Many of them are also used in traditional medicine for various purposes such as aromatherapy for the purpose of altering one's mood, cognitive, psychological and physical wellbeing . The volatiles constitutes were extracted from the aerial parts of Thymus altaicus by water steam distillation were analyzed by GCMS method. Fifty six compounds were separated. Their relative contents were determined by area normalization in which 47 volatiles were identified. The major volatile oils of T. altaicus are 3-cyclohexene-1methanol, α,α-4-trimethyl- (35.84%), benzene, 1-mehtyl-2-(1- methylethyl)(15.24%), β-myrcene (10.30%), thymol (7.94%), 1,4-cyclohexadiene, 1-methyl-4- (1-methylethyl)- (5.34%), caryophyllene (5.26%), 1,6-octadien-3ol,3,7- dimethyl(4.78%), 2,6-octadien1-ol, 3,7-dimethyl-(E)- (2.06%), 2,6octadien-1-ol,3,7- dimethyl-, acetate, (E)- (1.31%), caryophyllene oxide (1.23%). Materials and Method Plant material: Thymus altaicus was collected in Altay region of east Kazakhstan,in July 2012 and identified by Prof. Shen Guan Min from the Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences. The oils were isolated by water-distillation for 4 hrs and then dried over anhydrous sodium sulphate. GC-MS analysis:the aerial part of Thymus altaicus were analyzed byElectron Impact Ionization (EI) method on Perkin-Elemer Autosystem XL –TurboMass (Gas Chromatograph coupled to Mass Spectrometer) fused silica capillary column (30m x 2.5mm; 0.25 μm film thickness), coated with PE-5 ms were utilized. The carrier gas was helium (99.999%). The column temperature was programmed from 60°C (held for 5 m in), at 2°C/min to 180°C, at 3.5 C /min to 290°C. – 98 –

The latter temperature maintained for 40 min (Acquisition parameters full scan; scan range 40-350 amu). The injector temperature was 310°C. Injection: with a 0.1ul: detector ion source (EI-70eV). Samples were injected by splitting with the split ratio 1:60. Identification of the compounds:Identification of compounds was done by comparing the NIST and Wiley library data of the peaks and mass spectra of the peaks with those reported in literature. Percentage composition was computed from GC peak areas on PE-5 ms column without applying correction factors[152-154]. Volatile oils from the aerial parts of Thymus altaicuswere analyzed by GC-MS. Fifty six compounds were separated. Their relative contents were determined by area normalization. The yield from whole herbs of Thymus altaicus was found to be 5.53 %. Table 1 report the composition of the volatile oils of the aerial parts of T. altaicus. Forty seven components have been identified in the volatiles (Table 4.2) of T. altaicus which the major constituents are 3-cyclohexene-1-methanol, α,α,4-trimethyl- (35.84%), benzene, 1-mehtyl-2-(1- methylethyl)- (15.24%), β-myrcene (10.30%), thymol (7.94%), 1,4-cyclohexadiene,1-methyl- 4-(1-methylethyl)- (5.34%), caryophyllene (5.26%), 1,6-octadien- 3-ol,3,7-dimethyl- (4.78%), 2,6-octadien- 1-,3,7-dimethyl-, (E)- (2.06%). According to the report 3-cyclohexene-1-methanol, α,α,4-trimethyl-(α-terpineol) (35.84%) shows antioxidant effects and antiseptic is present in many extracted oils of various plant species, acts as an antihypernociception and anti-inflammatory [155]. And second major volatile constituent benzene, 1-mehtyl-2-(1- methylethyl)- (15.24%) showed antimicrobial and antibacterial activates [156-157]. β-myrcene (10.30%) and essential oils containing this terpenoid compound are widely used as a fragrance in cosmetics, as a scent in household products, and as a flavoring additive in food and alcoholic beverages [158]. Furthermore, it was reported that β-myrcene is an analgesic substance and the active principle of lemongrass (Cymbopogon citratus Stapf) 'abafado', an infusion made with the pan covered in order to prevent the loss of volatile constituents [159]. Lemongrass 'abafado' is widely used in Brazilian folk medicine as a sedative and as a remedy for gastrointestinal disorders [160]. – 99 –

Table 4.2 The volatile constituents of aerial parts of Thymus altaicus

5

Cont ent (%） 6

C10H16

136

0.72

16.17

C10H16

136

0.32

18.46

C10H16

136

0.77

20.96

C10H16

136

0.20

21.81

C10H16

136

0.60

24.92

C10H16

136

10.30

26.10

C10H16

136

0.45

26.93

C10H16O

152

0.09

27.56

C10H16

136

0.42

28.43

C10H18O

154

0.66

31.66

C10H16

136

5.34

34.10

C10H14

134

15.24

34.63

C10H16

136

0.05

14

Eucalyptol 1,4-Cyclohexadiene,1-methyl-4-(1methylethyl)Benzene, 1-methyl-2-(1-methylethyl)Bicyclo[4.1.0]hepta-2-ene, 3,7,7trimethyl 1-Octen-3-ol

