Secondary metabolites

Secondary Metabolites by Prabhu Thirusangu

1. Secondary Metabolites By Prabhu Thirusangu , Molecular Biomedicine Laboratory, Sahyadri Science College Kuvempu University

2. 2 Primary metabolites : Molecules that are essential for growth and development of an organism . Examples: 1.Carbohydrates 2.Proteins 3.Lipids 4.Nucleic acids 5.Hormones Secondary metabolites : molecules that are not essential for growth and development of an organism. Metabolites

3. 3 Secondary metabolites are derived from primary metabolites

4. 4 Why secondary metabolites? are biosynthetically derived from primary metabolites. They are more limited in distribution being found usually in specific families. Chemical warfare to protect plants from the attacks by predators, pathogens, or competitors Attract pollinators or seed dispersal agents Important for abiotic stresses Medicine Industrial additives

5. 5 Possibly over 250,000 secondary metabolites in plants Classified based on common biosynthetic pathways where a chemical is derived. Four major classes: Alkaloids , glycosides , phenolics , terpenoids Secondary metabolites

6. 6 Alkaloids Most are derived from a few common amino acids (i.e., tyrosine, tryptophan, ornithine or argenine , and lysine) Compounds have a ring structure and a nitrogen residue. Indole alkaloids is the largest group in this family, derived from tryptophan Widely used as medicine

7. 7 Terpenoids Terpenes are generally polymers of 5-carbon unit called isoprene Give scent, flavors, colors, medicine... Three plant hormones are derived from the terpenoid pathway.

8. ALKALOIDS

9. WHAT ARE ALKALOIDS? These are commonly applied to basic nitrogenous compounds of plant origin that are physiologically active. Organic nitrogenous compounds with a limited distribution in native nature.

10. Characteristics: They are bitter in taste. Derived from amino acids.The amino acids that are most often serve as alkaloidal precursors are: phenylalanine, tyrosine, tryptophan, histidine , anthranilic acid, lysine and ornithine . Alkaloids form double salts with compounds of mercury, gold, platinum and other heavy metals. These salts are obtained as precipitate which are microcrystals .

11. Insoluble or sparingly soluble in water, but the salts formed on reaction with acids are usually freely soluble. Most are crystalline solids although a few are amorphous.

12. Free alkaloids are usually soluble in polar solvents like ether, chloroform Some alkaloids are liquid because of lacking of oxygen in their molecules. ( e.g coniine, nicotine, spartenine )

13. Sources and Occurrence of Alkaloids Alkaloids can occur in plant kingdoms; among the angiosperms, Leguminosae , Papaveraceae , Ranunculaceae , Rubiaceae , Solanaceae , Berberidaceae are outstanding alkaloid-yielding plants.

14. Uses of Alkaloids in Plants: Poisonous agents which protect plants against insects and herbivores End products of detoxification reactions representing a metabolic locking-up of compounds otherwise harmful to the plants. For regulatory growth factors Reserve substance capable of supplying nitrogen or other elements necessary to the plant’s economy

15. Naming for alkaloids From the generic name or the genus of the plant yielding them ( e.g vinblastine and vincristine . atropine) The specific name or species of the plant yielding alkaloids ( e.g belladonnine )

16. From their physiologic activity ( e.g emetine, morphine) From the discoverer ( e.g pelletierine ) ~ All names of alkaloids should end in “- ine ”. ~ A prefix or suffix is added to the name of a principal alkaloid from the same source. (quinine, quinidine , hydroquinine )

17. Pharmacologic action of Alkaloids: Analgesic (morphine, codeine) Narcotics (strychnine, brucine which are central stimulant) Anti malarial ( quinine) Anti pyretic Anti cancer ( vincristine ) Mydriatics (atropine) Anti inflammatory Miotics ( physostigmine , pilocarpine ) Ephedrine (rises in blood pressure, bronchodilator) Reserpine (produce fall in excessive hypertension)

