Production of secondarymetabolites-200422175353.ppt

1. PLANT SECONDARY METABOLITES
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. CONTENTS
Introduction
Secondary metabolites
Pathway for production of secondary metabolites
Why they are produced?
Yield enhancement
Elicitation
Secondary metabolite release and analysis
Factors affecting product yield
Applications
Conclusion
References

3. METABOLITES : Introduction . . .
The chemical compounds produced by plants are collectively
called as phytochemicals.
Primary metabolites – participating in nutrition and metabolic
processes inside the plant.
Secondary metabolites – those chemical compounds that do
not participate in metabolism of plants but influencing the
ecological interactions between the plant and its environment.

4. Primary metabolites –
participating in nutrition
and metabolic processes
inside the plant.
Secondary metabolites –
influencing the ecological
interactions b/w the plant
and its environment.

5. SECONDARY METABOLITES
• The useful natural products are synthesized through
secondary metabolism, hence they are also known as
secondary metabolites.
• The secondary metabolites include alkaloids, terpenoids,
tannins, glycosides (steroids and phenolics) and saponins.

6. • Their chief applications are in Pharmaceuticals, in food
flavoring and perfumery. According to one estimate
4300 different flavor compounds have been identified in
food.
• Certain flavors consist of one or few related compounds
such as 2-isobutylthiazole (tomato flavors), methyl-ethyl
cinnamates (strawberry), methyl anthranilate (grape),
benzaldehyde (cherry), menthol (mint), safranal (saffron
).

7. PATHWAY FOR PRODUCTION OF SECONDARY
METABOLITES

8. WHY THEY ARE PRODUCED?
Secondary Metabolites are produced in plants is mainly for:
Disease resistance (from fungus, bacteria, viral, and pests),
For pollination and
To combat in extreme conditions of stress. The stress may be
biotic or abiotic [Drought, Cold, Temparature, etc.]

9. IF THE SECONDARY METABOLITES ARE PRESENT IN
CALLUS CULTURES, Process involved…
a)Initiation and Establishment of Callus Cultures
b)Initiation and Establishment of suspension cultures
c)Growth Studies
d)Selection & Screening of high yielding Cell Lines.

10. YIELD ENHANCEMENT...
Biotransformation
Immobilization
Two phase system culture
Hairy root Cultures

11. BIOTRANSFORMATION
The conversion of one chemical into another (ie.
Substrate into a product) by using biological systems
as biocatalysts is regarded as Biotransformation.
The biocatalyst may be free or immobilized.
In Biotransformation, it is necessary to select cell lines
that possess the enzymes for catalyzing the desired
reactions.
Eg: Hydroxylation, Reduction & Glycosylation.

12. IMMOBILIZED CELL CULTURE
The cells are physically immobilized by entrapment, Besides
Individual cells, it is also possible to immobilize aggregate cells
or even calluses.
The Common entrapment methods are:
1.Entrapment of Cells in Gels
2.Entrapment of Cells in Nets/Foams
3.Entrapment of Cells in hollow Fiber Membranes

13. Plant Culture speci
es
Immobilized Method Substrate Product
Catheranthus roseus in Agarose Cathenamine Ajmalicine
Digitalis lanata in alginate Digitoxin Digoxin
Capsicum frutescens in polyurethane
foam
Sucrose Capsaicin
Catheranthus roseus in alginate, agarose,
carrageenin
Sucrose Ajmalicine
Petunia hybrida in hollow fibres Sucrose Phenolics
Morrinda citrifolia in Alginate Sucrose Anthaquonine
Solanum aviculare Attachment
polythene beads
Sucrose Steroid Glycosides
Synthesis of metabolites using immobilized cell of some plant species:

14. HAIRY ROOT CULTURE
Hairy roots are induced by genetic insertion of T-DNA of Ri
plasmid lodged in Agrobacterium rhizogenes into wounded
tissue causing the growth of very fine adventitious roots.
The wounded region produces a phenolic compound called
acetylsyriongone.
Vir genes are involved in the transfer of T-DNA into the plant
cells.

15. Plant Species Secondary metabolite(s)
Nicotiana tabacum Nicotine, Anatabine
Atropa belladonna Atropine
Datura stramonium Hyoscyamine
Lithospermum erythrorhizon Shikonin
Catheranthus roseus Ajmaline, Serpentine
Mentha vulgaris Monoterpenes
Solanum laciniatum Steroid alkaloids
List of plant species for Hairy root cultures:

16. TWO PHASE SYSTEM CULTURE
Strategy – to provide an artificial site for Secondary
Metabolite accumulation.
The cells are kept apart from the product by a separation
phase (liquid) placed in a reactor.
[Eg: Dextran & PEG are used for Separation of Phenolic
Compounds].
Example – Sanguinarine from cultures of California poppy
cells.

