Unit I Metabolic pathways in higher plants & their determination Pharmacognosy & Phytochemistry II B. Pharm. Vth Semester Biosynthetic Pathways Metabolic pathways

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Pharmacognosy & Phytochemistry II
B. Pharm. Vth Semester
Metabolic pathways in
higher plants & their determination
Unit I
Dr. Amit Gangwal
HoD, Pharmacognosy
I/C: Publicity Wing
Criterion 7 I/C, NAAC

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CO Code Course Outcomes
CO504.1 Understand basic metabolic pathways and formation of different
primary & secondary metabolites through different
pathways besides learning how these pathways are traced.
CO504.2 Explain basics related to chemistry, extraction, classification and
various properties pf varied secondary metabolites like alkaloids,
glycosides, phenolics and others.
CO504.3 Explain basics related to chemistry, extraction, classification and
various properties pf varied secondary metabolites like iridoids,
resins, volatile oils etc. and explain and perform isolation,
identification, analysis of various individual secondary metabolites
from plants.
CO504.4 To summarize the modern extraction techniques and examine
applications of latest techniques in the isolation, purification and
identification of crude drugs.

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8. What is Biosynthesis?
 Process of forming large molecules from smaller
subunits within living organism, done by mainly
enzymes.
 Also known as anabolism- simple molecules join to
form macromolecules.
◦ E. g. formation of carbohydrates through photosynthesis
in chloroplast through conversion of light energy into
chemical energy. (synthesis of glucose from
H2O+CO2)
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• Gush
• Hour
• Roguish
• Rough
• Rush
• Shrug
• Sigh

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14. Metabolism
 What is Metabolism?
 Metabole: Greek word: Means Change
 Metabolic pathway: is a series of steps in biochemical
reactions that help to convert molecules or substrates like
sugar into different usable materials.
 All these reactions take place inside cell, enzymes which
are protein molecules, break down or build up molecules.
 Enzymes are catalyst to these metabolic reactions.
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Anabolism: synthesis of larger/complex molecules –
requires energy (Glucose → glycogen)
Catabolism: Met. Reac. in cell that degrade substrate into
smaller/simpler products- releases energy (Glucose to
smaller molecules)
Anabolic: Small molecules join into larger ones. Hence
Energy is required
+ Energy
Catabolic: Large Molecules are broken down into the smaller one
and hence energy is released
+ Energy

16. Metabolic pathways
 Two types:
 Anabolic Pathway: Utilization of energy for
biosynthetic reactions. Utilizes NADH, ATP etc.
 Metabolites: intermediates, small mol. products of
metabolism
 Primary metabolites- responsible for primary function
for plants’ own growth and development)
 Catabolic Pathway: release energy by breaking down
molecules into simpler molecules. Ex. Cellular
respiration: Sugar is taken in by cells and broken down
to release energy.
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18. Metabolic pathways
 Primary metabolites: carbohydrates, lipids, proteins, amino
acids, nucleic acids, cellulose etc.
 Not responsible for therapeutic activity
 Primary metabolic pathway: pathways followed for
production of primary metabolites
 Secondary metabolites: not responsible for growth of
plants
 Secondary metabolites: synthesized for adaptation by plants
under stress conditions, toxic subs, for attracting
pollination, for defense etc.
 They are therapeutically active.
Ex: Alkaloids, glycosides, tannins, terpenoids etc.
 They are the derivatives of primary metabolites.
 Synthesis of secondary metabolites: pathway known
Secondary metabolic pathway.
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19. Metabolic pathways
 Primary Metabolic pathways:
1. Glycolysis
2. TCA/ Kreb’s / Citrate pathway/Citric acid cycle
(Tricarboxylic Acid Cycle)
3. Pentose phosphate pathway/ Hexose monophosphate
pathway
 Primary metabolic pathway of carbon
1st step is photosynthesis
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Energy
Water
CO2 Sugar
O2
6CO2 + 6H2O C6H12O6 +6O2
Photosynthesis

