Phenolics

2
CHAPTER THREE
PHENOLICS
GENERALITIES
The shikimate and the acetate pathways can combine to further expand the cinnamic acid derivatives. Flavonoids are examples of secondary metabolites of mixed biosynthetic origin (see below). In order to simplify the classification of these numerous types of compounds, we will use a nomenclature based on their carbon skeletons: an aromatic ring is symbolised by C6, a side chain of n carbon atoms, even when it is part of a heterocyclic ring, is represented by Cn. For example, benzoic acids are considered as C6-C1, cinnamic acids and coumarins as C6-C3, and flavonoids as C6-C3-C6. This classification will be used to highlight the biogenetic relationships between the phenolics and to provide a basis for the plan of this chapter.
COOH COOH
I-
Definition Biosynthetic outlines

HO OH OH OH
Phenolics form a large group of substances characterised by at least one aromatic ring substituted by at least one hydroxyl group. This OH can be free or engaged in another function, such as ester, ether, or glycoside. Yet, not all compounds with a phenolic functional group can be classified among phenolics. It depends on their biogenesis: morphine, thymol, or taxiphylline, for example, feature a phenol group; however, they are respectively classified as alkaloid, monoterpenoid, and cyanogen heteroside, since they are the direct result of biosynthetic routes specific of these types of compounds.
HO
shikimic acid
hydroxycinnamic acids
phenylpropane derivatives
COOH COOH O OH COOH OH OH OH OH OH
H
O H H N CH3
O--D-glucose CN
quinones benzoic acids

COOH COOH O
OH
chorismic acid
HO
HO
morphine
thymol
taxiphylline
Plant phenolics arise from two main aromatization pathways: (i) the shikimate pathway, which can be summarised by the following sequence: monosaccharides aromatic aminoacids cinnamic acids plant phenolics derivatives such as benzoic acids, acetophenones, lignans, lignin, coumarins (Scheme II-1) (ii) the acetate pathway, which can be summarised by the following sequence: acetate units polyketides1 (cyclisation) chromones, isocoumarines, orcinols, depsides, xanthones, quinones (Scheme II-2).
COOH OH O
Scheme II-1:
simple phenols
COOH
isochorismic acid
o-succinyl benzoic acid
The shikimate pathway. This simplified diagram highlights the relationships between various types of phenolics originating from shikimic acid.
reduction In addition, a simple derivative, the 3-hydroxy-3-methylglutaric acid, is the starting point of the biosynthesis of the vast group of terpenoids and steroids.
1
The polyketide intermediates can evolve in different directions: cyclisation will lead to phenolics with a distinctive substitution pattern. However, the polyketides are also the precursors of the fatty acids through
Prof. Dr. JFF WEBER
UITM, Faculty of Pharmacy
jffweber@salam.uitm.edu.my
CH3-CO-SCoA
acetyl coenzyme A +
n HO2C-CH2-CO-SCoA
CH3-(CO-CH)n-1-CO-SCoA cyclisation
poly--ketothioester
mevalonyl coenzyme A Example 1

O O O O O O CO-SCoA O HO
quinones chromones isocoumarines etc...

OH
provided that the pH of the aqueous solution is lowered at a value below the pKa of the compounds, as schematised opposite: This acidity of phenolics is also responsible for their great reactivity, as further explained below.
2- Phenolic Oxidative Coupling
OH O
A phenolate ion can easily be oxidised in a phenoxy radical, which can exist under three mesomeric forms:
O
poly--ketothioester Example 2
SCoA O O O OO R
anthraquinone
OH O
O -e
HO
poly--ketothioester
Scheme II-2:
isocoumarine
The acetate pathway. The poly--ketothioester actually results from the condensation of an acetyl coenzyme A unit with a varying number of mevalonyl coenzyme A (which loses one carbon atom). This intermediate is very reactive and can undergo cyclisation according to a number of patterns. Two of them are given as examples of the diversity of this biogenetic pathway.
These radicals are highly reactive and can react reaction on each other according to a radical mechanism known as the phenolic oxidative coupling. Depending on the mesomeric forms involved, this coupling leads to an ether linkage or a biphenyl linkage. Once the coupling itself is accomplished, the quinonoid rings rearomatise. Below, two examples of such couplings are shown:
O O O H O
re-aromatisation
HO O
biphenyl ether
II-
Properties of Phenolics:
O O H O H O
re-aromatisation
1- Acidity of Phenols
HO HO
biphenyl
The hydrogen of a phenolic group is quite mobile; in alkaline medium it is easily removed to yield phenolate (or phenoxide) ion which is stabilised by resonance as shown below:
O H OH O O O O
This acidity explains why phenolics are easily extracted by organic solvent alkaline aqueous solutions. On the other hand, phenolics in aqueous acidic water solution are extractible by a nonmiscible moderately polar solvent
PHM 501: PHARMACOGNOSY Part II : Special Pharmacognosy Chapter two: Phenolics
organic solvent
PHENOLICS
These reactions can be intermolecular, thus leading to the formation of new rings, or intermolecular and resulting in oligomers or polymers. When such a reaction occurs during the biosynthesis of natural compounds, it is enzymatically controlled, leading to just the suitable compounds. However, the phenolic oxidative coupling is one of the main reactions involved in the degradation of phenolic compounds, generating complex insoluble polymers known as phlobaphenes.
alkaline water
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III- Extraction and Assays

1- Extraction of Phenolics
Phenolics do have a slight acidic character, as explained above. Therefore their extraction can be facilitated by the use of media at varying pH. This is the basis for the extraction in acidic medium (Scheme II-4).
Powdered drug H2O/OH-
Phenolics are supposed to be rather polar compounds and one can anticipate that they should be best extracted using polar solvent. In fact, a number of so-called phenolics are in fact in ether or ester forms, in which case the polarity of these compounds is considerably reduced. A general scheme for the phenolic extraction would start with a hydro-alcoholic solvent and can be referred to as extraction in a neutral medium (Scheme II-3).
Powdered drug ROH/H2O
Exhausted powder Phenoxide/carboxylate ions in aqueous solution 1) H+ 2) Organic solvent
Exhausted powder
Aqueous phase
Hydroalcoholic solution
Phenols/carboxyic acids in organic solution
Reduced pressure ROH Aqueous solution
Scheme II-4: Principle of the extraction of phenolics in a basic medium
2- Characterisation of Phenolics
Non-polar solvent Polar solvent

Esters in organic solution Free phenolics in organic soluion
Phenolics can be detected through coloured reactions with help of a few general reagents, such as ferric chloride (FeCl3), phosphomolybdate/phosphotungstate reagents, and few more These reagents are far from being absolutely specific and may react with a number of other metabolites. A standard procedure includes the use of ferric chloride on a crude hydro-alcoholic extract, which provides a colouration varying from dark blue to dark green. An other common test for phenolics is the Gibbs reaction, in which 2,6-dibromo-N-chloro-p-benzoquinoneimine is converted into a coloured indophenol derivative by a coupling reaction with the phenolics present in the extract (Scheme II-5).
Glucosides in aqueous soluion
Scheme II-3: Principle of the extraction of phenolics in a neutral medium
PHM 501: PHARMACOGNOSY
Part II : Special Pharmacognosy Chapter two: Phenolics
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Br O Br N Cl
HO R' O
Br N Br OH R'
There are some reactions which are more specific of certain groups of phenolics and can be used to characterise them in a crude drug and/or in an extract: For example, let us mention the following tests: - Bate-Smith reaction, for tannins; - Cyanidine test, for flavonoids; - Borntrger reaction, for anthracenosides. These reactions will be examined more in depth when reviewing these different categories of phenolics. HPLC, Quantitation ****
2,6-dibromoquinone chlorimide
dibromoindophenol (coloured)
Scheme II-5: Principle of the Gibbs test.
Other colour tests are based on the same principle of forming a coupling derivative between phenolics and the a diazonium salt, thus leading to a coloured azobenzene (Scheme II6).
HO R' R N N R N N R' OH
p-substituted diazonium salt
azobenzene (coloured)
Scheme II-6: Principle of coloured reactions between phenolics and diazonium salts.
TLC is usually performed on an alcoholic or hydroalcoholic extract, on silica gel with fluorescent indicator or on micro-crystalline cellulose (for anthocyanins). Visualisation procedures are not necessary for compounds as deeply coloured as anthocyanins. For other phenolics, the plate can be observed under UV light at 254 nm. Since the phenolic ring has a max usually around 280 nm (varying according to substitution and conjugation), it quenches the fluorescence of the indicator. A number of phenolics are fluorescent at 365 nm, due to conjugation associated with a more or less rigid structure. This fluorescence can be increased, depending on the circumstances, by exposing the plate to ammonia vapours, or by spraying it with a Lewis acid solutions, such as AlCl3. A number of visualisation reagents were suggested to provide coloured spots. The abovementioned ferric chloride (FeCl3), phosphomolybdate/phosphotungstate reagents can be used, although they are not very sensitive. A very CHO CHO common practice is based on the use of vanillin, anisaldehyde and other aromatic aldehydes in acidic medium which react upon OCH3 heating with the phenolics (and actually with OCH3 OH many other types of compounds) to yield vanillin anisaldehyde spots of various bright colours.
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BENZOATE DERIVATIVES [ C6-C1 ]