48.93

C8H16O

128

0.31

15

Terpineol, Z-, beta-

50.54

C10H18O

154

0.05

16

Camphor

56.01

C10H16O

152

0.58

17

1,6-Octadien-3-ol, 3,7-dimethyl 1,6-Octadien-3-ol, 3,7-dimethyl-, acetate Germacrene d

57.82

C10H18O

154

4.78

58.84

C12H20O2

196

0.91

60.75

C15H24

204

0.03

61.27

C12H20O2

196

0.16

62.14

C11H16O

164

0.10

63.13

C15H24

204

5.26

64.16

C10H16O

152

0.04

Pea k No. 1

Constituents

tR (min)

2

3

Molecular Formula 4

l-R,α-pinene Bicyclo[3.1.0]hex-2-ene, 2-methyl-5(1-methylethyl) Camphene

16.03

8

β-Pinene Bicyclo[3.1.0]hexene, 4-methylene-1(1-methylethyl) β-Myrcene Cyclohexene, 1-methyl-4-(1methylethylidene) 2,3-Dehydro-1,8-cineole

9

Limonene

10

1 2 3 4 5 6 7

11 12 13

18 19 20 21 22 23

Bornyl acetateBenzene, 2-methoxy-4-methyl-1-(1methylethyl)Caryophyllene 2-Methyl-6-methylene-oct-3,7-dien-2ol

– 100 –

MW

1

2

3

4

5

6

24

Pulegone

67.17

C10H16O

152

0.16

25

α-Caryophyllene

69.10

C15H24

204

0.29

26

69.89

C10H16O

152

0.22

71.40

C10H18O

154

35.84

72.74

C15H24

204

0.11

73.14

C12H20O2

196

0.10

74.06

C10H16O

152

2.06

75.57

C12H20O2

196

1.31

32

2-Octadienal, 3,7-dimethyl-, (Z)3-Cyclohexene-1-methanol-α,α-4trimethyle Naphthalene, 1,2,34,4A,5,6,8Aoctahydro-7-methyl 2,6-Octadien-1-ol, 3,7-dimethyl-, acetate, (Z)2,6-Octadienal, 3,7-dimethyl-, (E)2,6-Octadien-1-ol, 3,7-dimethyl-, acetate, (E)2,6-Octadienal, 3,7-dimethyl-, (Z)-

78.86

C10H16O

152

0.80

33

2,6-Octadien-1-ol, 3,7-dimethyl-, (E)-

82.50

C10H18O

154

0.91

34

Benzenemethanol, α, α- 4-trimethyl-

82.87

C10H14O

150

0.12

35

Cis-Z-α-bisabolene epoxide

89.47

C15H24O

220

0.32

36

Trans-Z-α-bisabolene epoxide

89.76

C15H24O

220

0.12

37

94.18

C15H24O

220

1.23

98.20

C15H24O

220

0.05

39

Caryophyllene oxide 12-Oxabicyclo[9.1.0]dodeca-3,7-diene, 1,5,5,8-tetramethyl Cuminol

101.6

C15H14O

150

0.71

40

Thymol

107.0

C10H14O

150

0.62

41

Tau-muurolol

107.8

C15H26O

222

0.15

42

α-Farnesene

108.4

C15H24

204

0.02

43

Phenol, 2-methyl-5-(1-methylethyl)-

109.0

C10H14O

150

0.27

44

α-Cadinol Tetracyclo[6.3.2.0(2,5).0(1,8)]tridecan9-ol, 4,4-dimethyl 1H-3A,7-methanoazulene, octahydro1,4,9,9-tetramethyl Aromadendrene oxide-(2)

110.8

C15H26O

222

0.39

115.3

C15H24O

220

0.21

117.6

C15H26

206

0.08

120.2

C15H24O

220

0.23

27 28 29 30 31

38

45 46 47

Problems 1. Definition: What is terpenoid, show structures, and make short statements on some important terpenoids. 2. Classifications of terpens, detail discuss and write related structures of each type. – 101 –

3.

Extraction and isolation of terpens from plant resources, and to draw block scheme, indicate the applied solvents and methods. 4. Please describe the methods of obtaining essential oil from plant resources. Make short statements for each method. 5. Describe the classification of sesquiterpens, write structures of some important sesquiterpens and biological activities. 6. Definition: Diterpenoids, explain with biosynthesis, structure and properties. 7. Structures of abietic type of terpen, distribution in plant material, and biological activities. 8. Definition: Triterpenoids, explain with biosynthesis, structure and properties. 9. Distribution of ursolic acid and oleanolic acid in plant material, they belong to which class of natural products, structure, and biological activities. 10. Compare the carotenoids with other terpen compounds, show their structure, and explain about properties.

