Role Of Natural Products

What nutritionists and physicians define as phytochemicals are called secondary metabolites or natural products by plant biologists (Croteau et al., 2000). Classifying certain metabolites as either primary or secondary is sometimes difficult. For example, chlorophyll, while being necessary for primary metabolism, is classified as a tetraterpene, which is normally considered a secondary metabolite. In general, primary metabolites have key roles in the physiological processes central to plants, such as photosynthesis, respiration, lipid metabolism, and amino acid and nucleic acid synthesis (Table 9.1). In this paper, phytochemicals are defined as secondary metabolites, distinct from vitamins and micronutrients. Secondary metabolites are divided into three groups based on their biosynthesis: terpenoids, alkaloids, and the phenylpropanoids and associated phenolic compounds (Table 9.1). Natural products are synthesized from many of the same intermediaries, such as phosphoenolpyruvate (PEP), pyruvate, and acetyl-CoA (Figure 9.1). Plants produce more than 25,000 terpenoids, 12,000 alkaloids, and 8,000 phenolic compounds, and many secondary metabolites are unique to individual taxa. It is thought that these compounds, while not directly involved in primary plant metabolism, have evolved from interactions with other organisms: herbivores, pathogens, pollinators, and other plants. For example, plants, in response to insect herbivory, release elevated levels of volatiles, which serve as a signal to insect predators and para-sitoids by the insect-damaged plants (Paré and Tumlinson, 1997). The exact function in plants of the vast majority of natural products is largely unknown and their role in human health has been provided by epidemiological studies and bioassays. In this respect, they differ from vitamins and micronutrients, whose metabolic functions and physiological consequences of deficiencies are well defined. Yet, phyto-chemical mining and discovery is a very active field in the medicine and pharmaceutical industry. For strategies on discovery of bioactive compounds, the reader is referred to Duke et al. (2000).

Plant biologists can manipulate the concentration of a phytochemical by one of two ways: traditional plant breeding or bioengineering. Both methods have in common the necessity to have a clear understanding of basic physiology and biochemical pathways through which plant improvement is realized. With a bioengineering approach, the biosynthetic pathway, target genes, and metabolic flux controls must be clearly elucidated. Plant breeding offers the advantage that if genetic variation exists for the chemical of interest between interbreeding species or cultivars, then the concentration of that compound can be increased in elite horticultural lines.

TABLE 9.1

Major pathways involved in primary and secondary metabolism I. Primary Metabolism

A. Respiration

  • Glycolysis - primary pathway for the breakdown of glucose, and all carbohydrates that can be converted to glucose. Glucose is broken down to pyruvate.
  • Enzymes localize to mitochondria.
  • Phosphoenolpyruvate serves as a substrate for aromatic amino acids.
  • Pyruvate serves as a substrate for aliphatic amino acids.
  • Acetyl-CoA serves as a substrate for fatty acids and isoprene derivatives.
  • Pentose phosphate pathway - glucose phosphate is converted to pentose and CO2.
  • Tricarboxylic acid cycle - (Krebs cycle, citric acid cycle) - release of reduction equivalents from activated acetate units.

B. Carbohydrate Synthesis and Metabolism

  • Carbohydrates are classified as either structural or storage polysaccharides.
  • Synthesis from carbon dioxide (carbon-linked reactions) or by gluconeogenesis.
  • Carbon-linked reactions (Calvin cycle) produce 3-phosphoglycerate (3PGA) or phos-phophenolpyruvate (PEP), which is converted into oxaloacetate (OAA).
  • Enzymes localize to the chloroplast.
  • Light reactions - (water oxidations) produce O2, ATP, and NADPH.
  • Enzymes localize to the chloroplast.
  • Gluconeogenesis is the net process by which lipids (triglycerides) are converted to sucrose.
  • Process occurs in the glyoxysome, mitochondria, spherosome, and the cytoplasm.

C. Lipid Metabolism

  • Lipids are classified as fatty acids, neutral lipids, and polar lipids.
  • Glyoxylate cycle - converts fats (acetate units) to sugars; occurs in glyoxysome, cytosol, and mitochondria.
  • Enzymes localize to plastids.
  • Acetyl-CoA is the initial substrate for synthesis of the carbon backbone of all fatty acids.