IV- Structures, Origin and Properties
1- Structures
V-
Salicylic Acid DerivativesContaining Drugs
Queen-of-the-Meadow (Meadowsweet): Filipendula ulmaria (L.) Maxim., (= Spirea ulmaria L.), Rosaceae
There are two slightly different official crude drugs in the Pharmacopoeias: i) the flowering tops ii) the dried flowers. Botany The queen-of-the-meadow is naturally occurring in Europe. It is a herbaceous perennial plant, with alternate leaves. The flowers are small, yellowish-white and grouped in irregular corymbs.
Phenolic acids that are hydroxylated derivatives of benzoic acid are quite common. They can be found either in a free state or as esters or glucosides. Examples of such compounds include monotropitoside (methyl salicylate glycoside). Incorporated in this group, are the aldehydes corresponding to these acids (such as vanillin) or the corresponding alcohols (e.g.: salicylin).
COOCH3 O Glc Xyl
CHO
CH2OH O
Glc
OCH3 OH
monotropitoside
vanillin
salicylin
Gallic acid and its dimers are constituents of hydrolysable tannins and will be covered in a separate chapter.
2- Properties - Characterisation
Usually these phenols are soluble in polar organic solvents. They are also soluble in alkaline solutions like sodium hydroxide, sodium carbonate, or sodium bicarbonate solutions. Thus, they can be extracted with an organic solvent from a slightly acidic aqueous medium. These compounds are unstable and quite sensitive to oxidation: it is necessary to avoid extreme pHs, light, and heat during extraction and analysis. Chromatographic analysis can be performed using various techniques: - TLC, on silica gel or cellulose. - HPLC, on reverse phase. - GC (for aglycones only, though), after silylation, in order to stabilise and increase the volatility of these compounds.
Composition of the drugs These drugs contain various types of phenolics: Flavonol glycosides Hydrolysable tannins Glycosides of phenolic acids such as: Xyloglucoside of methyl salicylate = monotropitoside Xyloglucoside of salicylaldehyde
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Steam distillation of the drugs yields an essential oil containing methyl salicylate and salicylaldehyde (which are the result of the hydrolysis of the glycosides during the distillation process).
COOCH3 OH
CHO OH
Willow, Salix alba L., Salix spp, Salicaceae
methyl salicylate
salicylaldehyde
The willows are trees that are common in damp regions all over Europe. The bark of these tress displays a high content of salicylin1, its derivatives, and related compounds. They have the same indications as the queen-of-the-meadow.
Pharmacological properties and uses So far, there are no clinical trial and only a few of animal testing. However, the presence of salicylates justifies the traditional claims of these drugs as analgesic and antipyretic1. Therefore they are used to treat rheumatism, arthritis, feverish states, headache, toothache. In addition, they are used as diuretic.
Wintergreen (= tea berry): Gaultheria procumbens L., Ericaceae
Wintergreen = North American plant. Drugs = the leaves and the essential oil obtained from the leaves. This oil is rich in methyl salicylate (the plant itself is rich in monotropitoside).
Leaves used as anti-inflammatory; analgesic; counter irritant. Wintergreen oil is used in North America in the formulation of oral hygiene and cosmetic products. It is often replaced by synthetic methyl salicylate.
The name of aspirin was formed by Dr. Friedrich Bayer (a German chemist, 1825-1880, co-founder of the giant chemical company of the same name).
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The salicylic acid and the salicylates obliviously owe their names to these plants.
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VI- Balsams and Benzoins A balsam is an oleoresin containing large amounts of benzoic acid, cinnamic acid and their esters, and other derivatives.
Peruvian Balsam, Myroxylon balsamum (L.) Harms, var. pereir (Royle) Harms (= M. pereir (Royle) Klotzsch = M. peruiferum L.), Fabaceae
Botany Myroxylon balsamum var. pereir is a tree growing wild in central America, especially in San Salvador. The drug The tree exudes the balsam after it has been successively beaten, stripped and scorched with a torch. The balsam is soaked up with rags and extracted with boiling water. The commercial balsam is the water insoluble part of the exudate which sinks (due to its density which is more than 1 kg.dm-3) and is recovered by decanting. It is a blackish brown syrupy liquid, with strong vanilla-like smell, insoluble in water, soluble in polar organic solvent, not miscible with oils. Composition of the drug This drugs contains mainly benzoate and cinnamate derivatives: 6-8% free benzoic and cinnamic acids 50-60% esters, chiefly composed of benzyl benzoate and cinnamate, as well as cinnamyl cinnamate. Alcohols: benzylic and terpenoid alcohols.
Properties and uses This balsam is an antiseptic and a vulnerary (helps the process of tissue repair). These traditional claims have been confirmed by experimentation. As it is irritating when taken orally, it is only used externally, mainly in dermatology for the treatment of minor wounds such as burns, cracks, erythema, pruritus, ulcers. It is used either as an active principle or as an excipient for topical preparations. It is also used in the acute attack of piles and other anal disorders for the symptomatic treatment of pain, pruritus, and feeling of congestion. Peruvian balsam is also used in cosmetic, hygiene products, and perfumes. A disadvantage: it can induce contact dermatitis in some people.
Tolu Balsam, Myroxylon balsamum (L.) Harms. (= M. toluiferum H., B. and K.), Fabaceae
Myroxylon balsamum is a tall tree from South America and West Indies. The composition of the balsam is close to the former one:
Free benzoic and cinnamic acids (15-25%) Benzyl benzoate Resinous fraction (mono- and sesquiterpenoids)
Tolu balsam is considered as an antiseptic and expectorant. It is an ingredient of some coughing syrups.
The Benzoins:
The term benzoin derives from the Arabic term (14th century): Luban Jawi (Java incense) which was subsequently altered over time as follows: Banjawi, Benjui, Benzui, Benzo, Benzoin, and even Benjamin.
Aldehydes: vanillin. There is also a resinous fraction, that is a terpenoid residue, more or less polymerised.
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Sumatra Benzoin = Kemenyan, Styrax benzoin Dryander and S. paralleloneurus Perkins, Styracaceae
CLASSIFICATION OF THE PHENYL PROPANOID DERIVATIVES
Both species grow wild in Malaysia and Indonesia. In addition, they are cultivated in Sumatra. The balsam obtained from these two plants contains large proportion of free benzoic and cinnamic acids (over 20 %). It is rarely used at present days by the pharmaceutical industry.
Siam Benzoin, Styrax tonkinensis Craib., Styracaceae
[ C6-C3 ++]
A large number of chemical types are biogenetically related to the phenyl propanoids. Some of the most important ones are shown below in table II-1. They are grossly arranged by increasing degree of molecular size, i.e. by the size of the adduct. Most of the phenyl propanoid derivatives do not actually derive from cinnamic acid itself, but from a mono-hydroxylated derivative, para-coumaric acid, or from dioxygenated compounds, such as caffeic acid or ferulic acid. In rare instances, precursors with less common substitution pattern can be the starting point of the biosynthesis of phenolics.
COOH HO
HO HO COOH
H3CO HO COOH
Styrax tonkinensis is a wild tree from Thailand, Laos, and Northern Vietnam. The composition of this balsam is similar to the former ones: it is mainly made of coniferyl benzoate (60-80 %) and free benzoic acid (10-20 %). The Siam benzoin is mostly used in cosmetology, perfumery and food industry. Its pharmaceutical applications are as antiseptic and vulnerary for external use.
****
p-coumaric acid
caffeic acid
ferulic acid
It should be noted however that the hydroxylation pattern of the end product may not depend on just that of the precursor, but secondary hydroxylation/dehydroxylation/ O-methylation reactions may take place during the biosynthesis. Further modifications can be brought about by prenylation (addition of an isoprenyl side chain) and O-glycosylation (frequent) or C-glycosylation (rare), thus leading to the large array of known phenolic substances.
TABLE II-1: Classification of the Phenyl Propanoid Derivatives
No
Process Esterification
COMPOUND CLASS
C6-C3 O
ROH
HO CINNAMATE
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Cyclisation
C6-C3
OH - CO2
C6-C2-C6
OH
ring hydroxylation
HO
COOH OH
lactonisation
HO O O
HO O O O OH OH OH STILBENOIDS OH HO OH
benzo--pyrone COUMARINS
G
+ 3 C2 (acetates)
Condensation
(C6-C3)2
HOOC
2
O O
OH
COOH
2'
x2
R R R R
OH
C6-C3-C6
LIGNANS
OH
HO
xn
(C6-C3)n
LIGNINS
OH O FLAVONOIDS (sensu lato)
Chain elongation
C6-C2-C5
OH OH
Miscellaneous
C6-C7-C6
+ 2 C2 O O OH
HO O O STYRYLPYRONES
HO R O O DIARYLHEPTANONOIDS
OH R'
OH O O O
C6-C1-C6
OH HO O OH
C6-Cn
O OH (CH2)n CH3
oxidation
C6-C1
+ 3 C2
J
OH O XANTHONES
R HO
ARYLALKANONES
****
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CINNAMATE DERIVATIVES [ C6-C3 ]

