Transcript phytochemical - Portal UniMAP

PHYTOCHEMICAL
• Define the properties and classification of
phytochemicals.
• Compare the types of phytochemicals in
plants
Introduction
• Plant metabolism
– Primary
• directly involved in normal growth, development, and
reproduction of plant species
• Eg.
– Secondary
• not directly involved in those processes, but usually has important
ecological functions like defences against predators, parasites and
diseases, for interspecies competition, and to facilitate the
reproductive processes
• Eg. coloring agents, attractive smells, etc
Both: famous for their beneficial effects on human health
What is phytochemical?
• Secondary metabolite
• Phyto = plant + chemical
– Natural substance in plant
– Bioactive compound
– Not vitamin
• phytochemicals are classes of compounds that are only found in
plants that do not also fall into the category of essential nutrients.
– phytochemicals are compounds that we ingest when we eat plants,
but they are the parts that aren’t absolutely needed by the body – in
other words, they are not nutritionally essential.
• Generally, the arils of fruit contain large amounts of organic acids,
sugars, minerals, and vitamins, but the peels contain higher amount
of phenolic compounds than the flesh.
Phytochemical classes
Classification are based on
• Biosynthetic origin
• Solubility properties
• Presence of certain key functional groups
Phenolic compound
•Recognized by their
hydrophilic compounds
•Common origin from
the aromatic precursor
shikimic acid
Organic acids, lipid
and other classes of
compounds
Terpenoids
•Share lipid properties
•Biosysnthetic origin
from isopentenyl
phytophosphate
Sugar and their
derivatives
•Water-soluble
carbohydrates
Nitrogen
compounds
•Recognized by their
positive responses to
either ninhydrin or the
Dragendorff reagent
Macromolecules
•Easily separated from
other constituents by their
high molecular weights
Methods of extraction and isolation The plant material
• Ideally – fresh plant tissue and should be plunged into boiling alcohol
within a minutes of its collection
• Alternatively – may be dried before extraction
– Drying under controlled conditions to avoid chemical changes
– Dried as quickly as possible without using high temp, in a good air
draft
• Take note for the nature of the compounds eg:
– Essential oil – sensitive to temp change and decreased over time–
avoid drying
– Flavonoids and alkaloids – remarkably stable with time
– Tannin – better to extract from vacuum-dried fresh leaves rather than
air-dried
• Free of contamination and disease – not affected by viral, bacteria or
fungal infection
• Botanical identity of the plant – not to be mistakes – taxonomy expect
Methods of extraction and isolation The extraction
• No precise mode of extraction – no right or wrong method of extraction
• In general – ‘kill’ the plant tissue
– Prevent enzymic oxidation or hydrolysis
– Plunging fresh leaf or flower tissue
– Suitably cut up where necessary
• Boiling ethanol/ alcohol – good all-purpose solvent for preliminary
extraction
• Plant material can be macerated in a blender and filtered
• Extraction will be assumed completed when its completely free of colour
– repeat extraction
• Classical chemical procedure for dried material – Soxhlet apparatus
• The extract obtained is clarified by filtration and then concentrated in
vacuo – rotary evaporator
• For volatile compounds – needs special precaution and apparatus.
Methods of separation
• Chromatography
–
–
–
–
Paper chromatography (PC)
Thin layer chromatography (TLC)
Gas chromatography (GC)
High performance liquid chromatography (HPLC)
• The choice of technique depends on the solubility properties and
volatilities of the compounds
–
–
–
–
PC – applicable to water-soluble compounds
TLC – separating lipid-soluble compounds
GC – volatile compounds
HPLC – less volatile compounds and polar compounds
• Capillary electrophoresis, liquid-liquid extraction, droplet countercurrent chromatograhy (DCCC), affinity chromatography, differential
ultracentrifugation
Methods of identification
• Isolation and purification
• First to determine the class of compound
• Then to find out which particular substance it is
within that class
• Methods
–
–
–
–
UV and visible spectroscopy
Infrared spectroscopy (IR)
Mass spectroscopy (MS)
Nuclear magnetic resonance spectroscopy (NMR)
Phenolic compounds
Introduction
• Wide range of plant substances which possess in common
an aromatic ring bearing one or more hydroxyl substituent
• Phenolic substances tend to be water-soluble, since they
are usually located in the vacuole.
• The flavonoids form the largest group but simple
monocyclic phenols, phenylpropanoids and phenolic
quinones all exist in considerable numbers.