18. TYPES OF ALKALOIDS

19. True or hetero cyclic alkaloids Pyridine- Piperidine alkaloids pyrrole & pyrrolidine alkaloids Tropane alkaloids Quinoline alkaloids Isoquinoline alkaloids Indole alkaloids Imidazole alkaloids Norlupinane alkaloids Steroid alkaloids Purine alkaloids

20. CHEMICAL CLASSIFICATION OF ALKALOIDS TRUE ALKALOIDS PYRROLE & PYRROLIDINE DERIVATIVES e.g. Hygrine , Cocca species

21. Pyrrolizidine Derivatives e.g. senecionine , seneciphylline

22. Piperidine & pyridine derivatives Areca Arecoline Hydrobromide Lobeline Nicotine

23. TROPANE DERIVATIVES e.g. Atropine hyoscine cocaine Hyoscyamine Scopolamine Coca

24. QUINOLINE DERIVATIVES e.g. Quinine, quinidine , cinchonine CINCHONA BARK

25. Quinine Dia -stereo-isomer of quinidine It occurs as white, odourless, bulky crystals or as a crystalline powder. It darkens when exposed to light and effloresces in dry air. It is freely soluble in alcohol, ether and chloroform but slightly soluble in water.

26. Uses Antimalarial For treating of chloroquinine resistant falciparum malaria combination with pyrimethamine and sulfadoxine or tetracycline or clindamycin . It has a skeletal muscle relaxant effect. It is widely used for the prevention and treatment of nocturnal recumbency leg cramps.

27. ISOQUINOLINE DERIVATIVES e.g. Morphine, codeine, berberine , emetine

28. APORPHINE (REDUCED ISOQUINOLINE-NAPTHALENE) DERIVATIVES e.g. boldine

29. I NDOLE DERIVATIVES e.g. ergometrine , ergotamine, reserpine , vincristine , vinblastine

30. IMIDAZOLE DERIVATIVES e.g. Pilocarpine , isopilocarpine

31. NOR LUPINANE DERIVATIVES e.g. Cytisine , lupanine

32. PURINE DERIVATIVES e.g. Theophylline , caffeine, theobromine

33. STEROIDAL DERIVATIVES e.g. protoveratrine , solanidine

34. PSEUDO ALKALOIDS DITERPENES e.g. Aconitine , aconine , hypoaconitine PROTO OR NON HETERO CYCLIC ALKALOIDS ALKYLAMINES e.g. Ephedrine,pseudoephedrine , colchicine

35. C 17 H 19 NO 3 a component of blackpepper (Piper nigrum ) has been used in various traditional medicine preparations an insecticide. has various effects on human drug metabolizing enzymes, and is marketed under the brand name, Bioperine , PIPERINE

36. Quinine , molecular formula C 20 H 24 N 2 O 2 is a white crystalline quinoline alkaloid. Quinine is extremely bitter, and also 　 possesses antipyretic, analgesic and anti-inflammatory properties . has strong anti malarial properties, quinine in therapeutic doses can cause various side-effects, e.g. nausea, vomiting and cinchonism , and in some patients pulmonary oedema . It may also cause paralysis if accidentally injected into a nerve. Non-medicinal uses of quinine include its uses as a flavouring agent in tonic water and bitter lemon.

37. VINCRISTINE A CANCER KILLER

38. Vinca alkaloids The Vinca alkaloids are a subset of drugs that are derived from the periwinkle plant, Catharanthus roseus .

39. VINCRISTINE Periwinkle Structure

40. Serpentine Molecular Formula: C 21 H 22 N 2 O 3 Isolated from Rauwolfia serpentina   To treat High blood pressure to treat insect stings and the bites of venomous reptiles

41. Terpenoids Isoprene: Farnesol : Chlorophyll: β -Carotene

42. TERPENES The chemist Leopold Ruzicka ( born 1887) showed that many compounds found in nature were formed from multiples of five carbons arranged in the same pattern as an isoprene molecule (obtained by pyrolysis of natural rubber). He called these compounds “ terpenes ”. C C C C C . isoprene natural rubber D isoprene unit head tail C C C C C

43. The Biological Isoprene Unit The isoprene units in terpenes do not come from isoprene . They come from isopentenyl pyrophosphate. Isopentenyl pyrophosphate (5 carbons) comes from acetate (2 carbons ) via mevalonate (6 carbons ).