Few drugs may be classified under this category, even though they usually contain, in addition to caffeic acid derivatives, a number of other chemical types. Therefore, this classification is purely arbitrary. A number of cinnamate derivatives are actually part of essential oils, along with volatile terpenes. Drugs which contain them will be reviewed in the section devoted to essential oils. As a result, only minor drugs are included in this group. As examples of such a drugs, let us consider the plants mentioned below, which are used in phytotherapy both locally and overseas:
Big-flowered Java tea = misai kucing, Orthosiphon aristatus (Blume) Miquel [= O. staminus Benth. = O. spicatus (Thumb.) Bak.], Lamiaceae
Composition The leaves are rich in potassium salts, around 3% (dry weight). As most Lamiaceae, this drug yields an essential oil (0.02-0.06%), rich in sesquiterpenoid hydrocarbons. Di- and triterpenoids can also be isolated from the drug. Phenolics form the largest group of constituents: some 20 compounds have been so far identified: Flavonoids: di-, tri-, tetra- and pentamethylated compounds. Caffeic esters: they form the bulk of the polyphenolic content. They comprise notably the following compounds, together with their derivatives:
mono- and dicaffeoyltartaric acid. rosmarinic acid (resulting from the condensation of 2 caffeoyl units). lithospermic acid (resulting from the condensation of 3 caffeoyl units).
HO O O COOH COOH
HO HO OH OH HOOC O O COOH O O
HO
Botany The big-flowered Java tea originates from South East Asia (Burma, Thailand, Malaysia, Indonesia, Philippines) and is even found in Australia. It is cultivated in and exported from Indonesia. It is a perennial plant with opposite and irregularly dentate leaves. Its flowers are white or lilac and have exert stamens twice as long as the tube of the corolla (hence, the vernacular Malay and French names). The Drug The drug consists of the leaves. They are diamond-shaped, with a short and purplish petiole. The upper side is dark green, the underside is lighter and slightly pubescent.
HO
monocaffeoyltartaric acid
HO
O HO HO O O OH OH OH
dicaffeoyltartaric acid
HO CO2H O O O HO OH CO2H OH
rosmarinic acid
lithospermic acid
Properties and Uses Only few pharmacological studies were performed. In South East Asia, it is traditionally used as diuretic and for kidneys and bladder disorders. In Western countries it is used as an infusion or in capsules to enhance renal digestive elimination functions, notably the excretion of water. It is used also as an adjunct in weight loss programs.
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Echinacea (coneflower), Echinacea purpurea, E. angustifolia, E. pallida, Asteraceae
Botany Echinacea species are perennials (0.5-1m high), which belong to the sunflower family and which originate in eastern North America.
and the U.S. for prevention and treatment of upper respiratory tract infections, and Echinacea purpurea herb is believed to be the most potent echinacea species for this indication. Less commonly, echinacea has been used for wounds and skin problems, such as acne or boils. Studies indicate that echinacea does not appear to prevent colds or other infections. Studies to date have not proven that echinacea shortens the course of colds or flu. For example, two National Centre for Complementary and Alternative Medicine (NCCAM)-funded studies did not find a benefit from echinacea, either as Echinacea purpurea fresh-pressed juice for treating colds in children, or as an unrefined mixture of Echinacea angustifolia root and Echinacea purpurea root and herb in adults.
Inhibitors of the HIV-1 integrase reaction
Few caffeic acids derivatives were recently shown to inhibit in vitro the integration of the HIV-1 virus into the CD4 cells. Among them are chicoric acid, and the following compounds:
OH HO HO COOH OH OH
3,5-dicaffeoylquinic acid
The drug The aboveground parts of the plant and roots of echinacea are used fresh or dried to make teas, squeezed (expressed) juice, extracts, or preparations for external use. Composition Alkylamides and polyacetylenes Polysaccharides Caffeoyl esters, such as chicoric acid, a diester of the tartaric acid:
OH HO HOOC O O H H COOH O O OH OH
HO
O O OH
O O
(Baccharis genistelloides, Asteraceae)
H3C O
O O O COOH OH OH
OH
1-methoxyoxalyl-3,5-dicaffeoylquinic acid
(synthetic)
O O OH
O O
* * *
chicoric acid
Properties and Uses Traditionally used for a range of infections and malignancies, the roots and herb (above ground parts) of echinacea species have attracted recent scientific interest due to purported "immune stimulant" properties. Oral preparations are popular in Europe
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COUMARINS [ C6-C3 ]
IStructures and Biosynthesis
leading to the furano- and pyranocoumarins. Depending on how these additional rings are positioned (as a consequence of whether prenylation occurred in position 6 or 8), one distinguishes linear or angular furano- and pyranocoumarins.
H3CO
The basic structure of the coumarins is the benzopyran-2-one nucleus. The simplest of all is the coumarin, which is not substituted. Most of them are substituted. in 7 by an OH. In addition OH or OMe groups can substitute the positions 6 and 8, while one OH might be glycosylated.
R
R
O O
6 7 8 1 2
suberosin (1 additional prenyl unit)
aurapten (O-geranyl derivative: 2 additional prenyl units)
R OH OH OH
R H H H : umbelliferone : aesculetin : aesculin
HO O O O
H
O R" O
R'
OH OCH3 OGlc
OH
coumarin
OH OH : fraxetin
lomatin (angular pyranocoumarin)
marmesin (linear dihydrofuranocoumarin)
These simple coumarins originate from the para-coumaric acid. It is first hydroxylated in ortho to the propenoic chain, yielding the 2-hydroxy-para-coumaric acid. The action of natural light induces the isomerisation of the olefinic double bond from the E (trans) to the Z (cis) configuration. The molecule then lactonises spontaneously into a coumarin as shown in the following scheme.
COOH
2'
Furanocoumarins may loose the isopropyl group of the prenyl group after a sequence of oxidation and retroaldolization reactions.
II-
Distribution
COOH HO OH
h
HO COOH OH HO O O
The simple coumarins are widely distributed, throughout the whole plant kingdom. The Fabaceae (= Leguminoseae) and Asteraceae (= Compositae) families display a wide range of structures. Yet, the most complex structures are found in members of the Apiaceae (= Umbelliferae) and Rutaceae families.
HO
A number of coumarins undergo O- or C-prenylation1, i.e. incorporate 1, sometimes 2 or 3 prenyl units. C-prenylation happens mostly in position 6 or 8. These isoprene chains are highly reactive and can form additional rings with the 7-OH, thus
III- Physico-chemical Properties, Characterisation Free coumarins are soluble in medium and high polarity organic solvents, such as diethyl ether, chlorinated solvents or alcohols, which can be used for the extraction of these compounds. Glycosides are best extracted by water or hydroalcoholic mixtures.
The prenylation of the molecule is due to the addition of DMAPP = dimethylallyl pyrophosphate, which is the building block of the terpenoids. It is interesting to note that these compounds are build through the three main biogenetic pathways, i.e. the shikimate pathway (the aromatic ring), the acetate pathway (the pyrane ring), and the mevalonate pathway (the furan ring).
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A general property of the lactone ring is its sensitivity to basic medium, which induces its opening. A decrease of the pH leads to the ring closure. However, there is a high risk of generating artefacts. Similarly, chromatography on silica gel leads often to artefacts, which can be avoided by using various organic gels as chromatographic support (PS-DVB, Sephadex LH20, Toyo pearls,). The UV spectrum of the coumarins depends much on the substitution pattern and is deeply altered when the solution is made alkaline. Coumarins are fluorescent. On a TLC plate, UV excitation makes these compound to emit various colours, which are enhanced when the plate has been exposed to ammonia vapours. Quantitation can be performed by spectrofluorometry, or best by HPLC, with UV detection or, for greater selectivity and sensitivity, fluorometric detection.
Sweet clover = Melilotus officinalis (L.) Pallas, Fabaceae
dicoumarol
OH
OH
IV- Drugs with simple coumarins Simple coumarins comprise compounds such as the coumarin itself, umbelliferone and its O-methyl derivative herniarin, aesculetin, aesculin (= esculin), 4methylaesculetin glycoside. They seem to have venous tonic and vascular protective properties. They are found in some traditional medicines of temperate countries such as the following:
Common horsechestnut, Aesculus hippocastanum L., Hippocastanaceae
The sweet clover is an erect herb, growing in the whole temperate part of the Northern hemisphere. The O O drug is made of the flowering tops. Upon aggression, the OO sweet clover produces some phytoalexins (substances biosynthesised upon stress, meant to resist to aggression). One of them is the dicoumarol, with anticoagulant properties. From this compound, warfarin and related drugs were developed. Traditionally used as antispasmodic, carminative; expectorant, ophthalmic, emollient, vulnerary, diuretic. In homeopathy, Melilotus officinalis is prescribed against head aches going along with high blood pressure and menopause.
The drug is the bark. In addition to coumarins, it contains several saponins. This drug will be reviewed more in details together with the saponin-containing drugs (chapter three, terpenoids).
V-
Drugs with furanocoumarins
The main furanocoumarins are psoralen, bergapten (= 5-methoxypsoralen = 5-MOP), methoxsalen (= 8-methoxypsoralen = 8-MOP = ammoidin = xanthotoxin), and isopimpinellin (5,8-dimethoxypsoralen).
R
5
R H OCH3
O O
R H H : psoralen : bergapten (5-methoxypsoralen)
OCH3 : methoxsalen (8-methoxypsoralen)
R'
OCH3 OCH3 : isopimpinellin (5,8-dimethoxypsoralen)
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1- Photosensitizing properties and their consequences The furanocoumarins are photosensitizers. In addition, they were shown to be mutagenic and carcinogenic. These toxicities can possibly be explained by the fact that these compounds can easily be involved in cycloaddition with pyrimidine bases of DNA, leading to its denaturation. This has two consequences: There are accidents, on a regular basis, in the form of acute dermatitis, and, in many cases, hyperpigmentation which may last a long time, resulting from the contact with furanocoumarin-containing plants or products followed by sun exposure.1 The photosensitizing property of psoralen and its derivatives found however a medical application. This is the PUVA therapy, named after the combined use of Psoralen and UVA radiations. It is indicated for the treatment of psoriasis, vitiligo and other dermatological disorders2. Drugs containing methoxsalen are Meladinine and Oxsoralen-ultra. 2- Furanocoumarin-containing drugs Plants with furanocoumarins belong mostly to the Rutaceae, Apiaceae, and Moraceae families. A number of them are also used as spices. (i) Rutaceous plants Most plants from the Rutaceae family contain some furanocoumarins, sometimes in significant amounts.
Citrus spp.
Citrus plants usually contain bergapten as well as other linear furanocoumarins. Some furanocoumarins also inhibit a wide range of cytochromes P450. Since they are present in substantial amount in grapefruit (Citrus sp.), the metabolism of drugs might be modified by large intake of grapefruit juice.
Bergamot, Citrus bergamia, Rutaceae
Bergamot oil is the most well-known resource for bergapten. The bergamot tree is mainly found in Calabria (Italy) and Ivory Cost. Bergamot is the industrial source for furanocoumarins. In cosmetology, the oil is used to induce sun tanning.
Rue, daun ingu and/or daun aroda, Ruta graveolens, Rutaceae
Plant originating from S. Europe, N. Africa, and Western Asia, but is no longer found in the wild. It is cultivated more or less all over the world. The drug is made of the leaves. It contains an essential oil (1%), rutin (a flavanoid glycoside, 7 to 8% in the dried leaves), and linear furanocoumarins (mainly psoralen, bergapten, methoxsalen and isopimpinellin). As traditional medicine, it is said to be antiinflammatory, analgesic, and to dispel stagnant blood. It is used for colds, fever, infantile convulsion, amenorrhea, infantile eczema, raumatic injury, snake and insect bites. At high doses, it is used as an abortive. (ii) Apiaceous plants Furanocoumarins are the most widespread in the Apiaceae family. So far these molecules have been identified in more than 35 species.
Typically, such accidents happen to children playing in meadows during beautiful summer days and coming into contact with furanocoumarin-containing plants while the sun is shinning abundantly. More commonly nowadays, some adults (essentially Caucasian) are keen to speed up the tanning process while laying on the beach. Some sun-tanning lotions incorporate extracts of furanocoumarin-containing plants, such as bergamot, in order to stimulate the melanin generation. An excessive application of these products followed by lengthy sun-bathes often ends up in disastrous results. In tropical countries, with very strong insolation, such preparations should not be used. The technique is as follows: 20-40 mg of a furanocoumarin are orally administrated to the patient. Two hours later, the skin area to be treated is exposed to a controlled quantity of UVA (long wavelength UV: 320-400 nm). The complete treatment requires about 20 sessions of exposures. Sessions are brief at the beginning and increase in duration in the course of the treatment.
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Angelica, Angelica archangelica L., Apiaceae Chinese angelica, dong quai, Angelica sinensis [Oliv.] Diels, Apiaceae
LIGNANS (C6-C3)2
IStructures:
A member of the Apiaceae Umbelliferae family, angelica produces white flowers that bloom in umbrella-like clusters in June-July. A typical plant grows to a height of approximately two meters.
Lignans, strictly speaking, have a skeleton resulting from the bonding between the carbons of the side chain of two 1-phenylpropane units: = 8-8 bond . Usually one or few additional bonds lead to a variety of structures. It is customary to distinguish 9 fundamental structural groups numbered 1, 2a, 2b, 2c, 3, 4a, 4b, 5, 6. Other condensation products of phenyl propanoid units are called neolignans. The bond between the 2 units involves none or only 1 carbon: Example: 3-3, 8-O4, 8-3, 8-1. The drug is made of the dried root. It contains an essential oil as well as simple coumarins and furanocoumarins. Western herbalists recommend angelica for the treatment of blood deficiency, (anemia), abdominal cramping (especially menstruation problems such as painful menstruation, amenorrhea), constipation (due to aged and debilitated), traumatic injury, arthritis. Traditional Chinese practitionners use angelica for its overall warming effect, i.e. to strengthen heart, lung, and liver meridians, as well as lubricate the bowel. It is considered a blood tonic. It is also used by women for menstrual pain and regulating the menstrual cycle. * * *
8'
O
8
O O
O O
type 1
type 2a
type 2b
type 2c
type 3
O O
O
type 4a type 4b type 5 type 6
The nine fundamental types of lignans
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H3CO
CH2OH
8
O
-e-
H3CO O
CH2OH
3 3' 3'
4'
7
O
HO H3CO HO
Coniferyl acohol
COOH H3CO COOH
3'
-e
OCH3
O OCH3 OH
-
Sinapic acid
Examples of neolignan skeletons
H3CO H
OH CH2O H C O OH O O O O
II-
Biosynthesis
Few studies were conducted, but the mechanism is quite clear: lignans are biosynthesized through a radical addition: Oxidation of a phenyl propanoid leads to a radical which can exist as one of 4 mesomers as mentioned previously. In theory a very large number of couplings are possible. Actually, only those mentioned above (8-8, 8-1, 8-3, 8-0-4, 3-3) are common. This is probably due to the fact that it is an enzyme-driven reaction. The results of such a radical additional is a quinone methide products. Re-aromatization induces one or more ring closures.
Podophyllotoxin
H3CO O OCH3 H3CO OH OCH3
Simplified biosynthesis of podophyllotoxin (type 4b)
III- Distribution Lignans are widely distributed: several hundred compounds are known from about 70 families of Angiosperms. Wood of gymnosperms contains derivatives with a C17 skeleton = norlignans. (C18 for normal lignans:) Neolignans are restricted to the primitive groups of Angiosperms: Magnoliales and Piperales.
IV- Biological Properties Lignans being phenolic compounds, they will display usually strong antioxidant properties, and will then be recommanded as antihepatotoxic. Some lignans are reputed to have estrogenic properties. A few lignans were shown to have strong antitumour activity and drugs were developed from naturally-occuring lignans.
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V-
Some Lignan-Containing Drugs
Pharmacological Properties Podophyllotoxin and peltatins exhibit anti-tumour properties: they are mitotic spindle poisons by inhibiting the polymerization of tubulin (the cell division is blocked at the beginning of the metaphase. However, these compounds are too toxic to be used in cancer chemotherapy. Semi-synthetic compounds were developed from structure-activity relationship studies, and led to 2 close derivatives: etoposide and teniposide. Both compounds are demethylated in 4, have the carbon-4 of R configuration (S in podophyllotoxin); the 4-OH group is glucosylated, and 2 OH of the glucose (4- and 6-OH) are blocked by acetalization as ethylidene (etoposide) or as thienylidene (teniposide). Actually etoposide and teniposide act by a different mechanism: they stop the cell cycle at the end of the S phase or at the beginning of the G2 phase. The also act on DNA by inhibiting the topoisomerase II. Uses The May apple was traditionally used by Amerindians as purgative, anthelmintic, emetic. Podophyllum extracts are nowadays only used to treat warts. Yet, preparation containing such extract are contra-indicated in case of pregnancy. Example of such a preparation: Posafilin = Podophyllum extracts + salicylic acid. The resin, or podophyllin, is used only for the extraction of podophyllotoxin. However, another species is frequently used for this purpose: Podophyllum hexandrum Royle (= P. emodi Wall.) from the Himalaya, which is much richer. P. hexandrum was used by the traditional Chinese practitioners for more than 2 000 years. Podophyllotoxin is only used:

R O HO OH O O O O O
May Apple = Mandrake, Podophyllum peltatum L., Berberidaceae
Botany and Drug The may apple is a small plant, with an aerial stem of about 30 cm, which bears two (and only two) leaves and one trimerous white flower. This plant grows wild in the eastern U.S.A and Canada, in damp and shady forests. The drug is made of the rhizome. Chemical Composition The drug contains 3-6 % of resin, formerly known as podophyllin. This resin chiefly contains lignans of the type 4b (1-aryltetrahydronaphtalene): Podophyllotoxin: 20 % - and peltatins: 5 and 10 % respectively a few close derivatives.
OH O O O O O O O O OH
(R)
O
R = CH3 : Etoposide R = Thienyl : Teniposide

H3CO OH
OCH3
H3CO OH
OCH3
H3CO OR
OCH3
Podophyllotoxin
R = H : -Peltatin R = CH3 : -Peltatin
Note should be taken that these compounds have a lactone ring which is transfused to the cyclohexane moiety. The resulting strained structure is reason for the molecule instability. In even slightly alkaline medium, the carbon-2 epimerizes at (via the enolate) resulting in a cis-fused ring. This derivative is a stable but inactive compound.
for the treatment of external condylomas, in alcoholic solution, to obtain the above-mentioned semi-synthetic derivatives: etoposide and teniposide.
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Etoposide: in embryonic carcinomas of testicles, lymphomas, leukaemias, small cell lung carcinomas, etc, in combination with other anti-tumour drugs. Teniposide: Treatment of lymphomas, leukaemias, brain and bladder tumours, in single-drug or multiple-drug chemotherapy. Both compounds exhibit haematological toxicity.
Schizandra, Wu-wei-zi, Schizandra chinensis (Turcz.) Baillon, Schizandraceae.
Clinical trials were undertaken to assess the value of this plant against hepatitis B: actually, the extract could not eradicate the virus. However active principle may not be the lignans! P. niruri L. = Pokok dukung anak, amin buah, keman jolok: it displays similar antihepatotoxic properties:, and thus is used for the same indications, i.e. liver diseases.
***
The plant is a creeper from northern China. The drug is made of the fruits. The seed contains around 30 lignans with a type 5 skeleton : dibenzocyclooctane. Traditional Chinese medicine uses it as tonic, antitussive, antidepressant, antineurotic, antipsychotic. Pharmacological experiments showed that the alcoholic extract is antihepatotoxic, and that the lignans are lipid peroxidation inhibitors (this is, however, a common activity of numerous phenolics and it can be related to the physicochemical properties of the phenol groups). Schizandra chinensis The extract of this plant is used in China to treat hepatitis of various aetiologies.
Ph ll
Phyllanthus species, Euphorbiaceae
Several species are sources of folk remedies in numerous countries. P. amarus Schum and Thonn. (bhumyamlaki) is used in Southern India to treat jaundice and other liver diseases. It contains numerous phenolics of different types: diarylbutane lignans and neolignans flavonoids hydrolysable tannins one alkaloid. The lignans are antihepatotoxic. More interestingly, the aqueous extract of the drug inhibits in vitro the DNA polymerase of the hepatitis B virus.
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The following chapters are related to compounds which have a mixed biogenetic pathway as they include:

Ginger, halia, Zingiber officinale Roscoe, Zingiberaceae
a phenyl propane unit from the shikimate pathway; additional carbons that elongates the propane chain and which originate from the acetate pathway.
The drug is the rhizome. Chemical composition It contains an essential oil dominated by sesquiterpenes with notably ar-curcumene and xanthorrhizol.
R
DIARYLHEPTANOIDS AND ARYLALKANONES [ C6-C7-C6] AND [ C6-Cn ]
R=H
: ar-curcumene
R = OH : xanthorrhizol
O (CH2)n
diarylheptane arylalkanone
CH3
The pungent principles are arylalkanones called the gingerols: They are actually a group of Zingiber officinale substances different by the length of the side chain: it can comprise 7 to 10, 12, 14 or 16 carbons corresponding respectively to the [3]-, [4]-, [5]-, [6]-, [8]-, [10]-,or [12]-gingerols. Together with these compounds are the corresponding ketones (= gingerones).
O H3CO OH (CH2)n CH3
n = 1- 4 : n= 6 n= 8 n = 10 : : :
[3-6]-gingerols [8]-gingerols [10]-gingerols [12]-gingerols
These two types of compounds have little in common excepted that they are specific to several genera of the Zingiberaceae family. They are the colouring substance of turmeric (kunyit) and pungent principles of ginger (halia). No proprietary drug was ever derived from these compounds, but a number of Zingiberaceae are used in traditional medicines, especially throughout Malesia. Zingiberaceae is a large family which include about 50 genera around the world, in tropical and sub tropical areas. There are 20 genera in Malaysia, with 150 species. We shall limit one study to two genera: Zingiber and Curcuma.
HO
In addition, the dried drug contains also the dehydration products, the shogaols. Actually, there are a great number of varieties, races and cultivars of ginger. In a number of cases, their botanical status is still uncertain and their chemical differences have to be really assessed.1
1 Example: Halia bara, or red ginger, seems to be a distinct variety, showing at least some differences in its essential oil composition. Halia padi, a short ginger, which seems to have lost its blooming capability, is used for traditional postpartum treatment of women mainly in Indonesia, Johore and Melaka; it may not be even a variety, probably just a race, with apparently no significant difference in composition with the standard ginger.
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Pharmacological Activities and Uses: Ginger has been used since remote times in India and China. Pharmacological experiments showed a number of activities. Some of the gingerols were shown to be cholagogue1 and hepatoprotective. Crude extracts have antiulcer effects and stimulate the gastrointestinal mobility. As a result, the rhizome powder is an effective antinausea preparation. It is used in China against motion sickness. In traditional Malay medicine, ginger is used internally as stomachic. A short variety, halia padi, is used in postpartum treatment (see note 1 previous page)
Bonglai, Zingiber cassummunar, Zingiberaceae
Pharmacological Properties and Uses: The crude extracts was shown to be hepatoprotective. Curcumin is antiinflammatory. Turmeric is traditionally used as choleretic and cholagogue. The food industry uses curcumin as a permitted non toxic colouring agent.
Temu lawak, C. xanthorrhiza Roxb., Zingiberaceae
Temu lawak is a species close to turmeric, with a close chemical composition, including hydrogenated curcuminoids. It has the same traditional usage. * * *
This ginger is also used in postpartum treatment.

Turmeric, kunyit, Curcuma domestica Val. (= C. longa L.), Zingiberaceae
Turmeric is an other spice, widely used in traditional medicines. The rhizome makes the drug, although the leaves are sometimes also used in traditional medicine. Composition: Turmeric is, like ginger, rich in an essential oil with monocyclic sesquiterpenes. The colouring principles are the curcuminoids (diarylheptanoid derivatives). The chief component is curcumin alongside with desmethoxy, bisdesmethoxy and dihydrocurcumin.
O H3CO HO O OCH3 OH
curcumin
Curcuma domestica
1 Compound which induces an increased release of bile from the gall-bladder. This term should not be confused with choleretic which characterises a compound which increases the secretion of bile by the liver.
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OH HO O HO R OH OH O OH O O OH
FLAVONOIDS [ C6-C3-C6 ]
Flavanones (dihydroflavones)
Flavanonols (dihydroflavonols)
R = H : dihydrokaempferol R = OH : taxifolin (dihydroquercetin)
I-
Generalities
3' 2' 8 7 1 O 1' 2 3 5 4 4'
R = H : naringenin R = OH : eriodictyol
1- Introduction
2-phenylchromanones
Flavonoids, taken in the broad sense, form a very large group of substances: around 3 000 of them are known. The basic structure is a C6-C3-C6 which is cyclised (apart from some exceptions) in a 2-phenylbenzopyran = 2-phenylchromane.
B
5' 6'
A
6
Anthocyanidins and anthocyanins1 have a positively charged 2-phenylbenzopyrilium skeleton = flavylium. (Anthocyanidins= aglycones, anthocyanins = heterosides).
OH HO O
Anthocyanidins
R OH
R = H : pelargonidin R = OH : cyanidin
2- Structural Classification
Basically, their differ by the degree of oxidation of the pyran ring: The most oxidised = flavonoids in the strict sense. Basic ring = 2-phenylchromone = 2-phenyl benzopyrone. Depending on the substitution in position 3, two groups are distinguished: flavones and flavonols. They exists under both aglycone and heteroside forms.
OH HO O HO O OH
OH
2-phenylbenzopyryliums = flavyliums
With a totally reduced pyran ring: flavan-3-ols and flavan-3,4-diols.