• Several important groups of polymeric materials in plants –
the lignins, melanins and tannins – are polyphenolic
• Function of some classes of phenolic compound – eg. The
lignins as structural material the cell wall; the anthocyanins
as flower pigments,
• Phenolic compounds are all aromatic, so that they all show
intense absorption in the UV region of the spectrum
Phenols and phenolic acids
• The free phenols and phenolic acids are best
considered together since they are usually identified
together during plant analysis
• Acid hydrolysis of plant tissue releases a number of
ether-soluble phenolic acids
• These acids are either associated with lignin
combined as ester groups or present in the alcoholsoluble fraction of the leaf, maybe present in the
alcohol-soluble fraction bound as simple glycosides.
• Free phenols are relatively rare in plants
• Simple phenols are included in here because their
identification is important to determining the
structure of flavonoid.
Phenylpropanoids
• Naturally occurring phenolic compounds which
have an aromatic ring to which a three-carbon sidechain is attached.
• Derived biosynthetically from the aromatic protein
amino acid phenylalanine and they may contain
one or more C6-C3 residue.
• The most widespread -hydroxycinnamic acids –
which are important not only as providing the
building blocks of lignin but also in relation to
growth regulation and to disease resistance.
• Four hydroxycinnamic acids are common in plants –
ferulic acid, sinapic, caffeic and p-coumaric acids.
Flavonoid pigments
• Derived from the parent substance flavone – more
general
• Classes of flavonoid
• Mainly water-soluble compounds
• Can be extracted with 70% ethanol and remain in
the aqueous layer, following partition of this
extract with petroleum ether.
Properties of different flavonoid classes
Flavonoid class
Anthocyanins
Proanthocyanidins
Flavonols
Flavones
Distribution
scarlet, red, mauve and blue
flower pigment; also in leaf
and other tissue
mainly colourless, in
heartwoods and in leaves of
woody plants
mainly colourless
copigments in both cyanic
and acyanic flowers;
widespread in leaves
as flavonols
Characteristic properties
water-soluble, visible max
515-545 nm, mobile in
BAW on paper
yield anthocyanins (colour
extractable into amyl
alcohol) when tissue is
heated for 0.5h in 2 M HCl
after acid hydrolysis, bright
yellow spots in UV light on
Forestal chromatogram;
spectral max 350-386nm
after acid hydrolysis, dull
absorbing brown spots on
Forestal chromatogram;
spectral max 330-350nm
Properties of different flavonoid classes
Flavonoid class
Glycoflavones
Distribution
as flavonols
Biflavonyls
colourless; mainly confined to the
gymnosperms
Chalcones and
aurones
yellow flower pigments;
occasionally present in other tissue
Flavanones
colourless; in leaf and fruit
(especially in Citrus)
Isoflavanons
colourless; often in root; only
common in one family, the
Leguminosae
Characteristic properties
contains C-C linked
sugar; mobile in water
unlike normal flavones
on BAW chromatograms
dull absorbing spot of
very high Rf
give red colours with
ammonia (colour change
can be observed in situ),
visible max. 370-410
give intense red colours
with Mg/HCl;
occasionally an intense
bitter taste
mobile on paper in water;
no specific colour tests
available
Flavonoid
• Flavonoid are phenolic - change in colour when treated with
base or ammonia.
• Flavonoid contain conjugated aromatic systems - show intense
absorption bands in the UV and visible regions of the spectrum.
• Flavonoid generally present in plants bound to sugar as
glycosides and any one flavonoid aglycones may occur in a
single plant in several glycosidic combinations.
• When analysing flavonoids, it is usually better to examine the
aglycones present in hydrolysed plant extracts before
considering the complexity of glycosides that may be present in
the original extract.
• Flavonoids are present in plants as mixtures and it is very rare
to find only a single flavonoid component in a plant tissue.
• In addition, there are often mixtures of different flavonoid
classes.