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24. Metabolic pathways
 Sugar or glucose enters Glycolysis cycle &
finally converted to 2 pyruvate.
 After glycolysis, pyruvate in presence of O2
(aerobic) is converted to Acetyl CoA.
 In TCA cycle - Oxidation of Acetyl CoA takes
place.
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Glycolysis
Glucose
Hexose Kinase
ATP
ADP
Glucose 6 Phosphate
Phospho glucose isomerase
Fructose 6 Phosphate
Phospho Fructose kinase
ATP
ADP
Fructose 1, 6 Bis Phosphate
GLAP DHAP
Trios PO4 isomerase
1,3 Bisphospho Glycerate
2 NAD+ + 2Pi
2 NADH
3 Phosphate Glycerate
Phospho glycerate kinase
2 ATP
2 ADP
2 Phosphate Glycerate
Phospho glycerate mutase
Phospho enol pyruvate
enolase
Pyruvate
Pyruvate kinase
2 ATP
2 ADP
aldolase
GLAP hydrogenase
DHAP: di hydroxy acetone phosphate
GLAP: Glyceraldehyde 3 Phospahte

26. Citric acid Cycle
CITRIC ACID CYCLE
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27. Metabolic pathways
 Sugar or glucose alternatively enters pentose phosphate
pathway (HMP)
 It helps in formation of NADPH from fatty acids,
synthesis of ribose for nucleotide & nucleic acid
(oxidative pathway)
 Helps in formation of erythrose 4
phosphate, used in synthesis of
aromatic amino acids through
shikimic acid pathway.
(non oxidative)
↓ precursor for lignin,
& other biosynthetic pathways.
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HMP SHUNT PATHWAY
Glucose 6 Phosphate
NADP+
NADPH + H+
Mg+2 Glucose 6 P
dehydrogenase
6 Phosphoglucanolactone
Glucanolactone
hydrolase
6 Phosphogluconate
NADP+
Mg+2
CO2, NADPH + H+
Phosphogluconate
dehydrogenase
Ribulose 5 phosphate

28. Shikimic acid Pathway
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31. SHIKIMIC ACID PATHWAY
 It is an intermediate from carbohydrate for
biosynthesis of phenyl propane derivative
(C6-C3 unit) ex. Tyrosine & phenylalanine
 Precursor for synthesis of shikimic acid is
phospho enol pyruvate- (from Glycolysis) &
erythrose-4-phosphate an intermediate of PPP/
HMP
 The N required in pathway is obtained from –
other amino acids- Glutamine, glycine, serine.
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32. Role of Shikimic acid
 Precursor for biosynthesis of some phenolic,
phenyl propane derivatives- flavonoid,
coumarin, tannins, lignin
 Precursor for indole, indole derivative &
many alkaloids & other aromatic metabolites
 Gallic acid biosynthesis from 3 de-
hydroshikimate (intermediate)
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 In the genesis of aromatic building blocks of lignin & in
formation of few tannins, vanillin, phenylpropane units of
flavones & coumarins and also in the formation of
alkaloids.

33. SHIKIMIC ACID PATHWAY
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Phosphoenol pyruvate (Glycolysis) + erythrose-4-PO4 (PPP)
2-keto, 3-deoxy-7-phospho-D-gluco heptanoic acid
DAHP synthase
3-dehydro- quinic acid (DHQ)
DHQ synthase
3-dehydro-shikimic acid
3-dehydro quinate dehdratase
dehydration
Shikimate dehydrogenase Reduction
Shikimic acid
Shikimic acid-3-PO4
Shikimate kinase Catalyses ATP dep.
phosphorylation
5-enol-pyruvylshikimate-3-PO4
Coupling, loss of H2O
EPSP synthase
Phosphoenol pyruvate
Chorismic acid
Chorismate synthase
Claisen rearrangment
Chorismate
mutase
Prephenic acid
p-hydroxy phenyl pyruvate
Oxidative
decarboxylation
Retention of -OH
Prephenate
dehydrogenase
Tyrosine & α-
ketoglutarate
Transamination
Glutamate as
N2 source
Phenyl pyruvic acid
Tryptophan
Aldol condensation
Anthranilic
acid
Phenylalanine