OH HO O HO O OH
R OH
R OH
R OH
OH
OH
OH
Flavan-3-ols
R = H : afzelechin R = OH : catechin
Flavan-3,4-diols
R = H : leucopelargonidin R = OH : leucocyanidin
OH
OH
Flavones R = H : apigenin R = OH : luteolin
Flavonols R = H : kaempferol R = OH : quercetin
2-phenylchromanes
2-phenylchromones
The reduction the 2,3 double bond leads to the corresponding groups of flavanones and flavanonols (= dihydroflavonols). Here also, they can be found both as aglycones and heterosides.
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Flavan-3-ols can polymerise and yield the proanthocyanidins or condensed tannins (see below). Chalcones and dihydrochalcones are bicyclic derivatives, with the pyran ring opened.
OH HO
Furthermore, flavonoids are good UV filters, thus protect the plant from the damaging effect of UV radiation. This explains why flavonoids are universally present in leaf cuticle and epidermal cells. Tannins, are part of the defence mechanisms used by plants against fungi and bacteria.
4- Biosynthesis
Chalcones
R
R = H : isoliquiritigenin R = OH : butein
There are a few more groups which are related to the flavonoids, the isoflavonoids and the neoflavonoids, where aryl migration took place during the biosynthesis. They are respectively 3- and 4-arylbenzopyrans.
O
The triacetate origin of the ring A is shown by the phloroglucinol substitution pattern, which is very common among flavonoids. This triacetate moiety actually originates from the consecutive condensation of 3 units of malonylcoenzyme A with a substituted cinnamyl CoA. The first compound made is a chalcone, which is easily converted into a flavanone (actually this reaction can be spontaneous, depending pH). A series of oxidation, reduction, substitution by OH groups, and other modifications, leads to the various classes of flavonoids.
Isoflavonoids
Neoflavonoids
All the above structures can be glycosylated and/or, less frequently, prenylated.
3- Distribution - Location - Biological role
With the exception of algae, flavonoids are virtually universal plant pigments. The glycosidic forms are water soluble and accumulate in vacuoles. They concentrate in epidermal cells, notably in the case of flowers (pigments of the petals), or spread in both epidermis and mesophyll. Flavonoids are present in the cuticle, always as free aglycones, with partial or total methylation of their hydroxyl groups, in order to increase the lipophilicity. Flavonoids appear to play different roles: Their basic role is to provide pigmentation: flavonols and chalcones are yellow,, anthocyanins are red, orange, blue or purple. They are thus responsible for the colour of the flowers and sometimes of other parts of the plant.
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OH CoAS O O R O O OH O O OH
46
5- Physicochemical Properties
HO CoAS
malonyl CoA
R = H : coumaroyl CoA R = OH : caffeoyl CoA

OH
OH HO OH
HO
a i gr lm ary
n tio
OH O
de R hy dr og en
FLAVANONES
ati on
OH
(i) Solubility and Extraction Glycosides are usually soluble in water and in alcohols. Yet, some of them are only sparingly soluble (hesperidin, rutin). The solubility of the aglycones depends greatly on their degree of methylation. Flavonoids are usually extracted by hydro-alcoholic or a water-acetone mixture, most often at high temperature. The organic solvent is evaporated under reduced pressure and leaves an aqueous solution. Lipophilic flavonoids of the cuticle are directly extracted by solvents of medium polarity (CH2Cl2), but must be separated from waxes and fats extracted simultaneously.
Powdered drug
CHALCONES
ISOFLAVONES
FLAVONES
OH OH HO O
HO
R OH
R OH
H2O / ROH or H2O / acetone Heating Exhausted powder Reduced pressure
dehydrogenation
OH O
OH
FLAVANONOLS
FLAVONOLS
ROH or acetone
OH HO O HO R OH OH OH OH OH O OH
Aqueous solution
Crystallisation
FLAVAN-3,4-DIOLS
FLAVAN-3-OLS
Petroleum ether
Flavonoid permethyl ethers + chlorophylls and lipids
Crystallised flavonoids
OH HO O
HO OH OH OH OH O OH OH HO
OH
R OH
Diethyl ether
Free aglycones
O HO OH OH O OH O OH R OH
OH OH
HO R
OH O OH O
Ethyl acetate
Less polar glycosides
HO
R HO R HO R
OH HO
OH
ANTHOCYANIDINS
PROANTHOCYANIDINS
Most polar glycosides
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(ii)
Characterisation
The "cyanidin reaction" is a coloured reaction specific to the flavonoids stricto sensu: a flavonoid solution is added with conc. HCl and magnesium turnings. The so-called nascent hydrogen induces a partial reduction of the flavonoid into the corresponding flavylium ion, which is deeply coloured in red, orange or purple, depending on the substitution pattern of the original flavonoid.
Mg + 2 HCl
OH HO O
MgCl2 + H2
OH
Flavonoids, due to their phenolic groups, are antioxidant and free radicals scavengers. Free radicals are produced during anoxia and inflammation. The main species produced are the hydroxyl radical (OH) and the superoxide radical anion (O2-). The presence of radicals cause a reaction cascade, where lipids auto-oxidise. Under normal circumstances, these reactions are interrupted by enzymatic systems (superoxide dismutase, catalase, glutathion peroxidase). Flavonoids and many other phenols like vitamin E (= tocopherols) react with free radicals to prevent the degradation of the phospholipids of the cell membrane. Flavonoids are enzyme inhibitors. In vitro experiments showed that a number of flavonoids inhibit a variety of enzymes. Some oxygenases are inhibited: this could be linked to the free-radical scavenging property. It could possibly explain the anti-inflammatory and anti-allergic properties attributed to some drugs known to contain flavonoids. Flavonoids also inhibit some enzymes involved in the vascular tonicity. Actually, the main property attributed to flavonoids is a so-called "vitamin P" activity: i.e. as venous tonic, decreasing the capillary permeability and fragility. Is there any direct relationship with the above-described properties? It's far from being clear. i- In vitro experiments usually are not comparable with in vivo conditions. For example, glycosides are used in most tests. Yet, it is most likely that glycosides are hydrolysed in the gastrointestinal tract before any absorption takes place. ii- Flavonoids are good heavy metal complexants. As a result, enzymes with a heavy metal as co-factor will be unspecifically inhibited. iii- The concept of vitamin P activity is not universally recognised: The FDA denies any activity to flavonoids. On the other hand, some European countries use them widely. In fact, the clinical efficiency of most flavonoids has still to be proven. Only a small number of molecules or titrated extracts have proved superior to placebo according to clinical trials of current standard.
OH OR
H2 nascent
HO
OH OH
OH
OH
(Quercetin & its glycosides cyanidin, red) TLC is carried out on a classical manner, on silica gel, with the mobile phase made of mixtures such as: - formic acid, water, methylethylketone and ethyl acetate (10:10:30:50 v/v) - formic acid, glacial acetic acid, water and ethyl acetate (7.5:7.5:18:67 v/v) Visualisation is obtained by various means: - Sometimes visualisation is unnecessary, as the compounds are already coloured. - Exposure of the plate to ammonia vapours induces a colour change of the flavonoid spots. - Spraying with AlCl3, anisaldehyde, diazoted sulphanilic acid, etc (general phenol reagents). (iii) Quantitation Quantitation methods include colorimetric and spectrophotometric techniques, as well as HPLC with UV detection. Protometry in non aqueous medium is also proposed occasionally. (iv) Biological Properties
There is a very large number of biological activities that where documented, most of the time, from in vitro experiments.
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II-
Chief Flavonoids (Stricto Sensu) on the Market
Buckwheat, Fagopyrum Polygonaceae
esculentum
Poench
and
F.
tataricum
(L)
Gaertn.,
1- Rutin (= Rutoside)
(i)
Structure
HO O
OH
Rutin is the quercetin-3-rutinoside1 or quercetin3-rhamnoglucoside. (ii) Sources
OH O-Glc-Rha
This is a pseudo-cereal originating from China and cultivated in Europe for its edible starchcontaining akenes. Rutin can be extracted from the leaves which contain 2-3 % (up to 8 % in improved varieties). Extraction is here a little more complicated, as there is the need to eliminate the leaf pigments and some photosensitising agents (fagopyrins).
OH
rutin Both quercetin and rutin are very common flavonoids, probably the most common ones. In spite of this wide distribution, it is most of the time in concentration not high enough and/or in mixture with other flavonoid derivatives, making its purification difficult. There are only three sources for industrial extraction:
Fagopyrum esculentum
Red stringybark = Eucalyptus macrorrhyncha F. Muell., Myrtaceae
Japanese pagoda tree = Scholar tree = Sophora japonica L., Fabaceae.
This is a tall tree from Central and Northern China and cultivated in temperate countries as ornamental species. The drug is made of the flower buds, containing 15-20 % of rutin and only traces of two others flavonoids. It is probably the best source for rutin.
Australian species whose leaves contain up to 10 % rutin.