D. Amino Acid Synthesis

  • GS/GOGAT pathway functions as the primary assimilation of inorganic N and secondary assimilation of free ammonium.
  • Enzymes localize to plastids.
  • Synthesis of glutamine, glutamate, asparagines, aspartate.
  • Aromatic amino acid pathways: important precursors of primary and secondary metabolism.
  • Enzymes localize to plastids.
  • Phenylalanine and tyrosine serve as precursors of alkaloids, flavonoids, isoflavonoids, hydroxycinnamic acid, and lignin.
  • Tryptophan serves as a precursor for indole phytoalexins, indole alkaloids, and indole glucosinolates.
  • Aspartate-derived amino acid pathway, leads to lysine, threonine, and methionine.
  • Required in human diets.
  • Enzymes localize to chloroplasts, mainly, and the cytosol.
  • Methionine incorporated into proteins; S-adenosylmethionine used for transmethylation of lipids, pectins, chlorophyll, and nucleic acids.
  • Branched-chain amino acids.
  • Includes threonine, isoleucine, valine and leucine.
  • Isoleucine and valine are synthesized in chloroplasts.
  • Acetohydroxy acid synthase (AHAS) is a key enzyme in valine and isoleucine synthesis, and a target for herbicides.

E. Purine and Pyrimidine Synthesis

  • Pyrimidine nucleotides are synthesized from the orotic acid pathway.
  • Amino donor is glutamine.
  • All enzymes used in pyrimidine synthesis localize to plastids.
  • Purine nucleotides are synthesized directly from 5-phosphoribosyl-1-pyrophospho-phate by sequential addition of purine precursors that include glycine, amide groups from aspartate and glutamine, and methenyl and formyl tetrahydrofolates.
  • Synthesis occurs in the cytosol.
  1. Secondary Metabolism
  2. Terpenes and terpenoids
  • Classification of terpenes is based on a basic branched C5 isoprene unit.
  • Isopentenyl diphosphate (IPP) is the fundamental precursor for terpenoids.
  • Plants emit about 15% of their fixed carbon into the atmosphere as isoprene.
  • Most terpenoids are produced, stored, and emitted in specialized structures, such as glandular trichomes, flower petals, and resin ducts.

# of C5 units

Class Name

Examples

1

hemiterpenesa

isoprene

2

monoterpenesa

volatile essences of flowers, essential oils, pyrethrin

3

sesquiterpenesb

essential oils, abscisic acid

4

diterpenesa

gibberellins, phytoalexins, taxol, skolin

6

triterpenesb

sterols, brassinosteroids, oleanolic acid (surface

waxes)

8

tetraterpenesa

carotenoids accessory pigments (photosynthesis)

> 8

polyterpenesb

plastoquinone, ubiquinone, dolichol, rubber

10

meroterpenes

partially derived from terpenoids;

cytokinins, vincristine, vinblastine

a Synthesized in plastids.

b Synthesized in cytosol and endoplasmic reticulum.

a Synthesized in plastids.

b Synthesized in cytosol and endoplasmic reticulum.

B. Alkaloids

  • All alkaloids contain nitrogen, most are basic. Accumulate in actively growing tissue, epidermal and hypodermal cells, vascular sheaths and latex vessels; present in vacuoles.
  • Often stored in tissues other than where synthesized.
  • Formed from L-amino acids (tryptophan, tyrosine, phenylalanine, lysine, arginine) alone or with steroidal, secoiridoid, or other terpenoid-type moiety.
  • Aromatic amino acids.
  • Phenylalanine and tyrosine give rise to peyote and morphine alkaloids, colchicines, and betalains.
  • Tryptophan gives rise to indole phytoalexins, indole glucosinolates, and indole alkaloids.

C. Phenylpropanoids

  • Derived from phenylpropanoid (C6C3) and phenylpropanoid-acetate (C6C3-C6) skeletons.
  • Most phenolic compounds are derived from phenylpropanoids and include lignins, lignans and flavonoids.
  • Major classes of plant phenols are given below.

# of C atoms

Class

Example

Source

6

phenols

catechol

Galutheria leaves

7

phenolic acids

p-hydroxybenzoic acid

widespread

8

phenylacetic acids

2-hydroxyphenylacetic

Astilbe leaves

acid

9

hydroxycinnamic acids

caffeic acid

ubiquitous

phenylpropenes

myristicin

Myristica fragrans

coumarins

6-7,dimethoxycoumarin

Dendrobium densiflorum

8-methoxypsoralen

Heracleum

mantegazzianum

isocoumarins

hydrangenol

Hydrangea macrophylla

chromones

eugenin

Eugenia aromatica

10

napthoquinones

juglone

Juglans nigra

13

xanthones

mangiferin

widespread

14

stilbenes

resveratol, lunularic acid

Vitis vinifera, liverworts

anthraquinones

emodin

rhubarb

15

flavonoids

flavones, catechins,

soybean, green tea

isoflavones

18

lignans

pinoresinol

conifers

neolignans

eusiderin

Magnoliaceae

30

bioflavonoids

amentoflavone

gymnosperms

From Goodwin, T.W. and Mercer, E.I. 1983. Int. Plant Biochem, 2nd ed., Pergamon Press, NY, 677 pp.