44. Terpenes Terpenes are natural products that are structurally related to isoprene. H 2 C C CH 3 CH CH 2 or Isoprene (2-methyl-1,3-butadiene)

45. The Biological Isoprene Unit CH 3 COH O 3 HOCCH 2 CCH 2 CH 2 OH CH 3 OH O Mevalonic acid H 2 C CCH 2 CH 2 OPOPOH CH 3 O O Isopentenyl pyrophosphate

46. Isopentenyl Pyrophosphate H 2 C CCH 2 CH 2 OPOPOH CH 3 O O Isopentenyl pyrophosphate or OPP

47. Isopentenyl and Dimethylallyl Pyrophosphate Isopentenyl pyrophosphate is interconvertible with 2-methylallyl pyrophosphate. OPP OPP Dimethylallyl pyrophosphate has a leaving group (pyrophosphate) at an allylic carbon; it is reactive toward nucleophilic substitution at this position. Isopentenyl pyrophosphate Dimethylallyl pyrophosphate

48. Carbon-Carbon Bond Formation The key process involves the double bond of isopentenyl pyrophosphate acting as a nucleophile toward the allylic carbon of dimethylallyl pyrophosphate. + OPP OPP

49. After C—C Bond Formation... + OPP The carbocation can lose a proton to give a double bond.

50. After C—C Bond Formation... + OPP OPP The carbocation can lose a proton to give a double bond. H – +

51. After C—C Bond Formation... OPP This compound is called geranyl pyrophosphate. It can undergo hydrolysis of its pyrophosphate to give geraniol (rose oil).

52. OPP OH Geraniol H 2 O After C—C Bond Formation...

53. From 10 Carbons to 15 + OPP OPP Geranyl pyrophosphate + OPP

54. From 10 Carbons to 15 + OPP H – + OPP

55. From 10 Carbons to 15 OPP This compound is called farnesyl pyrophosphate. Hydrolysis of the pyrophosphate ester gives the alcohol farnesol .

56. Cyclization Rings form by intramolecular carbon-carbon bond formation. OPP OPP + E double bond Z double bond

57. CLASSIFICATION OF TERPENES 58 TYPE OF NUMBER OF ISOPRENE TERPENE CARBON ATOMS UNITS hemiterpene terpene sesquiterpene diterpene triterpene tetraterpene C 5 C 10 C 15 C 20 C 30 C 40 one two three four six eight hemi = half di = two Sesqui = one and a half tri = three tetra = four NOTE:

58. Hemiterpenes  consist of  a single isoprene  unit. Isoprene itself is considered the only hemiterpene, but oxygen-containing derivatives such as  prenol  and  isovaleric acid are hemiterpenoids . Monoterpenes  consist of  two isoprene  units and have the molecular formula C 10 H 16 . Examples of monoterpenes are:  geraniol,limonene  and  terpineol . Sesquiterpenes  consist of  three isoprene  units and have the molecular formula C 15 H 24 . Examples of sesquiterpenes are:  humulene,farnesenes ,  farnesol . Diterpenes  are composed of  four isoprene  units and have the molecular formula C 20 H 32 . They derive from  geranylgeranyl pyrophosphate. Examples of diterpenes are  cafestol ,  kahweol ,  cembrene and  taxadiene  (precursor of  taxol ). CLASSIFICATION OF TERPENES

59. Sesterterpenes , terpenes having 25 carbons and  five isoprene  units, are rare relative to the other sizes, example:   geranylfarnesol . Triterpenes  consist of  six isoprene  units and have the molecular formula C 30 H 48 . The linear triterpene   squalene , the major constituent of shark liver oil, is derived from the reductive coupling of two molecules of  farnesyl pyrophosphate. Squalene is then processed biosynthetically to generate either  lanosterol  or  cycloartenol , the structural precursors to all the steroids. Sesquarterpenes  are composed of  seven isoprene  units and have the molecular formula C 35 H 56 . Sesquarterpenes are typically microbial in their origin. Examples of sesquarterpenes are ferrugicadiol and tetraprenylcurcumene . CLASSIFICATION OF TERPENES