2- Uses and Derivatives
Rutin is indicated for venous insufficiency, varicoses, haemorrhoids. To increase the solubility of rutin, some derivatives were developed. The most common one is troxerutin = 3,4,7-tris-(hydroxyethyl)-rutin = O-(hydroxyethyl)-rutoside.1 Below are some examples of some proprietary drugs which include rutin or related derivative: - Rutin used in association with aescin: Essaven. - Rutin used in association with Ginkgo extracts: Ginkor.
O O O OH OH
HO
O O Glc Rha
OH
troxerutin
- Rutin or its derivative used alone: Varemoid, Venoruton.
Rutinose = disaccharide: -L-Rha-(16)--D-glucose

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3- Citroflavonoids
III- Flavonoid - Containing Drugs

St Mary Thistle, Blessed Milk Thistle, Silybum marianum (L) Gaerth, Asteraceae
The citroflavonoids are flavonoids obtained from the fruits of various Citrus. species. (i) Sources A number of Citrus species, varieties and hybrids are cultivated for their fruits (oranges, grape-fruits, lemons, mandarins,) whose endocarp is edible. The pericap ("the skin") is very rich in flavonoids (and also in essential oil). (ii) Chemical composition
OCH3 Rha-Glc-O O OH
The main components are listed below: Flavanone glycosides, such as: Hesperidin (hesperitin 7-O-rutinoside) Neohesperidin Naringin Eriodyctin Eriocitrin Flavone glycosides Diosmetin (diosmin-7-O-rutinoside) Rutin (= rutoside) They are extracted as calcium and magnesium salts.
Botany, the Drug: It is a strong herb, with thorny leaves, purple flowers in capitules; it originates from around the Mediterranean sea and Western Asia. It is also cultivated in China. The crude drug consist of the fruits which are black akenes.
OH
hesperidin = hesperitin 7-O-rutinoside

OCH3 Rha-Glc-O O OH
Silybum marianum Composition The active ingredient is silymarin: it is actually a mixture of flavano-lignans (condensation products of a flavanonol and a phenylpropanoid alcohol). The main compound is silybin. Other related components include silychristin and silydiamin.
O CH2OH OCH3 OH
OH O HO O CH2 OH O OH OCH3
OH
HO
diosmetin
OH OH O
(iii) Uses Citroflavonoids are used often in mixtures, sometimes as pure products or as semi-synthetic derivatives. They are used as sole ingredients, or in association with vitamin C, methylaesculetin, ruscosides (saponins), vincamine (alkaloid), papaverine (alkaloid) The indications for the citroflavonoids are exactly the same as those for rutin : venous insufficiency and haemorrhoids. In Malaysia, one proprietary drug with citroflavonoids is registered, Daflon 500 (diosmin 50 mg + hesperidin 450 mg).
OH
silybin
H3CO HO O HO O O OH OH O OH
silychristin
silydiamin
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Pharmacological Activities - Uses This drug was shown to be antihepatoxic. Therefore, it is proposed for chronic liver diseases. It often associated with other products. For example: - Hepatofolk Planta: association with curcuma extracts. - Simepar: association with vitamins. IOther drugs
ANTHOCYANINS [ C6-C3-C6 ]
Structure and Physico-chemical Properties
Many other plants contain flavonoids. They are used in traditional medicines or in phytotherapy. But they contain also other secondary metabolite and it is difficult to asses the role played by flavonoids, if any.
These compounds occur in natural sources always as glycosides and always bear a 3-OH group. The aglycones (also called anthocyanidins) do not include many different structures. The six most common ones are shown below:
R1 OH HO O R2 OH OH
R1 = R2 = H : pelargonidin R1 = OH ; R2 = H : cyanidin R1 = OCH3 ; R2 = H : peonidin R1 = R2 = OH : delphinidin R1 = OCH3 ; R2 = OH : petunidin R1 = R2 = OCH3 : malvidin
The 3-OH group is always linked to a sugar; the most common anthocyanins are 3-monosides or 3,5-diglycosides. Other glycosylation patterns are known, but there is always at least one free OH, in order to allow the formation of the coloured quinoid structures (see below). Many anthocyanins are acylated by phenylpropanoic acids which esterify one or several OH groups on the sugar moieties. In turn, these ester groups can be glycosylated, and so on. As a result, anthocyanins structures can become very complex, as it can be seen from the example shown below:
OH HO O O OH OH HO O HO O OH HO O OH O O O COOH OH O HO O O O OH OH OH OH O HO O O O OH OH O HO O O
O OH OH OH
OH O
Ternatin A2
Anthocyanins are conventionally drawn as cations: This representation is correct when the pH value is 4 or lower: in this state, the cationic structure of the
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anthocyanins is stable and these compounds appear orange, or red or purple. However, if pH is raised between 4 and 6; a proton is lost and the anthocyanins take a quinoid structure: they turn blue in colour. Raising again the pH leads to uncoloured compounds, the leucoderivatives, and ultimately to decomposition products1 :
4<pH<6 : Blue
O HO O O OH
II-
Pharmacological Activity and Uses
Just like the flavonoids, anthocyanins are credited of so-called vitamin P activities. As a consequence, they are similarly used for the treatment of the venous insufficiency and capillary fragility. Actually the main industrial use of anthocyanins is as permitted non-toxic colouring agents (syrups, candies, etc.).
III- Chief anthocyanins - containing drugs

OH OR OH
pH<4 : Red
OR OH HO O OH
OH
Bilberry: Vaccinium myrtillus L., Ericaceae, (fruit)
OH OR HO O OH
OH
pH >7-8 : Leucoderivatives
OH OR
OH
Phenoxides
OH OH HO O O OH OR OH OH HO OH O OH OR OH
Black currant: Ribes nigrum L., Grossulariaceae (fruit)

If R = H
pH >8 : alcoholates
Decomposition Products
Anthocyanins are soluble in water and alcohols. They are generally extracted by an alcohol with a small amounts of hydrochloric acid (0.1-1 %) or better of acetic acid or tartaric acid (to avoid hydrolysis of the sugar moieties.). TLC analysis is best performed on microcrystalline cellulose, with a mobile phase made of water, methanol and some mineral or organic acid. Purification can be performed on ion exchange resins.
However, the vacuolar pH does not vary to such extent. Anthocyanins are stabilised by the phenomenon of co-pigmentation: this is the association of groups (such as cinnamoyl groups which acylate the sugars) and/or other molecules, (such as flavonoids (stricto sensu) to produce large sandwich-like complexes. Part II : Special Pharmacognosy Chapter two: Phenolics
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Red vine: Vitis vinifera L., var. tinctoria, Ampelidaceae (leaves)
TANNINS
I-
Introduction
Tannins are polyphenols but, according to their chemical structure, they can be classified into two groups: Hydrolysable tannins: galloyl esters of glucose and derivatives. The leaves contain vitamin C , tannins and anthocyanins.
Roselle, rosel, Hibiscus sabdariffa L., Malvaceae
Condensed tannins (=Proanthocyanidins): polymerised flavan-3-ols (see below).

-
The origin is not fully known but it is believed to be originated from West Africa or from India. Roselle is an annual. The plant is small in size like schrubs and can grow to the height of 2 m. The drug is the calyx.
They are studied together as: They are often found together, in various proportions, in the same plant. They share a common property: they are tanning agents: they induce the transformation of animal skin (highly degradable material) into leather (very stable, long lasting material).1
II-
Structure
1- Condensed tannins = Proanthocyanidins [ (C6-C3-C6)n ]
As already mentioned, proanthocyanidins are oligomeric flavan-3-ols. The most common monomeric units are shown below: In Malaysia most of the roselle is being planted commercially by the smallholders in Terengganu. The planting has later expanded to some parts of Johor in the Peninsula Malaysia. The calyx from the Roselle flowers is processed to produce juice for drinking. It is very high in vitamin C. It can also be prosessed to make jam and jelly. Locally, this plant is said to lower blood pressure. This claim is currently under study. * * *
The tanning properties of some plants were known since the prehistory. Just recently, in the turn of the century, the tanning of skins by chromium salts almost completely replaced the vegetable tannins. They are still used for some special leathers.
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R1 OH HO
8 6 4
60
R1 OH HO
8 6 4
HO O
4
OH
OH OH O OH
OH OH HO
OH
O (R)
2 3
R2
O (R)
2 3
R2
HO HO
8
O OH
(S) OH
OH
(R) OH
OH
R1 = R2 = H : afzelechin R1 = OH ; R2 = H : catechin R1 = R2 = OH : gallocatechin
R1 = R2 = H : epiafzelechin R1 = OH ; R2 = H : epicatechin R1 = R2 = OH : epigallocatechin

HO
OH O
OH
OH HO HO O
OH
OH HO
8
OH OH
OH OH OH O OH
The basic structure includes a bond between C-4 and C-8 or C-6. As the C-8 is more reactive than the C-6, the linkage C-4C-8 is most favoured one.
OH HO O 4 HO HO 8 OH OH
OH OH HO
8 4 6
OH
O
O
OH O
OH
HO
OH
HO
O HO
OH
OH HO
OH O OH
HO OH
HO HO HO OH
OH HO OH
O
OH
O OH
OH OH OH HO OH
Example of procyanidin made of five flavan-3-ols units linked 48 or 46 (stereochemistry not shown).
Example of procyanidin with an additional interflavanic ether linkage
Example of 48 dimer
Example of 46 dimer
Some derivatives include an additional bond between the C-2 and the 7-OH group, thus forming an ether linkage. The polymer may be made of a single type of monomers or a mixture of several different flavan-3-ols.
The question of the chain length, or how long can this chain be? Actually nobody really knows for sure. The solubility of the proanthocyanidins decreases as their degree of polymerisation increases. Above 6 or 8 units, the polymer is hardly soluble; its precise study is almost impossible. Very long chains (up to 50 units) are thought to be stored in some cells. Proanthocyanidin crude extract: A condensed tannin extract is always a complex mixture of monomeric flavan-3-ols (not tannins), dimers, trimers,up to hexa- or octomers, respectively in decreasing amount. This is further complicated by the fact that there are 3 asymmetric centres on each unit and that a few OH groups may be esterified by galloyl groups. The number of combinations is therefore very high.
2- Hydrolysable Tannins
Hydrolysable tannins are esters of glucose and a variable number of phenolic acid molecules: - If the phenolic acid is gallic acid, these derivatives are termed gallotannins.
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HO OH HO HO O
HOOC COOH OH HO HO OH OH
- If the phenolic acid is hexahydroxydiphenic acid (=HHDP), these derivatives are termed ellagitannins The most common pentagalloylglucose.1 representative
OH HO OH
of
the
gallotannins
is
the
HO
HO O
O O O O HO O O O OR
HO
HO O HO HO O O O O O O O O O O
OH
HO
OH OH
OH OH HO OH
hexahydroxydiphenic acid
ellagitannins
HO OH
OH HO OH
OH
pentagalloylglucose
Further metabolism include oxidation of the HHDP or the opening of the HHDP ring, or even of the glucose ring. These already complex molecules can undergo intermolecular coupling to give large oligomeric ellagitannins of molecules weight between 2000 and 5000.
3- Complex Tannins
This molecule can be further modified by plants in one of two directions: Evolution towards heavier molecule by formation of side chains consisting of several gallic acid bonded by meta-despsidic2 linkage. Up to 11 moieties of gallic acid can be attached to the glucose. Formation of ellagitannins: Two adjacent galloyl moieties can undergo an oxidative coupling, leading to the formation of HHDP (hexahydroxydiphenoyl) esters. This happens most frequently between pairs of galloyl units attached in positions 2 and 3 and in position 4 and 6 of glucose moiety. The remaining OH groups of the glucose remain free or are esterified by a galloyl group.
Complex tannins result of the condensation of ellagitannins with a flavonoid moiety; either a flavanol, a proanthocyanidin or a flavone unit.
III- Distribution Proanthocyanidins are found in all groups of plants, including gymnosperms and ferns. Gallotannins and ellagitannins (simple or complex) are characteristic of Dicots.
IV- Physico-chemical Properties, Extraction, Characterisation, Quantification