From Goodwin, T.W. and Mercer, E.I. 1983. Int. Plant Biochem, 2nd ed., Pergamon Press, NY, 677 pp.

  • Benzopyranones
  • A group of defense-related compounds that include coumarins, stilbenes, styrlpy-rones, and arylpyrones.
  • Coumarins can cause internal bleeding, photophytodermatitus, and are used to treat skin disorders.
  • Stilbenes, styrlpyrones, and arylpyrones are derived from cinnamoyl-CoA and mal-onyl-CoA and flavonoids pathways.
  • Adapted from Goodwin T.W. and Mercer, E.I. 1983. Intr. Plant Biochem, 2nd ed., Pergamon Press, NY, 677 pp.; Mohr, H. and Schopfer, P. 1995. Plant Physiology, 4th ed., Springer-Verlag, Berlin, Heidelberg, 629 pp., Buchanan, B.B., Gruissem, W., and Jones, R.L. (eds.) 2000. Biochem, Molecular Biology of Plants, Am. Soc. Plant Physiology, Rockville, MD, 1367 pp. With permission.)

Complete biochemical knowledge of the pathway is not necessary. Although it is often research in the model systems that provide the requisite knowledge to improve cultivated species, the vast majority of unique and undiscovered phytochemicals are produced in noncultivated plants. Arabidopsis thaliana (mouse-ear cress or thale cress) serves as the model plant for plant biologists, and gene sequences of certain enzyme classes can be identified from the Arabidopsis database, with the caveat that genes encoding enzymes in natural product pathways are not closely linked in plants (Dixon, 2001). Conversely, the pathways for vitamins are widely conserved by both eukaryotes and prokaryotes.

Many phytochemicals have antioxidant properties, including vitamins C and E, 6-carotene, a variety of carotenoids, and plant phenols. It is generally thought that by increasing the dietary intake of phytochemicals with antioxidant properties, aging effects on cells and diseases can be delayed. Commercial preparations often consist of a mixture of antioxidants. For example, Pycnogenol® is a mixture of phenolic and

Polysaccharides

Hexose phosphate pool-^ Starch

X Glycosides

Polysaccharides

Hexose phosphate pool-^ Starch

X Glycosides

Figure 9.1 Primary metabolites, intermediate products and derived natural products. (Adapted from Goodwin, T.W. and Mercer, E.I. 1983. Intr. PlantBiochem, 2nd ed., Pergamon Press, NY, 677 pp.; Mohr, H. and Schopfer, p. 1995. Plant Physiology, 4th ed., SpringerVerlag, Berlin, Heidelberg, 629 pp.; Buchanan, B.B., Gruissem, W., and Jones, R.L. (eds.) 2000. Biochem, Molecular Biology of Plants, Am. Soc. Plant Physiology, Rockville, MD, 1367 pp. With permission.)

Figure 9.1 Primary metabolites, intermediate products and derived natural products. (Adapted from Goodwin, T.W. and Mercer, E.I. 1983. Intr. PlantBiochem, 2nd ed., Pergamon Press, NY, 677 pp.; Mohr, H. and Schopfer, p. 1995. Plant Physiology, 4th ed., SpringerVerlag, Berlin, Heidelberg, 629 pp.; Buchanan, B.B., Gruissem, W., and Jones, R.L. (eds.) 2000. Biochem, Molecular Biology of Plants, Am. Soc. Plant Physiology, Rockville, MD, 1367 pp. With permission.)

polyphenolic compounds (Dillard and German, 2000). With mixtures, the specific bioactive chemical is usually not known, and it is possible the efficacy of the isolated compounds would diminish if ingested separately, i.e., mixtures of bioactive compounds may act synergistically. Improvement of the phytochemical content of plants using breeding or bioengineering requires that specific compounds be identified and quantified. Any step in the biosynthetic pathway could potentially be a target for manipulation, and it is theoretically possible that several intermediaries could be increased along the pathway in addition to the target metabolite.

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