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38. Acetate Pathway
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39. Acetate Pathway
 Acetate required for synthesis of various
secondary metabolites in plants.
 Starting material is acetate, utilized as acetyl Co-A
(active form of acetate)
 Various straight chain or aromatic compounds are
synthesized by acetate pathway.
 Acetate Mevalonate Pathway (MAP) contributes to
1/3rd of secondary metabolites.
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40. Acetate Pathway
Glycolysis
Pyruvate
End product
Acetyl Co-A
TCA
Acetate mevalonate pathway Acetate melonate pathway-
mevalonic acid Malonyl Co-A
Higher terpenoids,
rubber etc.
Long chain fatty acids &
polyketides
Isoprenoids
Squalene
Steroids
Lipids, fats , waxes

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Acetate-Mevalonate Pathway
• The role of acetic acid in biogenetic pathways was discovered in
1950 after the discovery of acetyl co -enzyme A ( active acetate ).
• Later, it was discovered that mevalonic acid is associated with
acetate.
• Isopentenyl Pyrophosphate (IPP) and its isomer
Dimethylallyl Pyrophosphate (DMAPP) are produced by
mevalonic acid.
• IPP and DMAPP are the two chief intermediates that set the ‘active
isoprene’ unit as the basic building block of isoprenoid
compounds.
• Both of the units produce geranyl pyrophosphate (C10-
monoterpenes), which further associates with IPP to yield farnesyl
pyrophosphate (C15-sesquiterpenes).

43. Acetate Mevalonate Pathway
 Isopentenyl pyrophosphate & its isomer that is dimethyl allyl
pyrophosphate are universal precursors for synthesis of
isoprenoids/terpenoids/volatile oils/cholesterol/saponins/steroids.
 Isoprene unit (C5H8) - responsible along with other pathways for
biogenesis of anthraquinone, naphthaquinone, terpenoids etc.
 Isoprene units (C5H8)
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CH3
CH2
C CH CH2
C
CH3
CH2 C
H
CH2
1
2
3
4
5

44. Terpenoids are derived from isoprene units (C5H8)
which are joined in a head-to-tail or head-to-head
fashion.
C5
C10
C15
C20
C25
C30
C40
hemiterpenes
monoterpenes
sesquiterpenes
diterpenes
sesterterpenes
triterpenes
tetraterpenes
CH3
CH2
C CH CH2

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Iso Pentenyl Pyrophosphate + Di Methyl Allyl Pyro Phosphate (active isoprene units)
CONDENSE
Geranyl pyropyrophosphate C10 Monoterpenes e. g. Menthol, limonene,
geraniol, linalool, citral
Farnesyl PP C15
Sesquiterpenes e.g. termerone,
zingiberene, abscisic acid, santalone
IPP
C30
Triterpenoids
Squalene
Steroids, cholesterol
Solanine, diosgenin
IPP
Diterpenes Taxol
Polyterpenes Rubber
C40 Tetraterpenes
Carotenoids, lycopene, β carotene

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• Farnesyl pyrophosphate produces geranyl-geranyl
pyrophosphate (C20-diterpenes) by combining with another
unit of IPP.
• Farnesyl pyrophosphate multiplies with its own unit to form
squalene and its successive cyclisation produces
cyclopentanoperhydrophenanthrene skeleton that contains
steroidal compounds (like cholesterol and triterpenoids).
• Thus, two different skeleton containing compounds, i.e.,
steroids and triterpenoids are produced by the acetate
mevalonate pathway working through IPP and DMAPP via
squalene.
• A huge array of monoterpenoids, sesquiterpenoids,
diterpenoids, carotenoids, polyprenols, glycosides, and
alkaloids are also produced in association with other pathway.