Quercetin 3-O-glycoside 5-Orhamonside @
Quercetin 3-rutinoside
Anthocyanins
• Most important and widespread group of
coloring matters in plant
• These intensely coloured water-soluble
pigments are responsible for nearly all the
pink, scarlet, red, mauve, violet and blue
colours in the petals, leaves and fruits
• The anthocyanins are all based chemically on a
single aromatic structure, that of cyanidin, and
all are derived from this pigment by addition
or subtraction of hydroxyl group or by
methylation or by glycosylation
• 6 common anthocyanidins (anthocyanin
aglycones formed when anthocyanins are
hydrolysed with acid)
–
–
–
–
–
–
Magenta-colored (cyanidin)
Orange-red colored (pelagornidin)
Mauve, purple and blue colour (delphinidin)
Peonidin
Petunidin anthocyanin methyl esthers
Malvidin
• Occurs with various sugars attached – glucose,
galactose, rhamnose, xylose or arabinose
• The number of sugar units (mono, di- or triglycoside) and the position of attachment of
sugar(usually to the 3-hydroxyl, or to the 3and 5- hydroxyls.
• Anthocyanin occur acylated with either an organic acid such
as malonic or with aromatic acid such as p-coumaric acid
• Acylation is commonly through the sugar unit of the 3position and both type of acylation may be present in the
same molecule.
• Anthocyanin are unstable in neutral or alkaline
solution
• Stable in acid solution but the colour may fade
due to exposure to light
• Anthocyanin must therefore be extracted from
plants with solvents containing acetic or
hydrochloric acid and solution should be stored in
the dark and preferable refrigerated.
Flavonols and flavones
• Flavonols occur most frequently in glycosidic
combination (glycone/presence of sugar)
• 3 at common – kaempferol, quercetin, myricetin
• Flavones only differ from flavonols in lacking a 3hydroxyl substitution; this affects their UV
absorption, chromatographic mobility and colour
reactions
• 2 common flavones – apigenin and luteolin,
corresponding in hydroxylation pattern to
kaempferol and quercetin.
Minor flavonoids, xanthones and
stilbenes
• The chalcones, aurones, flavanones,
dihydrochalcones and isoflavones are
designated ‘minor flavonoids’ because each of
these classes is of limited natural distribution.
• Chalcones and aurones together comprise the
‘anthochlors’, yellow pigments which can be
detected by the fact that a change to orange or
red colour is observed when a yellow petal is
fumed with the alkaline vapour of a cigar or
with a vial of ammonia.
Tannins
• Occur widely in vascular plants – associated with
woody tissue
• By definition – they have ability to react with protein,
forming stable water-insoluble co-polymers.
• Industrially – tannins are substances of plant origin
which have ability to cross-link with protein that
capable of transforming raw animal skin into leather.
• In plant cell, tannins are located separately from
proteins and enzymes of the cytoplasm but when
tissue s damage, eg when animal feed, the tanning
reaction may occur, making the protein less accessible
to the digestive juices of the animal.
• Plant tissue high in tannins but largely avoided by
most feeders because of the astringent taste.
• Two types of tannins
– Condensed tannins – occur almost universally
in ferns and gymnosperms and widespread
among angiosperms especially woody
species.
– Hydrolysable tanins – limited to
dicotyledonous plants
– Both can occur together in the same plant
such as in oak bark and leaf.
• Condensed tannins or flavolans – formed
biosynthetically by the condensation of single
catechin (or gallocatechins) to form dimers and then
higher oligomers, with C-C linking one flavan unit to
the next by a 4 – 8 or 6 – 8 link.
• Most flavonols have between 2 and 20 flavan units
• Proanthocyanidin is used alternatively for condensed
tannins because on treatment with hot acid, some of
the carbon-carbon linking bonds are broken and
anthocyanidin monomers are released.
• Most proanthocyanidins are procyanidins, which
means that they yield cyanidin on acid treatment.
General structure of flavan-3-ol unit.
A) (+)catechin and (-)epicatechin monomers, B) procyanidin B2 dimer and C) procyanidin oligomers with C4-C8 linkage
(Contreras-Domínguez et al., 2006).
• Hydrolysable tannins
– Galloylglucose – glucose core is surrounded by 5
or more galloyl ester groups
– Ellagitannins – esters of hexahydroxydiphenic acid
with glucose attachment
Nomenclature
Structure
Molecular weight
range
Condensed tannins
- Proanthocyanindins
(or flavolans)
Oligomers of
catechins and
flavans-3-4-dials
1000 - 3000
Hydrolysable tannins
- Gallotannins
-
Ellagitannins
Prototannins
- Tannin precursors
Esters of gallic acid 1000 – 1500
and glucose
esters of
1000 – 3000
hexahydroxydipheni
c acid and glucose
Catechins (and
gallocatechins)
flavan-3,4-diols
200 – 600
Quinone pigments
• The natural quinone pigments range in colour from
pale yellow to almost to black.