17. ELICITORS -Induced Production of Secondary Metabolites
The phenomenon of stimulating the production of secondary
metabolites is called as Elicitation.
Elicitors are of two types:
Biotic Elicitors: Elicitors of biological origin are called biotic
elicitors [Polysaccharides, Proteins, Glycoprotein or cell wall
fragments from bacteria, fungi, etc]
Abiotic Elicitors: Elicitors of non-biological origin are called
Abiotic Elicitors [Metal ions, UV, etc]

18. Elicitor
Microorganism
Plant Cell Culture(s) Secondary
Metabolite(s)
Aspergillus niger Cinchona ledgeriana,
Rubia tincoria
Anthraquinones
Phythium
aphanidermatum
Catheranthus roseus Ajmalicine, Sctricosidine,
Catheranthine
Botrytis sps. Papaver sominiferum Sanguinarine
Fusarium sps. Apium graveolens Furancocumarins
Pencillium expansum Sanguinaria Canadensis Benzophenanthridine
Alkaloids
Phythium
aphanidermatum
Dacus carota Anthocyanins
List of Elicitors used to induce secondary metabolite
production in Plant cell:

19. SECONDARY METABOLITE RELEASE AND ANALYSIS
Separation and purification of products from plant cell cultures
are difficult, effort is made them to make it cost effective. The two
approaches are:
1.Production of secondary metabolite should be high as possible.
2.Formation of side products which interfere with separation
must be made minimal.
Once good quantity of the product is released into the medium,
separation and purification techniques (Eg: Extraction) can be
used for its recovery. This mainly depends on the nature of the
secondary metabolite.

20. FACTORS AFFECTING PRODUCT YIELD
(i) Tissue origin genetic character. Plant cells are genetically totipotent,
therefore, proper environmental conditions should be given so that any
cell may be induced to produce any substance according to the
characteristic of parental plants. However, it has been found that low
yielding plants produce high contents of products and therefore, such
plant parts should be chosen where there is the highest concentration of
desired product.
(ii) Culture conditions. Chemical composition of nutrient media influences
the potential synthetic machinery (i.e. biomass) and synthesis of
secondary products. A balance should be maintained between the
production of biomass and secondary product.
(iii) Selection and screening. Cell clones from better strains are selected
which is rather a difficult task. The more difficult work is to detect the very
small amount of desired product present in single cell or small population
of cells.

21. TERPENOIDS
C10 : many volatile fragrance
and herb or spice oils…
C15 : oils, phytoalexins, antibiotics,
antifeedants…
C20 : phytol, gibberellic acid,
taxol, phytoalexins…
C30 : brassinosteroids, phytoalexins,
antifeedants…
C40 : carotenoid, ABA…
C5 : oils, many volatile fragrance…
Polyterpenes : plastoquinnones,
rubber…

22. ESSENTIAL OILS (TERPENOIDS)

23. Two basic skeletons of Phenolic compounds
Phenylpropanoid &
Phenylpropanoid-acetate
skeleton

24. Erythose 4-P
PEP
Shikimic acid
Chorismic acid
Trp
Phe, Tyr Cinnamic acid
Coumaric aicd
Malonyl CoA
Acetyl CoA
Flavonoids
Simple phenolics
Complex phenolics
Glycolysis
Pentose phosphate pathway
chalcone
Biosynthesis of
phenolic compounds
Shikimic
Acid
pathway

25. chalcones
Phenylpropanoid and phenylpropanoid-acetate pathways

26. PHENOLIC COMPOUND

27. PHENOLIC COMPOUND
Lignin
phenylpropanoid group
branched polymer

28. NITROGEN-CONTAINING COMPOUND
1) Cyanogenic glycoside
Glucosinolates
(mustard oil glycosides)
2) Alkaloids
Latex ( Codenine)

29. 2) Alkaloids

30. Ornithine
cocaine
Ornithine
Aspartate
nicotine
Aspartate,
Glycine,
Glutamaine
caffeine
Tyrosine
Tryptophan quinine
morphine
Examples of amino acid origin for some alkaloids

31. Plant products Plant species Industry Industrial uses
Codeine (alkaloid) Papaver sominifera Pharmaceutical Analgesic
Diosgenin (steroid) Dioscorea deltoidea Pharmaceutical Antifertility agent
Quinine (alkaloid) Cinchona leitgeriana Pharmaceutical Antimalaria
Digoxin (glycoside) Digitalis lanata Pharmaceutical Cardiactonic
Scopolamine
(alkaloid)
Dhatura
stramonium
Pharmaceutical Antihypersensitive
Vincristine (alkaloid) Catharanthus roseus Pharmaceutical Antileukaemic
Taxol Taxus brevifolia Pharmaceutical Ovarian and
breast cancer
Pyrethrin Chrysanthemum
cinerariaefolium
Agrochemical Insecticide
Quinine (alkaloid) C. ledgeriana Food & drink Bittering agent
Jasmine Jasmium sp. Cosmetics Perfume
Saffron Crocus sativa Food Flavoring/coloring agent
APPLICATIONS

32. CONCLUSION

33. REFERENCES
Books:
•Introduction to plant tissue culture By M. K. Razdan 1st edition
1997
•Plant biotechnology By H. S. Chawla
•Biotechnology By Dr. U. Satyanarayan
•Plant cell culture By R. A. Dixon, Robert A. Gonzales - 1994
Websites:
•www.nature.com
•www.plantphysiol.org
•www.wikipedia.com
•www.kbiotech.com

34. Thank you . . .

35. Ask your queries . . .