– 102 –

5. LABORATORY WORKS FOR THE COURSE OF CHEMISTRY OF NATURAL COMPOUNDS

Laboratory work 1 Obtaining pectin from citrus fruits Pectin is found in the cell walls of plants and often rich found in fruits and vegetables. When it is heated with acid and sugar then taken jelly-like mass. Jelly properties of pectin used in food industry as marmalade, jam, and other manufacture of products. As raw material for pectin are apples, citrus fruit peel, sunflower heads, etc. is used.Pectin is a high molecular compound (molecular weight 200,000). Its structural basis of methyl alcohol installments esterified poligalakturon acid.

On the basis pectin with araban and galactan polysaccharides and partially can be linked with phosphoric acid. By repeated deposition of pectin with Alcohol, all the impurities can be removed. The necessary reagents and materials: Peel of citrus fruit ………. …50 g Hydrochloric acid (0.03N) …. 200 ml Alcohol ……………………..500ml Lead acetate, ammonia, sugar, citric acid. Orange, lemon or tangerine skin cut off with meat cutter, put in – 103 –

fabric, and deposition in glass with alcohol, for cleaning compound of essential oils, pigments, etc. closing glass to 60-700C and boiled in water bath for 1 hour. Then Buchner squeeze the fabric Buchner funnel, pour alcohol again. This operation, alcoholic extracts repeated until the color turns very pale yellow. Washed mass putted to 500 ml flask and pour 200 ml of 0.03 N hydrochloric acid, heat in boiling water bath for 1 hour. Hot solution filtrate through the cotton, wash waste filter with hot water twice. Cooled (indicator litmus) neutralize with ammonia, and then evaporate water to steam in 60-80 ml. Add the remaining syrup two volume of alcohol. Then centrifuge of resulting crude pectin. If necessary to take very clean product, dissolve the pectin with a small amount of warm water, alcohols settle again and over the sediment to hour glass, and air dry. After sharing of alcohol, measure the sediment and calculates the output. Qualitative reactions: Galakturon acid test (by Ehrlich). Dissolve in 3-4 ml of water pectin (on the edge of a spatula), add a few drops of 10% of basic lead acetate, and heated in boiling water bath. If the precipitate formed at the beginning, turns orange over time, then there are acid. Testing pectin’s jelly-like properties. Add to the pectin 50 ml of water, filled with porcelain plate, for a certain time due to swelling. After adding 25 g of powdered sugar, sand bath for 10-15 minutes thoroughly heated. Boiled with 1 m L of 40% citric acid to the mixture, pour the mix into the porcelain or plastic forms. Take 2-3 hours after the jelly is ready to taste. Questions: 1. Technique experiment: what will you learn? 2. What conclusions will you reach? Explain. 3. Please making discussion about chemical and biological properties of pectin.

Laboratory work 2 Qualitative analysis of polyphenols of plantmaterials Measure on the scale of 100 g of raw plantmaterials; add 80% aqueous-alcoholic solution, left for a day. Filter the solution and – 104 –

repeat the method on. Joint extract divided into three: one chat extract used for qualitative analysis of polyphenols with paper chromatography (extract 1), the remaining two (2 and 3 extract) used to study the acid phenol.

1- extract is concentrated under vacuum at 40°C. The solvent system for paper chromatography: 1. n-Butanol: Acetic acid: Water (BAW, 40: 12.5: 29); 2. 15% sulfuric acid. Identification phenols by using the following reaction: 1. Complex formation with 1% aqueous solution of ammonium iron alum (IAK). Phenols with ortho-dihydroxyl groups are colored green, and three rows of hydroxyl groups in blue. 2. The reaction of nitrogen (azo) addition, leading to the formation of dyed nitrogen (azo) additives. A) Reaction with dinitro sulfanyl acid. Before use, add 0.3% sulfanyl acid solution of 8% hydrochloric acid solution, several drops of a 5% solution of sodium nitride and stir. Chromatogram sprinkle this solution and then 20% sodium carbonate solution. B) Diazote n-nitroaniline. Mix 1.5 m l of 0.5% n-nitroaniline rastora in 2N HCl and 0.5 ml of an aqueous solution of sodium nitride. Chromatogram sprinkle this solution and then with 15% aqueous sodium carbonate solution. 3. Solution of 1% vanillin in concentrated hydrochloric acid. Meta-dioxy group (resorcinol, phloroglucinol) in phenols are painted in red. 4. The oxidation reaction of silver oxide. Stir in the ratio 1:1 solution of 0.1N silver in nitric acid, 5N aqueous ammonia – 105 –