60. Tetraterpenes  contain  eight isoprene  units and have the molecular formula C 40 H 64 . Biologically important tetraterpenes include the acyclic  lycopene , the monocyclic gamma-carotene, and the bicyclic   alpha- and beta-carotenes. Polyterpenes  consist of long chains of  many isoprene   units,eg , Natural  rubber . Norisoprenoids ,eg : C 13 -norisoprenoids 3-oxo-α-ionol present in  Muscat of Alexandria leaves and 7,8-dihydroiononederivatives, such as megastigmane-3,9-diol and 3-oxo-7,8-dihydro-α-ionol found in  Shiraz leaves (both grapes in the species  Vitis vinifera ) CLASSIFICATION OF TERPENES

61. TERPENES The number of C atoms is a multiple of 5, C 5 C 10 C 15 C 20 C 25 C 30 C 35 C 40 Each group of 5 C is an isoprene subunit They can be saturated or unsaturated Many contain O atoms as well. What they all have in common is 1 & 2 above.

62. 63 JOINING ISOPRENE UNITS The terms head-to-tail and tail-to-tail are often used to describe how the isoprene units are joined. C C C C C . an extra bond Head-to-Tail Head-to-Tail Tail-to-Tail

63. a- Phellandrene (eucalyptus) Menthol (peppermint) Citral (lemon grass) O H OH Representative Monoterpenes

64. Representative Monoterpenes a- Phellandrene (eucalyptus) Menthol (peppermint) Citral (lemon grass) O H OH

65. a- Phellandrene (eucalyptus) Menthol (peppermint) Citral (lemon grass) Representative Monoterpenes

66. Representative Sesquiterpenes a -Selinene (celery) H

67. a -Selinene (celery) H Representative Sesquiterpenes

68. Vitamin A OH Representative Diterpenes

69. Vitamin A OH Representative Diterpenes

70. Vitamin A Representative Diterpenes

71. Squalene (shark liver oil) tail-to-tail linkage of isoprene units Representative Triterpenes

72. Farnesol Used as Perfumes Pesticides Pheromones Anti- tumour agent Antibacterial drug

73. Structure of chlorophyll

74. Chlorophyll a & b Chl a has a methyl group Chl b has a carbonyl group Porphyrin ring delocalized e - Phytol tail

75. Structure of chlorophyll a and b

76. β-carotene - TETRATERPENE 77 β -carotene carrots tail-to-tail head-to-tail head-to-tail

77. β -carotene – a linear terpene 8 isoprene units 40 carbon atoms

78. 79 Taxol is a terpenoid "the best anti-cancer agent” by National Cancer Institute Has remarkable activity against advanced ovarian and breast cancer, and has been approved for clinical use. Taxol Taxus brevifolia Nutt.

79. 80 Camptothecin is an indole alkaloid, derived from tryptophan. Has anticancer and antiviral activity Two CPT analogues have been used in cancer chemotherapy, topotecan and irinotecan.

80. Phenolics Derived from aromatic amino acids, such as phenylalanine, tyrosin , and trytophan . All contain structures derived from phenol Some examples: Coumarins : antimicrobial agents, feeding deterrents, and germination inhibitors . Lignin: abundant in secondary cell wall, rigid and resistant to extraction or many degradation reagents Anthocyanins Flavones Flavnols Phenols are present in every plant they attract pollinators to the plant and even impact how these plants act with one another. .

81. 82 Glycosides Compounds that contain a carbonhydrate and a noncarbohydrate Glycosides are present in vacuoles in inactive form Glucosinolates : found primarily in the mustard family to give the pungent taste There are four type of linkages present between glycone and aglycone : C-linkage/ glycosidic bond, O-linkage/ glycosidic bond N-linkage/ glycosidic bond S-linkage/ glycosidic bond .