• Relatively little contribution to colour in higher plants
• Frequently present in bark, heartwood or root or
leaves tissue where their colours are masked by other
pigments.
• Bacteria, fungi and lichen – pigmented by quinones
• Identification – divided into 4 groups: bezoquinines,
naphthalquinone, anthraquinones and isoprenoid
quinones
Recap - flavonoid
• have the general structure of a 15-carbon skeleton
• which consists of two phenyl rings (A and B) and
heterocyclic ring (C)
• occurs with various sugars attached
Terpenoids
•
•
•
•
Introduction
Terpenoids – all based on the isoprene molecules
CH2=C(CH3)-CH=CH2
General formula (C5H8)n
Its range from essential oil components, the
volatile mono- and sesquiterpenes (C10 and C15),
less volatile diterpenes (C20), involatile
triterpenoids and sterols (C30 )and carotenoid
pigments (C40)
• Classification – based on n values
• Significant either in pant growth, metabolism or
ecology
General formula : (C5H8)n
Value of n (number Number of
of isoprene units) carbon
atoms
Name of class
Main type and occurrence
1
5
Isoprene
2
10
Monoterpenoids
3
15
Sesquiterpenoids
4
20
Diterpenoids
Detected in Hamamelis japonica
leaf
Monoterpene in plant essential oil menthol from mint
monoterpene lactones
tropolones
Sesquiterpenes in essential oils
sesquiterpenes lactones (common
in Compositae)
abscisins (abscisic acid)
Diterpene acids in plant resins
gibberellins (gibberellic acid)
5
6
25
30
Sesterpenoids
Triterpenoids
8
>8
40
>40
Tetraterpenoids
Polyterpenoids
Sterol
Triterpenes
Saponins
Carotenoids
rubber eg in Hevea brasiliensis
• Chemically, terpenoids are generally lipidsoluble
• Located in cytoplasm of the plant cell
– Essential oils sometimes occurs in special
glandular cells on the leaf surface
– Carotenoids – associated with chloroplasts in the
leaf and with chromoplasts in the petal
• Extraction – light petroleum, ether or
chloroform
• Separation – by chromatography on silica gel
or alumina using same solvent
• Difficult in detection on a microscale except carotenoids
– Because terpenoids are colourless
– No sensitive universal chromogenic reagent
• Isomerism is common: geraniol and nerol
• Mostly alicyclic compound because the cyclohexane ring
usually twisted in ‘chair’ form, different geometric
conformations are possible, depending on the substitution
around the ring – often difficult to determine
• Functions of plant terpenoids
– growth-regulating properties – abscisins (sesquiterppenoid)
and gibberellins (diterpenoid)
– Agents of communication and defense among insects
– Non-volatile terpenoids – sex hormones among fungi
– Carotenoids
• plant colour - pale yellow through bright orange to deep red
• Accessory pigments in photosynthesis
– Mono- and sesquiterpenes – distinctive smells and odours
Essential oils
• Volatile steam-distillable – characteristic scent, odour or smell
• Commercially important – natural perfumes, spices and flavoring
in food industry
• Chemically, terpene essential oils divided into 2
– Monoterpenes (C10) – boiling point 140-180 °C
– Sesquiterpenes (C15) – bp >200 ° C
• Mono – divided into three group depending on whether they are
acyclic, monocyclic, or bicyclic.
• Within each group, mono-maybe simple unsaturated
hydrocarbons (limonene) or may have functional groups and be
alcohols (menthol), aldehydes or ketones (menthone, carvone).
• Simple mono- are widespread and tend to occurs as majority of
essential oils.
• Flower and seed oils tend to have more specialized monopresent
• Sesqui- group according to the basic carbon
skeleton (same as mono-)
• Either acyclic, monocyclic or bicyclic.
• But within group – there are too many
different compounds known
• 2 special sesqui- because of the growthregulating properties – abscisic acid (hormone
controlling domancy in seed) and xanthinin
(auxin-antagonist)
• Isolation from plant tissues – both mono- and
sesqui- separated by extraction into ether,
petroleum or acetone.