1- Solubility - extraction
1 2
Galloyl mono- or diesters of glucose are not tannins, as they are too small to carry the tannins properties Depside = ester of an acid phenol by another molecule of the same acid phenol
Tannins dissolve in water to form colloidal solutions. Their solubility decreases with increasing of degree of polymerisation. They are also soluble in alcohols, acetone and ethyl acetate.
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70:30.
The best extraction medium from a crude drug is a mixture water/acetone

Powdered drug H2O / acetone
Tannins are heavy metals scavengers: they precipitate in the presence of heavy metals such as lead, tin, copper. Pb(NO3)2 is often used to eliminate from crude extracts unwanted tannins which would interfere with a characterisation or a quantification reaction.1 (ii) Hydrolysis
the Bate-Smith reaction (specific to proanthocyanidins):

Exhausted powder Reduced pressure
The Bate-Smith reaction allows detection of proanthocyanidins in a crude extract in just a couple of minutes. It is carried out as shown below:
anthocyanidins (red Proanthocyanidins + HCl/Butanol + O2 (air) colour)
acetone Aqueous solution
Ethyl acetate
The mechanism is as follows: In acidic conditions, the interflavanic bond between the two upper units is hydrolysed. The uppermost unit is released as a carbocation. In presence of air, this carbocation oxidises into a colourful anthocyanidin.
OH OH HO OH OH HO HO O OH HO OH OH OH R OH OH R HO O OH O HO O OH HO O OH OH OH
Aqueous solution Ethyl acetate
Reduced pressure
OH OH
Tannin-rich extract
Tannins are not stable in solution, are sensitive to heat and light. It is necessary to perform the extraction at room temperature and as fast as possible.
2- Characterisation
OH
The term proanthocyanidin derives from the fact that this type of tannins can generate anthocyanidins upon hydrolysis.
(i)
Precipitation Reactions
Tannins precipitate proteins and other macromolecules.1 This property is used to characterise these compounds by using a colloidal solution of gelatine which precipitates in presence of tannins of any kind.
bacterial or fungal enzymes to access the collagen terminal amines, where the degradation of any protein would start. This would then account for the durability of leather. Protein precipitation also explains the characteristic astringency of these compounds: tannins combine with glycoproteins of the saliva, which thus looses its lubricating power.
1
This is the basic principle of the tanning process. Skin is mainly made of collagen, arranged in fibers. More precisely, collagen fibers consist of bunches of fibrils, made themselves of several protein molecules. At the end of these fibrils, there is a small gap before the start of the next one. A recent hypothesis suggest that tannins would fill that gap, and bind to the proteins by both covalent and non-covalent bonds, making it impossible for
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For example, this precipitation is performed on Digitalis crude extracts prior to the characterization of the cardiac glycosides.
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O O HO HO O O OH OH
In hot acidic conditions, hydrolysable tannins release readily sugar and either gallic acid, HHDP acid, or both. HHDP very quickly lactonises into ellagic acid. The name of ellagitannins given to this group of substances actually derives from this property to yield ellagic acid upon hydrolysis.
(iii) Coloured reactions Proanthocyanidins and hydrolysable tannins are polyphenols; thus, they react with FeCl3 and yield a dark green or dark blue precipitate. Gallotannins give a pink colour with potassium iodate KIO3. Ellagitannins give a coloured reaction with nitrous acid (HNO2) in presence of acetic acid: From pink, the colour turns to purple and then blue. (iv) Quantitation There is no fully satisfying method to quantify the tannins: Proanthocyanidins can be measured by the Bate Smith reaction, but the colour is not proportional to the quantity of proanthocyanidins and the reaction is difficult to standardise. Spectrophotometry can be performed using phosphomolybdic or phosphotungstic acids which lead to a blue or green colour. This reaction is not specific to tannins, but quantifies all polyphenols. Skin powder is then used to selectively precipitate the tannins. A new spectrophotometric measurement gives a value for the non-tannins compounds. The quantity of tannins is obtained by subtraction. Many other methods are available, each with advantages and draw backs.
macromolecules, in vitro inhibition of enzymes should not be of a great surprise and is of little significance. Tannins also have the general properties of phenols: free radical scavengers and inhibitors of superoxide anions. Although not yet proved, this may be in part the explanation of the "French paradox".1 Just like flavonoids, proanthocyanidins are said to possess Vitamin P properties (decrease in capillary fragility and permeability, increase in vascular tone).
VI- Chief Tannin-Containing Drugs

Gallnut = Oak-gall, Quercus infectoria Olivier, Fagaceace
The tree, Quercus infectoria, originates from Middle East: Turkey, Syria, Iran. The drug is the gall which forms as consequence of the development of the larvae of an insect on the leaves of this oak. In reaction to the growth of the larva, there is a proliferation of the host tissues resulting in a round, hard and dense mass, the gall. Galls from few other trees can also be used. Galls contain hydrolysable tannins in high proportions: 50 70 % in the case of Q. infectoria. It is can be used externally as an astringent, for burns and dermatitis and as haemostatic agent.
Witch hazel, hamamelis, Hamamelis virginiana L., Hamamelidaceae
V-
Biological Properties and Uses
The witch hazel is a small tree from the north-east of the American continent. The drug consists of the leaves. It contains a small amount of an essential oil and high content of tannins both of proanthocyanidins and hydrolysable type. It is also rich in polysaccharides, consisting mainly of arabans and arabinogalactans. It is traditionally used internally or externally to treat the symptoms of the venous insufficiency and of haemorrhoids.2 Hamamelis is a frequent component of various cosmetic preparations.
Most of the biological properties of the tannins are linked to their affinity for proteins: They have clearly demonstrated antiseptics effects, both against bacteriae and fungi. Internally, tannins-containing-drugs are used as antidiarrheal, externally for dermatitis. There is a great number of scientific papers reporting the inhibitory effects in vitro of tannins on such or such enzymatic systems. As they tend to bind to any
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The French paradox In cosmetology, the drug is used to produce the Hamamelis Water or Distilled Witch Hazel Extract, presented as astringent, even though it doesnt contains any tannins.
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Pale catechu, gambier, Uncaria gambier (Hunter) Roxb., Rubiaceae
Uncaria spp. are creepers, easy to recognise as they bear special hooks opposite to the leaves. This plant is locally used as astringent and antidiarrheal.
Hawthorn, Crataegus spp., Rosaceae
ISOFLAVONOIDS [ C6-C3-C6 ]
Isoflavonoids originate from the transposition of the aryl group from C-2 to C3 of a benzopyrone nucleus.
O
2 3
The hawthorn is a thorny shrub, common in almost all the temperate areas of the northern hemisphere. It has a complex composition: triterpernoid acids, aromatic amines, trace of essential oil, 1-2 % flavonoids, 2-3 % proanthocyanidins. This crude drug is active on the myocardium: it seems to have positive inotropic and negative chronotropic properties, as well as hypotensive property. The improvement of mild cardiac insufficiency is improved upon long treatment. These activities may result of a synergy involving several components of the drug. It is traditionally used to treat abnormalities the cardiac rhythm in the adult. In Malaysia, one proprietary preparation based on hawthorn extract is marketed under the name of Crataegutt Forte.
Green tea, Camelia sinensis L. Kuntze, Theaceae
Isoflavonoids are mostly encountered in members of the Leguminosae family. By virtue of prenylation and ring closures, numerous structural variations are made possible.
Akar tuba, Derris elliptica, D. malaccensis, Fabaceae
Epigallocatechine gallate, anticancer properties
They are vines from South East Asia. They contain rotenone, with piscidal (toxic to fishes) and insecticidal properties. The roots are traditionally used to catch fishes in rivers. Rotenones of various Derris spp. are used in phytopharmacy (for the treatment of garden plants), and in veterinary for the extermination of ectoparasites of pets.
Soya, Glycine max, Fabaceae
HO O
H O H
H OCH3 OCH3
rotenone
Genistein
OH O OH
Estrogenic / antiestrogen properties
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QUINONES
Sundew, Drosera spp, Droseraceae
These plants are small carnivorous plants: The leaf blade is covered with long red trichomes with globulous apexes, which secrete a viscous, highly refractive liquid (hence its name). Small insects are trapped on these trichomes and digested.
I-
Introduction
O
O O
ortho-quinone
para-quinone
Quinones originate mainly from the polyketide pathway.1 This group comprises notably the benzoquinones, the naphtoquinones and the anthraquinones. Some quinones incorporate up to 8 rings, arranged linearly or not. Distribution: Quinones are found in all kind of plants, although they are rare in ferns. They were also isolated from some marine animals. D. capensis D. rotundifolia D. rotundifolia The drug consists of the whole plant. It contains mainly plumbagin (= 2-methylnaphtoquinone). The sundew tincture is antispasmodic and is used in syrups against spasmodic coughs.
Henna, pokok inai, Lawsonia inermis L., Lythraceae
II-
Naphtoquinones:
O
Henna is a shrub from Middle East cultivated in almost all the Muslim world, including Malaysia. It may also grow wild in abandoned areas. The drug consists of the leaves. Other parts of the plant may also be used. The main component is lawsone. Actually it is under a glycosidic form in the fresh leave but is released by hydrolysis upon drying. This quinone dissolves in a alkaline solutions to give intense orange colour.
O OH
Naphtoquinones are yellow or orange pigments characteristic of some Angiosperm families: Ebenaceae, Droseraceae, Bignoniaceae. Although their pharmaceutical interest is limited, let us mention just two plants:
lawsone
Actually there are few natural quinones which are biosynthesised via another pathway. This can be recognised from a substitution pattern different from what one may expect for a polyketide derivative. However none of them are of real pharmaceutical interest.
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Henna is mainly used (since the neolithic time) as a colouring and cosmetic ingredient for hair, nail, and skin. However, it is also a powerful fungicide and is
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O O OH
traditionally used for scurf and superficial wounds. Noteworthy, the decoction of roots is used as abortive.
[O] anthranol
III- Anthraquinones
O
8 5 9 10 1 4
anthrone dimerisation
O
anthraquinone
This is a rather small group of compounds, with quite limited structural variations. The botanical distribution is very narrow: Anthraquinones are found only in four families: Liliaceae (aloe), Polygonaceae (rhubarbs), Rhamnaceae (buckthorn and cascara), and Caesalpinaceae (sennas). Yet, all these compounds are laxative and have wide market.
1- Structures
dianthrone
Anthraquinones naturally occur both as free aglycones and as glycosides. Anthraquinone aglycones all bear hydroxy groups in 1 and 8, and a methyl, a hydroxymethyl or a carboxyl group in 3 . The other variation is the possibility of a hydroxy or methoxy group on the C-6. The main structures are shown below:
OH O OH
Anthrones are unstable; they occur always as glycosides. Free aglycones are necessarily anthraquinones. The sugar moieties of the glycosides are of common types: mainly glucose and/or rhamnose. bond). There are O- and C-glycosides (sugar linked to the aglycone by a carbon-carbon
R1 = CH3 ; R2 = H : chrysophanol R1 = CH3 ; R2 = OH : emodin R1 = CH2OH ; R2 = H : aloe-emodin R = COOH ; R = H : rhein