82. Cyanogenic glycosides All of these plants have these glycosides stored in the  vacuole , but, if the plant is attacked, they are released and become activated by enzymes in the  cytoplasm . These remove the sugar part of the molecule and release toxic  hydrogen cyanide . An example of these is  amygdalin  from  bitter almonds Cyanogenic glycosides can also be found in the fruit seeds (and wilting leaves) of many members of the  rose family  (including  cherries ,  apples ,  plums , bitter almonds,  peaches ,  apricots , raspberries , and  crabapples

83. Sources Plant resins [ A liquid compounds found inside plants or exuded by plants, But not  saps, latex, or mucilage, The resin produced by most plants is a viscous liquid, composed mainly of volatile fluid  terpenes resins do not serve a nutritive function. The toxic resinous compounds may confound a wide range of herbivores, insects, and pathogens; while the volatile  phenolic compounds may attract benefactors such as parasitoids or predators of the herbivores that attack the plants

84. Latex latex as found in nature is a milky  fluid  found in 10% of all flowering plants ( angiosperms ).  It is a complex emulsion consisting of proteins ,  alkaloids ,  starches ,  sugars ,  oils ,  tannins ,  resins , and  gums  that coagulate on exposure to air. It is usually exuded after tissue injury. In most plants, latex is white, but some have yellow, orange, or scarlet latex. It serves mainly as  defense  against  herbivorous   insects. Natural rubber  is the most important product obtained from latex This latex is used to make many other products as well, including mattresses ,  gloves ,  swim caps ,  catheters  and  balloons In chewing gum, and glues

85. Sources Plant sterol; The richest naturally occurring sources of phytosterols are vegetable oils and products made from them. They can be present in the free form and as esters of fatty acid/ cinnamic acid or as glycosides, Phytosterols , which encompass plant  sterols  and  stanols , are  steroid  compounds similar to  cholesterol  which occur in plants and vary only in carbon side chains and/or presence or absence of a double bond. Stanols are  saturated  sterols, having no double bonds in the sterol ring structure.

86. Sources Sapogenins sapogenins  are the  aglycones , or non- saccharide , portions of the family of natural products known as  saponins . Sapogenins contain steroid or other  triterpene  frameworks as their key organic feature. For example, steroidal sapogenins like  tiggenin ,  neogitogenin , and tokorogenin  have been isolated from the  tubers  of  Chlorophytum arundinacelum .  Some steroidal sapogenins can serve as a practical starting point for the  semisynthesis  of particular  steroid hormones .

87. Essential Oil An  essential oil  is a concentrated  hydrophobic  liquid containing volatile  aroma compounds  from plants. Essential oils are also known as  volatile oils ,  ethereal oils ,  aetherolea , They are used in  perfumes ,  cosmetics ,  soaps and other products, for  flavoring  food and drink, and for adding scents to  incense and household cleaning products Oil Tonnes Sweet orange 12,000 Mentha arvensis 4,800 Peppermint 3,200 Cedarwood 2,600 Lemon 2,300 Eucalyptus globulus 2,070 Litsea cubeba 2,000 Clove 2,000 Spearmint 1,300

88. Phenylpropanoids The  phenylpropanoids  are a diverse family of organic compounds that are synthesized by plants from the amino acid phenylalanine Phenylpropanoids are found throughout the plant kingdom, where they serve as essential components of a number of structural polymers, provide protection from ultraviolet light, defend against herbivores and pathogens, and mediate plant-pollinator interactions as floral pigments and scent compounds.

89. Plant-derived Insecticides

90. Outline: Plant-Derived Insecticides Important insecticides from plants - rotenoids - New World and Asia - pyrethrins - Near Eastern center -tobacco - New World Ryania speciosa , Flacourtiaceae Antifeedants - neem , Azadirichta indica , Meliaceae

91. Introduction Many insecticidal compounds are known from plants. Most plants make defensive compounds called allomones . Only a few are important commercially. Plant-derived insecticides have largely been replaced by synthetic materials, but there are some advantages to the naturally occurring materials. For example, these substances are biodegradable. Selectivity is needed. Compounds that are toxic to insects, but not toxic to mammals, are preferable, of course.