• Classical extraction procedure – steam
distillation
• Due to the volatility – ideal for separation by
GC
Diterpenoids and gibberellins
• Comprises of a chemically heterogeneous group of
compounds
• All with C20 carbon skeleton - based on four isoprene
units
• Very limited distribution compound
• Probably the only universally distributed diterpene is
acyclic parent compound of the series – phytol
(present as the ester attachment in chlorophyll)
• 3 classes of diterpenes – resin diterpenes,
toxic diterpenes and gibberellins
• Resin diterpenes
– Have protective function in nature
– Exuded from wood of trees or latex of herbaceous
plants
• Toxic diterpenes
– Poisonous
• Gibberellins
– Group of hormone – stimulate growth
– Gibberellic acid – the most popular gibberellin
Triterpenoids and steroid
• Compounds with carbon skeleton based on 6 isoprene
units
• Derived biosynthetically from the acyclic C30
hydrocarbon
• Relatively complex cyclic structure
• Most either alcohols, aldehydes or carboxylic acids.
• Colourless, crystalline, often melting, optically active
substance which generally difficult to characterize
because lack of chemical reactivity
• Widely used test – Liebermann-Burchard reaction
(acetic anhydride-conc H2SO4) – produces a blue-green
colour with most triterpenes and sterols
• Divided into at least 4 groups of compounds:
– True triterpenoid
– Steroids
– Saponin
– Cardiac glycosides
occur mainly as glycosides
• Functions
– occur especially in waxy coatings of leaves and on
fruits – protective function in repelling insect and
microbial attack
– Taste properties – bitterness
• eg. Limonin – the lipid-soluble bitter principle in Citrus
fruits
Carotenoids
• C40 tetraterpenoids
• Extremely widely distributed group of lipidsoluble pigment
• 2 principle function
– As colouring matters in flowers and fruits
– Accessory pigment in photosynthesis
• Eg in flowers – yellow colour (daffodil, marigold)
• Eg in fruits – orange or red (tomato, paprika,
palm oil)
• Over 600 known carotenoids but only a few are common
in higher plant
• Identification easy to resolve – by reference to the
common substance
• Split into two class –
– Purely hydrocarbon and contain no oxygen - based on
lycopene
• Chemical structure of lycopene consists of a long chain of 8 isoprene
units joined head to tall, giving a completely conjugated system of
alternate double bond – which is the chromophore giving it colour
• Cyclization of lycopene at one end give γ-carotene
• Cyclization of lycopene at both end give β-carotene
• β-carotene isomers (α- and ε-carotene) differ in the position of the
double bond in the cyclic end units
– Oxygenated derivatives (contain oxygen) – xanthophylls
• Monohydroxycarotenes - lutein, rubixanthin
• Dihydroxy – zeaxanthin
• Dihydroxyepoxy – violaxanthin
• Glycosides – very rare in higher plant – the
best known is water-soluble crocin
• Combined forms of carotenoid
– xanthophylls esterified with fatty acid
– Eg. palmitic, oleic or linoleic acid
• Assignment presentation – 31st March 2015
• Mid term test 1 – 13th April 2015
• Mid term test 2 – 11th May 2015
Nitrogen compound
Introduction
• Nitrogen compounds – nitrogen element
• Nitrogen compounds are usually basic, thus form salts with
mineral acids
• Can be extracted from plant tissue using weak acidic
solvents
• Can be precipitated from extracts by addition of ammonia
• Many nitrogen compounds are charge molecules, so
electrophoresis can be used for separation
• Detection technique
– spray reagent Ninhydrin – amino acids
– Dragendorff reagent - alkaloids
Amino acid
• Plant amino acid conveniently divided into two groups (although
the division between the two groups is not entirely sharp and
methods of identifying and separating both groups are essentially
the same)
– Protein amino acid
• Generally recognized to be twenty in number
• Found in plant and animal
• Glutamic, aspartic acids, glutamine and asparagine - present in larger amount
and represent a storage form of nitrogen
• Histidin, trytophan, cystein and methionine – low amount in plant tissue and
cannot be readily detected.
– Non-protein amino acid
• Only γ-aminobutyric acid regularly present in plants
• Their role in plant is not clear, although present in high concentration in seeds
• May be important as nitrogen storage material
• Amino acid are colourless ionic compounnds
• Solubility properties and high melting point –
zwitterions
• Water soluble
• Amino acid have difference charge properties,
amino acid mixture can be divided into
neutral, basic and acidic fractions by using
either electrophoresis or ion exchange
chromatograhy.