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The sapogenins occurs in glycosidic form of saponins. The neutral
saponins are steroidal derivatives possessing spiroketal side chain
and acid saponins have triterpenoid structure.
The pathway is similar for the biosynthesis of sapogenins. The
triterpenoid hydrocarbon squalene is formed after cyclisation of
triterpenoids in one direction and spiroketal steroids
in other direction.
The squalene, cholesterol and other steroidal compounds including
aglycone are formed in the following manner.

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Acetate-Malonate Pathway
• With the involvement of Acyl Carrier Protein (ACP), the
acetate pathway works functionally to produce fatty acyl
thioesters of ACP.
• These acyl thioesters build the important intermediates in
fatty acid synthesis.
• In a later stage, even numbers of fatty acids from n -
tetraenoic (butyric) to n -eicosanoic (arachidic acid) are
produced by these C 2 acetyl CoA units.
• Subsequent direct dehydrogenation of saturated fatty acids
produces unsaturated fatty acids.
• Enzymes play a crucial role in directing the position of newly
introduced double bond in the fatty acids.

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• The acetate pathway is important in the formation of various important
phytoconstituents like fatty acids, polyketides, prostaglandins, aflatoxin,
tetracycline and other various important phytoconstituents.
• For the biosynthesis of fatty acid, the acetyl CoA carboxylated to form
malonyl CoA by the presence of enzyme named acetyl CoA carboxylase.
• The energy requisite for this carboxylation is supplied by ATP and loss of
CO2 occurs.
• After this step reduction, dehydration and again reduction will occur.
• During both reductions process the electron is provided by NADH+ and H+
and the formation of butyryl ALP will occur.
• The coupling between malonyl ALP and butyryl ALP will occur and their
reduction is repeated again for whole chain.
• Malonyl CoA bind again with the fatty acid residue by increase the chain
with two carbon unit.
• The first end product is palmitic acid which has 16 carbon atoms.
• The chain is further elongated by various mechanisms.

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59. Amino acid pathway
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• Plants and bacteria can synthesize all 20 of the amino acids. Whereas
humans cannot synthesize 9 of them.
• These 9 amino acids must come from our diets and therefore they
are called essential amino acids.
• The essential amino acids are Histidine, Isoleucine, Leucine, Lysine,
Methionine, Phenylalanine, Threonine, Tryptophan, and Valine.
• The 11 amino acids are called non-essential amino acids like
Alanine, Arginine, Aspargine, Aspartate, Cysteine, Glutamate, Glutamine,
Glycine, Proline, Serine and Tyrosine.
• The non-essential amino acids are synthesized by simple pathways,
whereas biosynthesis of the essential amino acids are complex. All 3
aromatic amino acids are derived from shikimate pathway.

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• The carbon skeletons of amino acids are derived from different
intermediates of the central carbon metabolism (boxed in blue).
• According to their respective precursors, the amino acids are grouped
into five families derived from glutamate, serine, pyruvate, aspartate, or
chorismate.
• The nine amino acids that cannot be synthesized in animals are shown
in dark-green boxes, while those that can be synthesized but
additionally need to be taken up with the diet are in brighter boxes.
• Proteinogenic amino acids that can be sufficiently synthesized in
animals are in pale green boxes and non-proteinogenic amino acids
and other important intermediates are boxed in white. DAHP, 3-deoxy-
D-arabinoheptulosonate-7-phosphate.

69. Biosynthesis of Glycosides
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• The metabolic process of glycoside formation occurs in
two steps:
• In first step various types of aglycone are formed by
biosynthetic reactions whereas in second step coupling
of aglycone with sugar moiety occurs.
• In different types of glycosides interaction of nucleotide
glycoside occurs between UDP-glucose with alcoholic
or phenolic group of secondary compound aglycone
(called O-glycosides), through linkage with carbon (C-
glycosides), nitrogen (N-glycosides) or sulphur (S-
glycosides).