1 2
R2 O
R1
2- Physico-chemical properties and characterisation
Beside the anthraquinonic form, there is a partly reduced form, called anthrone, and its tautomeric form, anthranol. Anthrone and anthranol are said to be reduced forms. Anthraquinones are called "oxidised forms". In certain circumstances, such as drying, anthrones may combine as dianthrones and yield dimeric species. Both moieties may or may not have the same substitution pattern. They are respectively known as homo- or heterodianthrones.
Solubility: Anthraquinones are yellow compounds, sparingly soluble in cold water, soluble in organic solvents and alcohols. Glycosides are soluble in water and hydroalcoholic solutions. Hydrolysis: O-glycosides can be easily hydrolysed by refluxing in acidic medium. C-glycosides are hydrolysed only by an oxidant such as FeCl3, HNO3, etcThe same treatment applied to a dianthrone hydrolyses the 10-10 bond and oxidises both carbons, thus yielding two anthraquinones. Characterisation: "Free" anthraquinones (as aglycones) can be characterized by the Borntrger reaction: This reaction consists simply in exposing these compounds to an alkaline medium, usually alcoholic sodium or potassium hydroxide. A red colour is then observed. Actually, all natural anthraquinones are hydroxyanthraquinones. When dissolved in an alkaline medium phenol ate ions are formed which further
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expands the bond conjugation, thus producing a bathochromic shift in light absorption and hence giving the characteristic red colour. It must be noted that the Borntrger reaction works only anthraquinones. If the extract contains glycosides and/or derivatives with degree of oxidation (anthranol glycosides, anthrones, anthrones dianthrones, dianthrones glycosides,), the reaction will not work. These have to be converted into "free" anthraquinones by hydrolysis and simultaneously or successively. on "free" a different glycosides, derivatives oxidation,
Identification: The identification test is usually performed by TLC. Visualization of the chromatogram is based on the Borntrger reaction (by means of ammonia vapours), directly or after oxidation into anthraquinones of the dianthrones or the C-heterosides. This can be performed right on the plate in a few minutes, with nitric acid (a strong oxidising agent) at high temperature (oven or hot air blower). Quantitation: Quantitative analysis is performed by a spectrophotometric method, based on a variant of the Borntrger reaction which uses magnesium acetate in methanol. This gives a red colour more intense and stable than the one obtained with NaOH or KOH. There are a few preliminary steps: Extraction of the drug, performed by hydroalcoholic solution or water. Removal of free anthraquinones1 by an apolar organic solvent (diethyl ether) from the acidified aqueous medium. Oxidation of the anthrones (FeCl3, reflux) into anthraquinones. Hydrolysis of O-glycosides: (HCl, reflux) into free anthraquinones. Extraction by an apolar organic solvent, evaporation of the solvent. Dissolution of the residue in a methanolic solution of magnesium acetate (pink solution). Absorbance measured at 515 nm.
3- Pharmacological properties
Free anthraquinones are practically inactive. O-glycosides of monomeric anthrones are too active. They must be oxidised into anthraquinones by prolonged storage or heating of the drug. The active ingredients are water-soluble and not resorbed nor hydrolysed in the small intestine. They reach the colon unmodified. There, the intestinal flora hydrolyses the glycosides (-glucosidases) and reduces the anthraquinones. The really active form is the free anthrones. The glycosides can in fact be looked upon as pro-drugs. Anthrones increase the peristaltism, decrease the water, sodium and chloride resorption, increase the potassium secretion in the intestine by inhibiting the Na-K ATPase activity of the enterocytes.
4- Uses
Crude drugs are used for laxative herbal teas or preparations, such as powders, extracts. Anthraquinones-containing drugs should be used only occasionally. A prolonged used can induce dependence and sometimes colon damages. Thus the use of such drugs should follow a few rules: Anthraquinones-containing drugs should not be used for more than 8 to 10 days in a row. They are contra-indicated to children under 12 (unless medical prescription,) to breast-feeding women (excepted senna), as anthraquinones may pass into maternal milk. They should not be associated with any drug causing hypokalemia. Counselling is important: it should stress on dietary guidelines and on life style (exercise) which may prevent chronic constipation.
5- Main anthraquinone glycosides-containing drugs Sennas: Tinnevelly senna, Cassia angustifolia Vahl, Fabaceae Alexandria senna, Cassia senna L. (= C. acutifolia Del.), Fabaceae
The active principles are the O-glycosides of dianthrones and anthraquinones, as well as C-glycosides of anthrones. They are laxative.
Plant and drug

The sennas are low shrubs from desert areas, with composite leaves. The fruit is a flattened, dehiscent pod, containing 6 to 8 seeds.
Free anthraquinones have no pharmacological activities and should be removed or they would be incorporated with the active ingredients.
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C. alata = gelanggang besar, guajava, C. occidentalis = kacang kota, ketepeng hutan.
Cassia senna
Cassia angustifolia
C. obtusifolia
C. alata
C. occidentalis
Both species yield two drugs each, the fruits and the leaflets. Composition Both species have a very similar composition: The chief components of the dried crude drugs are the sennidins A, B, C, and D: They differ by their substituent in position 3 (COOH or CH2OH) and by the stereochemistry at C-10 and C-10' (R,R or R,S).
-D-Glc-O O OH
Buckthorn, Rhamnus frangula L., Rhamnaceae
This shrub grows in central Europe. The crude drug is the bark. It must be stored at least for one year to allow the anthranols to oxidise. Then the drug contains anthraquinones monosides (frangulin) and diglycosides (franguloside).
Cascara, Rahmnus purshianus D.C., Rhamnaceae
The cascara is a tree from West America. The crude drug is the bark. This plant is characterised by the presence of C-glycosides, the cascarosides.
R = COOH ; 10-R, 10-R : sennidin A
R COOH
10
H
10'
R = COOH ; 10-R, 10-S : sennidin B R = CH2OH ; 10-R, 10-R : sennidin C R = CH2OH ; 10-R, 10-S : sennidin D
Aloes Cape aloe, Aloe ferox Miller, Liliaceae Curacao aloe, A. vera (L) Blum, Liliaceae
-D-Glc-O
OH
A. ferox grows in South Africa while A. vera originates from North Africa. They bear thorny fleshly leaves arrange in rosette.
Senna extract is the active ingredients of Sennokot and other preparations. Local Cassia species C. obtusifolia = gelanggang kecil (Korean medicine: purging fire in the liver to improve the acuity of vision and moistening intestines to loosen the bowels)
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Rhubarb, Rheum palmatum L., R. officinale Baillon, Polygonaceae
Rhubarbs are large herbaceous plants from Eastern Asia.
These plants provide two different drugs: the aloe juice and the aloe gel. The juice, stored in the pericyclic cells, is obtained by incision of the leaves. It contains mainly C-glycosides monoglycosides (aloine) and diglycosides (aloinosides). The gel comes from the central mucilage cells. It is made of pectins, hemicellulose and common components, such as steroids, amino acids, enzymes. It is used in cosmetology as hydrating agent.
The drug consists of the subterraneous organs (rhizome with or without roots). The dried drug contains anthraquinone O-glucosides, anthranols or anthrone di-O,C-glucosides and sennosides.
* ** *
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Prof. Dr. JFF WEBER

Phenolics

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2

Definition Biosynthetic outlines

COOH COOH O OH COOH OH OH OH OH OH

quinones benzoic acids

o-succinyl benzoic acid

Prof. Dr. JFF WEBER

UITM, Faculty of Pharmacy

mevalonyl coenzyme A Example 1

quinones chromones isocoumarines etc...

Prof. Dr. JFF WEBER

UITM, Faculty of Pharmacy

III- Extraction and Assays

Exhausted powder Phenoxide/carboxylate ions in aqueous solution 1) H+ 2) Organic solvent

Phenols/carboxyic acids in organic solution

Reduced pressure ROH Aqueous solution

Scheme II-4: Principle of the extraction of phenolics in a basic medium

Non-polar solvent Polar solvent

Glucosides in aqueous soluion

Scheme II-3: Principle of the extraction of phenolics in a neutral medium

PHM 501: PHARMACOGNOSY

Part II : Special Pharmacognosy Chapter two: Phenolics

Prof. Dr. JFF WEBER

UITM, Faculty of Pharmacy

Scheme II-5: Principle of the Gibbs test.

p-substituted diazonium salt

PHM 501: PHARMACOGNOSY

Part II : Special Pharmacognosy Chapter two: Phenolics

Prof. Dr. JFF WEBER

UITM, Faculty of Pharmacy

BENZOATE DERIVATIVES [ C6-C1 ]

Salicylic Acid DerivativesContaining Drugs

PHM 501: PHARMACOGNOSY

Part II : Special Pharmacognosy Chapter two: Phenolics

Prof. Dr. JFF WEBER

UITM, Faculty of Pharmacy

Willow, Salix alba L., Salix spp, Salicaceae

PHM 501: PHARMACOGNOSY

Prof. Dr. JFF WEBER

Prof. Dr. JFF WEBER

UITM, Faculty of Pharmacy

CLASSIFICATION OF THE PHENYL PROPANOID DERIVATIVES

PHM 501: PHARMACOGNOSY

Part II : Special Pharmacognosy Chapter two: Phenolics

Prof. Dr. JFF WEBER

UITM, Faculty of Pharmacy

PHM 501: PHARMACOGNOSY

Part II : Special Pharmacognosy Chapter two: Phenolics

Prof. Dr. JFF WEBER

UITM, Faculty of Pharmacy

CINNAMATE DERIVATIVES [ C6-C3 ]

PHM 501: PHARMACOGNOSY

Part II : Special Pharmacognosy Chapter two: Phenolics

Echinacea (coneflower), Echinacea purpurea, E. angustifolia, E. pallida, Asteraceae

(Baccharis genistelloides, Asteraceae)

Prof. Dr. JFF WEBER

UITM, Faculty of Pharmacy

suberosin (1 additional prenyl unit)

aurapten (O-geranyl derivative: 2 additional prenyl units)

R H H H : umbelliferone : aesculetin : aesculin

lomatin (angular pyranocoumarin)

marmesin (linear dihydrofuranocoumarin)

PHM 501: PHARMACOGNOSY

Prof. Dr. JFF WEBER