Amino acid
Glycine
Alanine
Serine
Cysteine
Threonine
Valine
Leucine
Isoleucine
Methionine
Aspartic acid
Asparagines
Glutamic acid
Glutamine
Arginine
Lysine
Proline
Phenylalanine
Tyrosine
Tryptophan
Histidine
Ninhydrin colour
red-violet
violet
blue-violet
orange-brown
violet
yellow
grey-violet
Charge properties
neutral
neutral
neutral
neutral
neutral
neutral
neutral
neutral
neutral
acidic
neutral
acidic
neutral
basic
basic
neutral
neutral
neutral
neutral
basic
Amines
• Considered simply as the products of decarboxylation of
amino acid, formed by the reaction:
RCH(NH2)CO2H  RCH2NH2 + CO2
• Divided into three groups
– aliphatic monoamines
• Volatile compound eg methylamine (CH3NH2), n-hexylamine
(CH3(CH2)5NH2
• Have an unpleasant fish-like smell
• Function in flowers as insect attractants
– aliphatic polyamines
•
•
•
•
Less volatile
Still possess offensive odours
Eg putrescine, agmatine, spermidine, spermine
Function – growth-stimulating activity in relation to their effect on
ribosomal RNA
– aromatic amines
• Many of known aromatic amines are physiologically active and
sometimes classified as alkaloids
Alkaloids
• Largest single class of secondary plant substance
• No one definition of term alkaloid which completely
satisfactory
• Alkaloid generally includes ‘ those basic substance
which contain one or more nitrogen atom, usually in
combination as part of a cyclic system’
• Often toxic to man and many have dramatic
physiological activities; hence wide use in medicine
• Usually colourless, often optically active substance,
most are crystalline but a few liquid at room temp (eg.
nicotine)
• Simple test for alkaloid – bitter taste
• Many alkaloid are terpenoid in nature (eg. solanine)
and some are best considered as modified terpenoid
• Others are mainly aromatic compound (eg. colchine) –
containing their basic group as side-chain attachment
• Specific to one family or to a few related plants – name
of alkaloid types are often derived from plant source –
eg nicotine (Nicotina tabacum), atropine ( Atropa
belladonna)
• Mostly in angiosperm, generally absent or infrequent
in gymnosperms, ferns, mosses and lower plant.
• Function of alkaloid – still obscure / not clear
• Some reported to be involved as growth
regulation, insect repellents or attractants
• Theoretically – they act as a form of nitrogen
storage in plants
• 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 nicotine are water
soluble (with a solubility of ≥1g/L), whereas
others, including morphine and yohimbine are
highly water soluble (0.1–1 g/L).
• Alkaloids and acids form salts of various
strengths.
• These salts are usually soluble in water and
ethanol and poorly soluble in most organic
solvents.
• Extraction - no single method of the 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.
Crystals of piperine extracted from black pepper
Chlorophylls
• Essential catalysts of photosynthesis
• Occur as green pigment in all photosynthetic
plant tissue
• Occur abundantly in the chloroplast, often
bound loosely to protein but are readily
extracted into lipid solvent such as acetone or
ether
• Chemically, chlorophyll contain a porphyrin (tetrapyrrole)
nucleus with a chelated magnesium atom in the centre and
a long-chain hydrocarbon (phytyl) side chain attached
through a carboxylic acid group.
• Thus, the structure of chlorophyll b only differs from that of
a in having an aldehyde group instead of a methyl
substituent attached to the top righ-hand pyrrole ring
• Chlorophyll are relatively labile and during isolation it is
necessary to protect them from degradation eg.
– Active chlorophyllase enzyme removes the phytol side
chain – chlorophyllides
– Lost of central magnesium atom – protochlorophylls
• Determination of chlorophyll – better to extract fresh
tissue and make measurement immediately
• Alternatively – stored in the dark in acetone containing
trace of NA2CO3 at -20 to -30 °C
• General precaution rules – work in dim light to avoid
pigment losses
Fatty acids and lipids
• Mainly occurs in bound form, esterified to
glycerol as fats or lipid
• Comprise up to 7% of the dry weight in leaves in
higher plants and are important as membrane
constituents in chloroplast and mitochondria.
• Abundantly in seeds or fruit to provide plants
with a storage form of energy to use during
germination.
• Seed oil from plant – commercially used – olive,
palm, coconut etc
• Lipids are defined by their special solubility
properties and are extractable with alcohol or
ether
• 3 main classes – due to different fatty acid
residue
– Triglycerides
– Phospholipids
– Glycolipids
• Identification – requires the determination of
their fatty acid component
Case study