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The following two steps are involved in this process:
1. In first step, the uridine triphosphate (UTP) transferred an uridylyl
group to sugar-1-phosphate and forms UDP-sugar and inorganic
pyrophosphate. The enzyme which catalyzes this reaction is uridylyl
transferases.
UTP + Sugar-1-PO4 UDP- sugar + PP1
2. In second step, transfer of the sugar moiety from UDP to a suitable
acceptor (aglycone) occurs. This reaction is mediated by enzyme
glycosyl transferases and forms glycoside.
UDP-sugar + Acceptor (aglycone) Acceptor-sugar (glycoside) + UDP

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• The sugars of glycosides are monosaccharides (like
Rhamnose, Glucose, Frucose or deoxy sugars i.e. digitoxose
or cymarose).
• The aglycone moieties of cardiac glycosides are steroidal in
nature.
• These are derivatives of cyclopentenophenanthrene ring
which contains unsaturated lactone ring attached with C17, a
14-alpha hydroxyl group and a cisjucture of ring C and D.
• The anthraquinone glycosides are biosynthesized from
shikimic acid pathway in Rubiaceae family.
• The alizarin biosynthesis shows ring A is derived from shikimic
acid whereas mevalonic acid is included in ring C.

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It is very important to understand the biosynthesis of flavonoids due to
their diversity.
These flavonoidal molecules are biosynthesized by their precursor
which is three molecules of acetic acid and phenyl propane moiety.
It mainly involves the interaction of five different pathways which are
named as:
1. The Glycolytic pathway.
2. The Pentose phosphate pathway.
3. The Shikimate pathway that synthesizes phenylalanine
(An amino acid).
4. The phenylpropanoid metabolism that produces activated cinnamic
acid derivatives i.e. 4-coumaroyl-CoA and lignin (also the plant
structural component).
5. The diverse specific flavonoidal pathway.

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• The flavonoids are biosynthesized via condensation of the shikimic
acid and acylpolymalonate pathways.
• The phenyl propane (cinnamic acid derivative) synthesized from
shikimic acid which acts as a precursor in a polyketide synthesis.
• In this scheme additional three acetate residues are incorporated
into the structure and followed by ring closure.
• The plants biosynthesizes different classes of flavonoids like
flavonols, flavanones, flavones, flavanols or catechins, iso-flavones,
dihydro-flavonols, anthocyanidins, and chalcones through
subsequent hydroxylation and reduction.

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• In the biosynthesis of Cyanogenetic or Cyanophoric glycosides (e.g.
Prunasin) the amino acid phenylalanine acts as a precursor.
• In this biosynthetic pathway an aldoxime, a nitrile and a cyanohydrin
are involved as intermediate.
• The chiral centre in the mandelonitrile provides the opportunity for 2
β–glucosides to form.
• D-mandelonitrile glucoside is formed in Prunus serotina (wild
cherry) whereas L-mandelonitrile glucoside (isomeric samburgrin) is
found in Sambucus nigra.
• These compounds are not found in same species.

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83. Alkaloids
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Summary

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What do you understand by study of basic metabolic pathways and formation of
different secondary metabolites?
2. How secondary metabolites are produced from biosynthetic pathways?
3. What is Shikimic acid pathway? Draw its pathway.
4. How glycosides are biosynthesized? Draw its pathway.
5. Draw biosynthetic pathway of Flavonoids.
6. Draw biosynthetic pathway of Isothiocyanate aglycones.
7. Draw biosynthetic pathway of Cyanogenetic glycosides.
8. How secondary metabolites are obtained by Cholesterol metabolism?
9. Draw Acetate pathway.
10. Draw Amino acid pathway.
11. Draw general scheme for amino acid production through pathway.
12. Write a detailed note on utilization of radioactive isotopes in the investigation of
biogenetic studies.
13. What is the role of radioactive tracers? How they are detected?
14. What is autoradiography? Explain precursor product sequence.
15. What is competitive feeding? How precursors are administered in any pathway?
16. What is sequential analysis? Describe briefly.

90. Thanks
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