solution. Chromatogram sprinkle this solution and then dried in an oven at 105°C for 3-5 minutes. Phenols are defined spots. The procedure for determining the chromatogram: after drying in air chromatogram (under the hood), its viewing under UV light, tag number and the light of certain things. Then, one determines the chromatogram of 1% sodium IAK tick certain color patches determined diazo chromatogram second reagent, the third solution of silver in nitric acid to ammonia. 5. Ammonia solution flavones, flavonols, flavanonols painted in yellow color, and change in the orange or red when heated. Chalcones and aurons are colored red or purple color. Net catechins not colored. Anthocyanins with ammonia or sodium carbonate are painted in blue or purple. 6. 1% solution of vanillin in concentrated hydrochloric acid, catechins are painted in red-strawberry color (phloroglucinol and resorcinol). This reagent proanthocyanidins (flavan-3, 4-diol; dimers in the A and B groups are painted in the color of a strawberry. Flavones, flavonols and their glycosides are painted in bright yellow color. Preparation of acid phenol. 2 extract was concentrated to a small residue is then added in the same volume of 5% aqueous sodium bicarbonate solution. Phenol acid form salts are readily soluble in water. Phenols extradited ethyl acetate. The aqueous portion was oxidized to the acid environment and extradited phenol acid ethyl acetate. Washed several times in an isolation flask to separate minerals ethyl acetate extract. Evaporate the ethyl acetate extract to minimum volume and the residue examined by paper chromatography. To determine the phenol acids by paper chromatography is used (including listed above, the system of solventsto phenols) Parts 1 and 2 (two systemic chromatography) and 3 (solvent system): 1. benzene: acetic acid: water (6: 7: 3 the organic phase) 2. Sodium formate: formic acid: water (10: 1: 200) 3. 1-Butanol: acetic acid: water (CCU 8: 2: 2) Determination. 1. The chromatogram browsing the UV light. All oxykorich acid reflected in blue. – 106 –

2. The chromatogram splash JACQUES solution. Acid with a hydroxyl-colored in green and blue three color hydroxyl. 3. Sprinkles diazo sulfanyl acid and n-nitroaniline diazote. Questions: 1. Do your results agree with your expectations? explain! 2. Discuss how the presence of different functional groups impact Rf (why do some compounds move further up the plate than others?) 3. Look up t he compound structures, consider what factors influence movement on Paper chromatography or a TLC plate? solvent polarity or intermolecular forces.

Laboratory work 3 Isolation and identification catechine from green tea The tea leaves contain catechins and their galloesters. Major impurity components: L – epicatechin, L – gallocatechin, their esters with acids gal. For example, a composition tanids Ceylon tea leaves: L – epicatechin 6.5%, gallocatechin 24.2%, epicatechingallat 9%, 49% gallocatechingallat. Tannin contents are 22-24%in green tea, 14-17% in black tea.

(+)-Catechin 3-O-gallate (1)(+)-Catechin 3,5-di-O-gallate (2) (1) Cathchin C15H14O6, M/W 290 g/mol, (2) Gallocatechin C22H18O10, M/W 442g/mol.

Tea catechins are not hydrolyzed. When heated with dilute acids are converted to insoluble solids, called flobafens. They are active as – 107 –

vitamin P, i.e. are the drugs strengthen capillaries and also help to absorb ascorbic acid in the body. The Materials and Reagents: Green tea ……………… 100 g Ethylacetate ……………60 ml Lead acetate, iron chloride solutions, vanillin sulfuric acid. In a 500 ml flask was put not fermented green tea, add 300 ml of hot water and heat to reflux for about one hour. Cotton to filter the solution in the funnel, the residue treated with water a second time. Add to associate lead acetate solution until complete precipitation of lead tanate. Filter the resulting solution was saturated color. Add a 1% solution of sulfuric acid to give an acidic medium. Filter the resulting white precipitate. Extradited filtrate 3 times with ethyl acetate in 20 ml. The ethyl acetate portion pour in a porcelain dish in advance by measuring its weight, and dry in the bath to dry. The resulting tannin crumble, and measure the weight.Yield should be about3-4 g Tannin, obtained from green tea, soluble in water and alcohol, amorphous powder. Qualitative reactions: dissolve a portion of tannin in the water, pour into 3 tubes and analyzed by qualitative reactions to determine catechines contents. 1. Iron salts (1% or FAK or 1% FeCl3) - painted in black and green. 2. 1% vanillin dissolved in concentrated hydrochloric acid – red color. Questions: 1. Do your findings agree with your expectations? Explain. 2. What is catechin and its role in plants?

Laboratory work 4 Isolation rutin and quercetin from buckwheat’s leaves Rutin is a citrus flavonoid glycoside found in many plants including buckwheat, the leaves and petioles of Rheum species, and asparagus. Rutin has biological activity as P vitamin, if it is – 108 –

insufficient, blood roots became weak and capillaries are flow faster.

