1. PHYTOCHEMICAL

2. Define the properties and classification of phytochemicals.
Compare the types of phytochemicals in plants

3. Introduction Plant metabolism Primary Secondary
directly involved in normal growth, development, and reproduction of plant species
Eg. Amino acid, ethanol, water, lactic acid
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

4. 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.

6. Phytochemical classes

7. Class
Sub class

8. 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
Terpenoids
Share lipid properties
Biosysnthetic origin from isopentenyl phytophosphate
Nitrogen compounds
Recognized by their positive responses to either ninhydrin or the Dragendorff reagent
Sugar and their derivatives
Water-soluble carbohydrates
Macromolecules
Easily separated from other constituents by their high molecular weights
Organic acids, lipid and other classes of compounds

9. 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

10. 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.

15. 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 counter-current chromatograhy (DCCC), affinity chromatography, differential ultracentrifugation

16. 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)

17. Class
Sub class

19. Phenolic compounds

20. 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

21. 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 alcohol-soluble 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.

23. Phenylpropanoids
Naturally occurring phenolic compounds which have an aromatic ring to which a three-carbon side-chain 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.

25. 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.

26. Properties of different flavonoid classes
Distribution
Characteristic properties
Anthocyanins
scarlet, red, mauve and blue flower pigment; also in leaf and other tissue
water-soluble, visible max nm, mobile in BAW on paper
Proanthocyanidins
mainly colourless, in heartwoods and in leaves of woody plants
yield anthocyanins (colour extractable into amyl alcohol) when tissue is heated for 0.5h in 2 M HCl
Flavonols
mainly colourless copigments in both cyanic and acyanic flowers; widespread in leaves
after acid hydrolysis, bright yellow spots in UV light on Forestal chromatogram; spectral max nm
Flavones
as flavonols
after acid hydrolysis, dull absorbing brown spots on Forestal chromatogram; spectral max nm

27. Properties of different flavonoid classes
Distribution
Characteristic properties
Glycoflavones
as flavonols
contains C-C linked sugar; mobile in water unlike normal flavones
Biflavonyls
colourless; mainly confined to the gymnosperms
on BAW chromatograms dull absorbing spot of very high Rf
Chalcones and aurones
yellow flower pigments; occasionally present in other tissue
give red colours with ammonia (colour change can be observed in situ), visible max
Flavanones
colourless; in leaf and fruit (especially in Citrus)
give intense red colours with Mg/HCl; occasionally an intense bitter taste
Isoflavanons
colourless; often in root; only common in one family, the Leguminosae
mobile on paper in water; no specific colour tests available

28. 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.

29. Quercetin 3-O-glycoside 5-O-rhamonside @ Quercetin 3-rutinoside

31. 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

33. 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

34. Occurs with various sugars attached – glucose, galactose, rhamnose, xylose or arabinose
The number of sugar units (mono, di- or tri-glycoside) and the position of attachment of sugar(usually to the 3-hydroxyl, or to the 3- and 5- hydroxyls.

35. 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 3-position and both type of acylation may be present in the same molecule.

36. 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.

38. 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 3-hydroxyl 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.

41. 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.

43. 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.

44. 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.

45. 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.

46. 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).

47. Hydrolysable tannins
Galloylglucose – glucose core is surrounded by 5 or more galloyl ester groups
Ellagitannins – esters of hexahydroxydiphenic acid with glucose attachment

48. Nomenclature
Structure
Molecular weight range
Condensed tannins
Proanthocyanindins (or flavolans)
Oligomers of catechins and flavans-3-4-dials
Hydrolysable tannins
Gallotannins
Ellagitannins
Esters of gallic acid and glucose
esters of hexahydroxydiphenic acid and glucose
1000 – 1500
1000 – 3000
Prototannins
Tannin precursors
Catechins (and gallocatechins) flavan-3,4-diols
200 – 600

49. 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

52. 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

53. Terpenoids

54. 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

55. General formula : (C5H8)n
Value of n (number of isoprene units)
Number of carbon atoms
Name of class
Main type and occurrence 1 5
Isoprene
Detected in Hamamelis japonica leaf 2 10
Monoterpenoids
Monoterpene in plant essential oil - menthol from mint
monoterpene lactones
tropolones 3 15
Sesquiterpenoids
Sesquiterpenes in essential oils
sesquiterpenes lactones (common in Compositae)
abscisins (abscisic acid) 4 20
Diterpenoids
Diterpene acids in plant resins
gibberellins (gibberellic acid) 25
Sesterpenoids 6 30
Triterpenoids
Sterol
Triterpenes
Saponins 8 40
Tetraterpenoids
Carotenoids
>8
>40
Polyterpenoids
rubber eg in Hevea brasiliensis

56. sesquiterpenes
monoterpenes

58. Chemically, terpenoids are generally lipid-soluble
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

59. 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

61. 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 °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 mono- present

63. 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 growth-regulating properties – abscisic acid (hormone controlling domancy in seed) and xanthinin (auxin-antagonist)

65. 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

68. 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)

69. 3 classes of diterpenes – resin diterpenes, toxic diterpenes and gibberellins
Have protective function in nature
Exuded from wood of trees or latex of herbaceous plants

70. Toxic diterpenes Gibberellins Poisonous
Group of hormone – stimulate growth
Gibberellic acid – the most popular gibberellin

71. 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

72. 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

74. Carotenoids C40 tetraterpenoids
Extremely widely distributed group of lipid-soluble 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)

75. 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

79. Combined forms of carotenoid
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

83. Nitrogen compound

84. 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

85. 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

86. 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.

87. Amino acid
Ninhydrin colour
Charge properties
Glycine
red-violet
neutral
Alanine
violet
Serine
Cysteine
Threonine
Valine
Leucine
Isoleucine
Methionine
Aspartic acid
blue-violet
acidic
Asparagines
orange-brown
Glutamic acid
Glutamine
Arginine
basic
Lysine
Proline
yellow
Phenylalanine
grey-violet
Tyrosine
Tryptophan
Histidine

88. 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

89. 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

90. Many alkaloid are terpenoid in nature (eg
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.

92. 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

93. 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.

94. 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

95. 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

96. 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

97. General precaution rules – work in dim light to avoid pigment losses
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

98. 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

99. 3 main classes – due to different fatty acid residue
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

100. Case study