92. Rotenoids A series of compounds found in members of the genera Derris , Lonchocarpus , Tephrosia are known as rotenones . Commercially, rotenoids are isolated mostly from the roots of Derris elliptica in Indonesia and from Lonchocarpus These compounds are isolated by grinding the plant and extracting with solvents such as hexane or petroleum ether or chloroform. The compounds are oil soluble or lipids. They make up 1-20% of the dry weight of the roots.

93. Derris elliptica , Fabaceae Pacific Island Ecosystems at Risk (PIER) Photo by Agnes Rinehart

94. Rabo molle, Lonchocarpus muehlenbergianus , Fabaceae Libro del Arbol, Celulosa Argentina, Vol. 2, 1975

95. False indigo bush, Amorpha fruticosa , Fabaceae

96. Pyrethrins Another major series of compounds, the pyrethrins , come from species of the genus Chrysanthemum (some people put these species in Pyrethrum ) ( Asteraceae or Compositae ). These were used as far back as the 1st century B.C. by the Chinese. Insecticidal plants mostly are grown in countries with inexpensive labor and high elevations such as Kenya and New Guinea.

97. Pyrethrum, Chrysanthemum cinerariifolium , Asteraceae Courtesy Dr. Saifu Dossaji

98. Harvesting pyrethrum flowers in Kenya Courtesy Dr. Saifu Dossaji

99. Ryania speciosa ( Flacourtiaceae ) is also used occasionally and an insecticide. A mixture of diterpene , alkaloids is isolated and used for specialty insecticide uses. Because the extract is expensive, it is not commonly used. Other plant-derived insecticides

100. Ryania speciosa , flower and fruit, Flacourtiaceae

101. Tobacco , Nicotiana tabacum , Solanaceae Tobacco (which contains nicotine) is another major source of insecticides. Tobacco wastes are often extracted and used as a source of nicotine. Nicotine especially effective against aphids .

102. Tobacco, Nicotiana tabacum , Solanaceae ,

103. Calabar bean Calabar bean ( Physostigma venenosa , Fabaceae) is (or has been) a trial-by-ordeal drug in the Calabar coast of Nigeria. The active component is physostigmine, an acetyl choline esterase inhibitor. The structure of several commercial carbamate insecticides is patterned after the structure of the plant alkaloid.

104. Calabar bean, Physostigma venenosum , Fabaceae R. Bentley and H. Trimen, Medicinal Plants , London, Churchill, 1880

105. Antifeedants Antifeedants are compounds that prevent insect feeding. Although many are toxic, the insects usually don’t consume enough to be poisoned. Only one of these, neem, Azadirachta indica , Meliaceae, is commercially available. The active compound, azadirachtin, is a structurally modified triterpene.

106. Neem, Azadirachta indica , Meliaceae Courtesy Dr. Ramesh Pandey William M. Ciesla, Forest Health Management International, United States

107. 108 Natural Colors And Flavors Better & Safer Alternatives

108. 109 Natural Colors saffron Anthrocyanin Carotenoids Carotene Chlorophyll Curcumin Iron oxides Riboflavin Titanium dioxides

109. 110 Carotenoids Yellow & Red colors . Sources- S weet potatoes, spinach and tomatoes. Antioxidant - Cancer research.

110. 111 Carotene Yellow to Orange colors . Sources: Carrots, yellow or orange fruits. Rich in Vitamin A. Lycopene saffron

111. 112 Chlorophyll Green coloring agent . Occurs naturally in all plants

112. 113 Curcumin Extracted from turmeric. Coloring and medicinal uses Wound healing Antiulcer Anti inflammatory Antimicrobial & antiviral Hepatoprotective Antioxidant No toxicity.

113. 114 Riboflavin (Vitamin B 2 ) Yellow to Orange colors. Sources- Leafy vegetables, Eggs, Milk & Wheat. Safe & Beneficial for health.

114. 115 Sweet Orange Oil Sweet orange fruit peel Stress reducing agent Aroma therapy: Peace of mind

115. 116 Flavor Cocoa seeds Vennila Strawberry Cinnamon Cardamom Cloves Pepper areca