When it is hydrolyzed in comfortable conditions, i.e. by rhamnodiastaza or 10% of acetic acid, rutin aglycone is decomposed to quercetin or rarely occurring disaccharide – rutinose. When rutin is hydrolytically decomposed by mineral acids, disaccharide is resolved to D-glucose and L-rhamnose. Quercetin was found out in many plants’ peel. The reagents and materials: Dry leaves of buckwheat ……………20g Ethyl alcohol ………….......................150ml Ether …………………………………20ml 20g of buckwheat is occupied to the 250ml graduated flask and add 150ml of 70% of alcohol, during 1 hour to hold reflux condenser under water bath and heat it. Then solution is separated by Buhner funnel, and to be washed by 10ml of ether two times. Taken products are analyzed by paper chromatogram in one system with standards. Paper chromatogram is filled to buthanol-1; acetic acid; water (BAW, 40:12,5;29) solutions system. Yield 0.3-0.4 g. From water, rutin trihydrate is crystallized, m.p is 192 ͦ C. Rutin is precipitated by absolute methyl alcohol, m.p. is 198 ͦ C. Rutin is poorly soluble in water and acetone, not soluble in benzene, ester and chloroform. Quercetine or 3,5,7,3,4-pentaoxiflavone dissolves 0.4g of rutin in 2 ml alcohol, then add 20ml 1% of HCl. After 30-40 min, quercetin is precipitate, due to water and alcohol it’s crystallized in hydrate form. – 109 –

Identification reactions: 1. Rutin and quercetin give green color with iron chloride solution. 2. With concentrated sulfur acid give green fluorescence yellow color. Questions: 1. What is the experiment about? What will you learn? 2. Biological properties of rutin and quercetin? What differences and relations between them?

Laboratory work 5 Isolation nicotine from a cigarette (tobacco) Nicotine 3-(1 – methyl – 2-pirrolidinil)pyridine – the main alkaloid of tobacco and shag, meets in the form of citrate. As a nicotine is especially poisonous it do not use in medicine. In agriculture it is used as an insecticide. And also is the main material for receiving niacin and its derivate.

The reagents and materials: Shag or cigarette, g...... 100 Ether, ml............................... 100 Hydrated lime, g................... 15 Oxalic acid, g........................ 20-30 Sodium hydroxide, hydrochloric acid, 2% solution of silicontungsten acid, methyl iodine, methanol, picric acid, acetic acid, nbutyl alcohol, Dragendorff reagent. On the mortar thoroughly pulverized tobacco with slaked lime. The mixture was transferred into a flask and make steam distillation (until stopped to form a precipitate with 2% solution of silicontungsten acid). To the taken distillate add oxalic acid (powder) to form an acidic medium (checked with Congo indicator). The solution – 110 –

was poured into a porcelain dish and evaporated in a water bath to form syrup. In the cooled residue oxalate of nicotine and other salts tobacco alkaloid are precipitated. Filter the precipitate, squeeze and shift into a separating funnel. To divide the free alkali treats the residue with 30% solution of alkali and separate 3-4 times with ether. Ether is dried and distilled on potassium hydroxide. Nicotine is highly soluble in organic solvents and in water and just received nicotine odorless, colorless, and as oil. If his store he gradually turns to black. Methods for determination and separation of alkaloids Alkaloids in plants are in the form of salts. Quantitative determination of alkaloids consists of three parts: a) Extraction of alkaloids from plants materials. b) Purification of alkaloids from resins, pigments, oils, pectin. c) Determination of the purified alkaloids. The composition of some alkaloids includes sulfur. Many alkaloids contain oxygen are solid crystals, in some cases, amorphous and odorless. An alkaloid berberine – yellow, sanguinary – orange. Some alkaloids that do not contain oxygen-volatile liquids and they are distilled with water. Alkali alkaloids are readily soluble in organic solvents and do not dissolve in water. Because alkaline properties, they form salts with acids. These properties used in separation and purification. Table 5.1 Qualitative reactions to the main alkaloids: №

Names of reagents

1

2

Composition of reagents 3

Results of reactions 4

1

Mayer

Solution of mercury dichloride and potassium iodide

White or yellowish precipitate

2

Wagner – Bouchard

Potassium iodide iodine solution

Brown precipitate

Dragendorff

Solution of bismuth nitrate and potassium iodide in the presence of acetic acid

Yellow-red or brick-red precipitate

3

– 111 –

1

2

3 Cadmium iodide solution of potassium iodide

4 White or slightly yellow precipitate is soluble in excess solution

4

Marmet

5

Silicotungstic acid solution

White precipitate

6

Solution of phosphomolybdic acid

Yellow precipitate, changes over time in the blue or green

7

Solution of phosphotungstic acid

White precipitate

8

Solution of picric acid

Yellow precipitate

These qualitative definitions show the presence of alkaloids, and to determine the exact group of alkaloids should be carried out to determine the refined and pure alkaloids. For specific reactions of concentrated sulfuric and nitric acid, and sulfuric acid containing formalin (Marquis reagent), ammonium molybdate (Froehde reagent). In recent years, the structures of alkaloids to be studiedby using the methods of gas chromatography, UV, IR, and NMR spectroscopy. Most alkaloids are stored in A-order. Questions: 1. Do your results agree or disagree with your expectations? what do you expect? if your results do not agree, offer an explanation 2. What do y ou know about alkaloids? Draw the structure of the common alkaloids 3. How alkaloids are subdivided?

Laboratory work 6 Obtaining caffeine from tea in laboratory Caffeine is a main derivative of purine. Its amount in leaves of tea is 3%, in the seeds of coffee is 6%. Milled coffee farm treats the caffeine from dried tea leaves and branch pieces, and also from powders of tea. It can be also produced by artificial. In medicine caffeine is used as stimulant of heart works. – 112 –

The reagents and materials: Tea and tea powders ………………50 g Magnesium powder ………………….25 g Chloroform ……………………………….150 ml Hydrogen chlorine, ammonia, codeine and hydrogen double powder Small dried and cut tea or tea powders add magnesium powder (225g of magnesium oxide in 150 ml water), 250 ml of water and heat it about 10-15min. Connected water solution is washed by 25 ml of diluted sulfur acid (by kongo indicator check medium acid media) and in water bath it’s concentrated till it remains one part of three. Hot solution is separated by layer filter, divided by chloroform 5 times. To each extract spent 30ml of solvent. At the first chloroform extract is diluted till a few milliliters by alkaline, then is washed by water the same amount. Solvent is distilled in water bath. From sediments take the crude caffeine, it is divided by crystallization from 8-10 ml of water. Yield should be about 0.8-1 g. Distillation caffeine from dried tea. To put small amount of dried tea into clock glass and cover another one. To observe distillation of caffeine through accurately heating. It is cleaved to upper side of glass in thin, needle form. Qualitative reactions. To 10 mg of caffeine add 5% of hydrogen double powder, a drop of 25% HCl and to dry in water bath. Sediment is divided into 2 parts. The first part is immediately wetted by ammonia, it is colored to purple. The second part is colored to light blue by adding 3 drops of water and 5 mg of codeine. Questions: 1. What did you observe during qualitative reaction? How can you explain it? 2. What amount caffeine presents in tea and coffee? 3. Please discuss about chemical and biological properties of caffeine. – 113 –

Laboratory work 7 Isolation of Carotene from carrots Previtamin carotene A is found in nature in the form of three isomers. It is necessary for organisms to live. Carotene produced in the production of vitamin.

β-carotene

The necessary reagents and materials: Carrot, 5kg NaOH, 10g Carbon tetrachloride, 400 ml Petroleum ether, 200ml Methyl alcohol, 500ml Chloroform, 400ml Infusorial earth, 30g Magnesium oxide, 20g Taking of Coagulation: completely washed carrots by ground meat grinder. It is separated into the fabric, and each part can manually squeeze out the juice. Compressed puree takes into cauldron and added 1 L water and stirred for 40 min. of intense mixing with mechanical mixture. And then get caught again compresses solution. Add the juice of two part solution, the protein product of thermal coagulation.It is a mix of juice jug with a temperature of 60-70 °C water bath heated before.Hot juicy in no hur ry to settle. Coagulate output of 300-400 g wet. Crystal carotene: New infused coagulate is a separating funnel. 10 g NaOH and 100 g stirred tetrachlorate. Extraction 4 times, added solution is washed with distilled water, 30-40 degrees inert gas supply is kept in a vacuum. – 114 –

In the flask with a remainder like warm oil add 30g of dry infusorial earth. The flask is shacked and mixed so it is not adhere to the mass for an hour. During adsorption to get rid of grease hardly lathering mixtures it is filtered and mixed with petroleum ether (b.p. 50-60 ºC) Filtration is conducted under non strong pressure of inertia gas. In this purpose the flask is closed with lid with a hole. The hole is continued by ball bladder which is full of Carbon dioxide or nitrogen. Wash adsorbent 2 times. Precipitant is replaced into the flask equipped with reflux condenser. To fill 150 ml of methyl alcohol and to heat 2-3 min then hot mixture is separated by that filter. By two times washing we can make away with sterols. Carotene is divided by several portions of chloroform from adsorbent. Chloroform is distilled under the vacuum 30 ºC until dry state. The sediment is dissolved by heating in 10 ml of chloroform. To the solution add 200 ml of methyl alcohol rapidly. To filter precipitant by glass filter, wash by hot methyl alcohol and again dissolve in 10 ml of chloroform and add 200 ml hot methyl alcohol to precipitate it. Taken crystal form of carotene is dried in vacuum desiccator. Yield should be about 0,3-0,4 g. Questions: 1. How do you think the obtaining results are coincided with theoretical part? Explain your answer. 2. What kind biological activates you know about carotene? 3. Please write and explain the structure of carotene.

Laboratory work 8 Isolation and qualitative analysis saponins from plant materials There are three types of reactionsfor determining the saponins from plant resources: 1. Reactions based on physical properties of saponins; 2. Reactions based on chemical properties of saponins; 3. Reactions based on biological properties of saponins; – 115 –

Second group of qualitative reaction for saponin is deposition and color reaction. Saponins precipitate with barium and magnesium hydroxides, salts of iron, lead acetate. Also triterpene saponins precipitate in average salts, steroids in lead acetate. Alcohol extract of triterpene and steroid saponins precipitate in alcohol extract of cholesterols. For qualitative reaction of saponins will take 100 ml flask and add 1 g of plant. Then pour 1 ml of water, connect with reflux condenser and heat in water bath. After that filtered and taken solution. Then we use our solution for qualitative reactions. 1. Bubble formation reaction. Take 2 test tubes, to the first pour 5 ml of 0,1N HCl, to the second pour 5 ml of 0,1N NaOH. For each tube add 2-3 drops of plant extract and mix. We can see existing of triterpene saponins in plant by constant and big volume of bubble in tubes. If in the structure of plant exists steroid saponins bubble in alkali media will be more. 2. To the tube add 2 ml of water extract and lead acetate. Precipitating occur. 3. To 1 m l of saponin extract added alcohol solution of 1% cholesterol. We take white precipitation. 4. Lafont reaction. To 2 ml of water extract add 1 ml of ulfuric acid, 1 m l of ethanol, 1 drop of 10% solution of iron sulfate and heat. While heating blue-green color appear . 5. To 2 ml of water extract added 10% solution of NaNO3 and 1 drop of concentrated H2SO4. After that red-blood color is appearing. 6. Liebermann Burchard reaction. For this reaction investigating substance dissolved in acetic anhydride and added concentrated sulfuric acid (50:1). After some time that pink color will be changed to blue. 7. Hemolysis. To 1 ml of isotonic solution add 1 ml of 2% erythrocyte. Blood give light red color. – 116 –

8. Thin-layer chromatography. Aqueous or alcohol extract testedby chromatography on silica gel or aluminium oxide. For neutral saponins is used n-butanol – sulfuric acid – water, for acid saponins n-butanol – in various ratio water solution of ammonia. Steroid saponins in a chromatogram with 1% solution of threechloric antimony, concentrated sulfuric acid, and acetic anhydride give yellow gouts. At present the chromatographic method is more precise method of determination of saponins. Questions: 1. What did you actually observe from experiment? Report you actual findings. 2. Do your results agree with your expectations? yes or no why or why not? Be specific. If needed, identify possible sources of error 3. Give the explanation of saponins and their structures.

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6. THE EXAMINATION QUESTIONS FOR COURSE OF CHEMISTRY OF NATURAL COMPOUNDS 1. Explain: Historically important natural products, Distribution of plant resources in Kazakhstan. 2. Please explain: Kazakh traditional medicine. Development condition of pharmacology in Kazakhstan? 3. Please give definition for: Natural products chemistry, Natural products, and Phytochemistry. 4. Illustration: Major classes of natural products, Definition: Medicinal chemistry,Pharmacognosy. 5. Please making definition and explain: Metabolites, Primary metabolites and Secondary metabolites. 6. Please explain: Development of isolation and purification of natural products. 7. Chromatography – what does it mean? History of chromatography? 8. Principles and types of chromatography. Explain with applications. 9. Liquid chromatography, types, which kind of natural compounds and material could be analyzed by the method. 10. Making statements on: Adsorption chromatography, Partition chromatography, types, and applications. 11. Please explain: Paper chromatography, Thin layer chromatography, Column chromatography. 12. What is Rf value? What does it mean that difference of Rf value? Explain the affecting factors to Rf value. 13. Please Make illustration: Phenols, structures, classification and their biological properties. – 118 –

14. Explain detail: Flavonoids, and their classification, please show structures of each class. 15. Illustration: Extraction and isolation of flavonoids from plant resources, and draw the isolation block scheme, indicate the applied solvents and methods. 16. Please discuss: Chemical and biochemical properties of flavonoids, identification method. 17. Please make statements on Glycosides, their classifications, and structures. 18. Definition: Carbohydrate, Discuss about groups of carbohydrates, show structures with Fischer and Haworth projections explain detail with examples. 19. Make a account for flavonoids glycosides and write reaction of Hydrolysis of glycosides, related structures, and properties. 20. Please make explain: Monosaccharaides, properties, ringstraight chain isomerism, show structures and explain detail with examples. 21. Please give definition for Amino acids, write the structure, and explain the naming methods and properties with sample. 22. Making illustration and compare the essential and nonessential amino acids, and basic set of 20 amino acids. 23. Definition: What is Alkaloid, show structures, and make short statements on historical important alkaloids. 24. Please make detail discuss on: Classification of alkaloids, show related structures and make examples. 25. What are the chemical and biochemical properties of Alkaloids, and identification methods? 26. Extraction and isolation of alkaloids from plant resources, draw block scheme, please indicated the applying solvents and methods. 27. Explain Protoalkaloids, together with Pyrrolidine, Tropane, and Pyrrolizidine types alkaloids, discuss detail with examples. 28. Explain Piperidine, Quinolizidine, Isoquinoline, and Indolizidine types alkaloids, discuss detail with examples. 29. Explain Pyridine, Oxazole, Isoxazole and Indole alkaloids types alkaloids, discuss detail with examples. – 119 –

30. Definition: What is terpenoid, show structures, and make short statements on some important terpenoids. 31. Classifications of terpens, detail discuss and write related structures of each type. 32. Extraction and isolation of terpens from plant resources, and to draw block scheme, indicate the applied solvents and methods. 33. Definition: Diterpenoids, explain with biosynthesis, structure and properties. 34. Definition: Triterpenoids, explain with biosynthesis, structure and properties. 35. Definition: Fatty acids? What are the key chemical properties of fatty acids? 36. Please make a st atement on marine natural products, introduction, structure, properties. 37. Make an account of classification of glycosides, show with structures, and tell about physico-chemical properties of glycosides. 38. Introduction of anthocyanines and flavones, structures. Distributions in plant materials. Biological activates. 39. What is essential oil, distribution in plant material, main compounds, biological and medicinal activities? 40. Please describe the methods of obtaining essential oil from plant resources. Make short statements for each method. 41. Biosynthesis pathway of steroids, discuss about structure and properties. 42. What is Saponin, distribution in plant materials, structures and biological properties? 43. Distribution of ursolic acid and oleanolic acid in plant material, they belong to which class of natural products, structure, and biological activities. 44. Compare the structures of Cumarins, Chalcones and Aurones. There are any relations or not? Discuss about their properties. 45. Structures of abietic type of terpen, distribution in plant material, and biological activities. 46. Describe the classification of sesquiterpens, write structures of some important sesquiterpens and biological activities. – 120 –

47. Distribution of sesterterpenoids in plants, structures, biological activities. 48. Compare the carotenoids with other terpen compounds, show their structure, and explain about properties. 49. Distribution of catechin in the plants, isolation and determine methods in laboratory (please including with structure and properties). 50. At laboratory condition how to make extraction of polyphenols from plant materials and their qualitative analysis by the chromatographic method. 51. Extraction and isolation of pectin from citrus fruit peel in laboratory. Show the structure. 52. Qualitative and qualitative analysis of rutin and quercetin by method of chromatography in laboratory. Show the structures, and please write hydrolysis reaction of rutin. 53. Please write biosynthesis pathway: Condensation of isoprene units. Formation of acyclic terpenes. 54. Please show biosynthesis pathway of formation of monoterpens, sesquiterpenes and diterpens. 55. In laboratory how to get the polyphenol extraction from plant materials and how to qualitative analysis them. 56. Please show the methods of determining the amino acids, quantitative and qualitative analyses of them in laboratory. 57. Extraction and isolation carotenoid from carrot in laboratory. Show the structure, and make a sh ort statement about properties. 58. Natural compounds possessing antioxidant activities, distribution in plant kingdom, related structures and isolation methods. 59. Extraction and isolation nicotine from tobacco in laboratory. Show the structure, and make a sh ort statement about properties. 60. Determine the carbohydrates, quantitative and qualitative analyses by method of chromatography in laboratory. 61. Give an account for natural compounds which possessing cytotoxic (anticancer) activities, distribution in plant kingdom, related structures and isolation methods. – 121 –

62. Extraction and isolation caffeine from black tea in laboratory. Show the structure, and make a sh ort statement about properties. 63. Please make short statements for fatty acids, structure, and discuss the metabolism process of fatty acids. 64. Please discuss about natural compounds possessing vassorelaxant activities, distribution in plant kingdom, related structures and isolation methods.

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CHEMISTRY OF NATURAL COMPOUNDS Educational manual Stereotypical publication Computer page makeup and cover designer: N. Bazarbaeva IS No. 9132

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