PHENYLPROPANOIOS .1 Chemistry and distribution

Y R R=H, resorcinol

2.3 PHENYLPROPANOIOS .1 Chemistry and distribution

Phenylpropanoids are naturally occurring phenolic compounds which have an aromatic ring to which a three-carbon side-ehain is attached.

They are derived biosynthetically from the aromatic protein amino acid phenylalanine and they may contain one or more C₆

-e

3 residues. The most widespread are the 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. Among the phenylpro- panoids are included hydroxycoumarins, phenylpropenes and lignans.

Structures of some typical plant phenylpropanoids are illustrated in Fig. 2.4.

Four hydroxycinnamic acids are common, in fact almost ubiquitous, in plants: ferulic, sinapic, caffeic and p-coumaric acids. Their separation is simple and they are readily detected on paper chromatograms because they fluoresce (different shades of blue and green), in UV light. At least six other cinnamic acids are known, but these are relatively rare in their

Hydroxycinnamic acids

HO~

R }CH=CH-Co,H

R=H, p-coumaric acid R=OH, caffeic acid

HO~O

R~

R=H, umbelliferone R=OH, aesculetin R=OMe, scopoletin

HO~CH=CH-co,H

MeO

R

R=H, ferulic acid R=OMe, sinapic acid

~~O ~

Psora len

Bound Forms

H~~O

^OH

HO

h

^C02H

H0-«J-_~

^CH=CH-CO-O

Chlorogenic acid

GIC0f'(~O

Meo~

Scopolin Lignans and Phenylpropenes

MeO

HO

--<:J'F- h

^H-CH-CH2

/ I \

~ 0

CHrCH-c'H-«

t

^OH

OMe Pinoresinol

MeO

HO_~

h

^{CH -CH=CH}2 2

Eugenol

q

}CH,-CH=CH,

MeO

Myristicin

H~

MeO ^}CH=CH-CH, Isoeugenol Fig. 2.4 Structures of phenylpropanoids.

.~.

Phenylpropanoids

51

occurrence; examples are isoferulic (3-hydroxy-4-methoxycinnamic), 0-

coumaric and p-methoxycinnamic acids. Hydroxycinnamic acids usually occur in plants in combined form as esters; and they are obtained in best yield by mild alkaline hydrolysis, since with hot acid hydrolysis material is lost due to the fact that they undergo decarboxylation to the corre- sponding hydroxystyrenes. Caffeic acid occurs regularly as the quinic acid ester, chlorogenic acid, but isomers are known (e.g. isochlorogenic acid) and derivatives with sugars (e.g. caffeoylglucose) and with organic acids (e.g. rosmarinic acid, caffeoyltartaric acid) have also been described.

The different combined forms of caffeic acid are of particular interest from the chemotaxonomic point of view (Harborne, 1966; Molgaard and Ravn, 1988).

The most widespread plant coumarin is the parent compound, coumarin itself, which occurs in over twenty-seven plant families. Itis common in many grasses and fodder crops and is familiar as the sweet- smelling volatile material released from new mown hay. Hydroxy- coumarins are also found in many different plant families; the common ones are based on umbelliferone (7-hydroxy), aesculetin (6,7-dihydroxy) or scopoletin (6-methoxy-7-hydroxycoumarin). Rarer hydroxycoumarins are daphnetin (7,8-dihydroxy) from Daphne and fraxetin (6-methoxy- 7,8-dihydroxy) from the ash tree, Fraxinus. More complex coumarins occur in plants, for example, the furanocoumarins typified by psoralen (see Fig. 2.4), but these are generally restricted to a few families, such as the Rutaceae and the Umbelliferae (Murray et al., 1982). Lignans, dimeric C₆

-e

3 compounds such as pinoresinol, which is illustrated in Fig. 2.4, are mainly found in heartwoods. Some two hundred structures have been reported. Their identification is only briefly touched upon here.

One other group of phenylpropanoids must be mentioned - the phenylpropenes - because of their important contribution to the volatile flavours and odours of plants. The phenylpropenes are usually isolated in the 'essential oil' fraction of plant tissues, together with the volatile terpenes. They are lipid soluble, as distinct from most other phenolic compounds. Some structures are widespread, such as eugenol, the major principle of oil of cloves. Others are restricted to a few families.

Anethole occurs in anise and fennel (both Umbelliferae) and mYfisticin, first described as a principle of nutmeg,Myristica fragrans, Myfisticaceae, is also found in a number of umbellifers. Pairs of allyl and propenyl isomers (e.g. eugenol and isoeugenol, see Fig. 2.4) are known, sometimes OCcurring together in the same plant. Isomerization of the allyl to the propenyl form can be achieved in the laboratory, but only under drastic conditions (e.g. with strong alkali). Such isomerization is very unlikely to Occur during normal conditions of isolation (i.e. extraction with ether, etc.)

52 Phenolic compounds

2.3.2 Recommended techniques

(a) Hydroxycinnamic acids and hydroxycoumarins

These are usually detected together, as with the simple 'phenolics', after acid or alkaline hydrolysis of a plant extract. They can be extracted into ether or ethyl acetate and the extract is then washed, dried and taken to dryness. Chromatography of the residue either one-dimensionally on paper or two-dimensionally on plates of microcrystalline cellulose is equally satisfactory for detection. A typical TLC separation of cinnamic acids is illustrated in Fig. 2.5. Paper chromatographic data are given in Table 2.3 for the cinnamic acids and Table 2.4 for the coumarins.

po

t

c:

CD

"5>

l/)

7

Solvent 2 -

Fig. 2.5 Thin layer chromatography separation on microcrystalline cellulose of cinnamic acids. Solvent 1=benzene-acetic acid-water (6: 7: 3). Solvent2=15%

acetic acid in water. Key: I, caffeic acid; 2, gallic acid; 3, cinnamic acid; 4, p- coumaric acid; 5, o-coumaric acid; 6, sinapic acid; 7, ferulic acid. Compounds 1, 4, 5, 6 and 7 can be detected as different shades of blue under UV light and fuming with ammonia. Compounds 2 and 3 are dark absorbing spots in UV light. Com- pound 2 gives a blue colour with Folin reagent on fuming with ammonia.

Table 2.3 RFcolour and spectral data for hydroxycinnamic acids

R_{F (}Xl00) in* Colour

EtOH EtOH-NaOH

Cinnamic acid BAW BN BEW Water UV UV +ammonia Amax Am•1X

p-Coumaric 92 16 88 42,85 none mauve 227,310 335

Caffeic 79 04 79 26,69 ^blue light blue 243,326 decomposition

Ferulic 88 12 82 33,75 blue bright blue 235,324 344

Sinapic 84 04 88 62 ^blue blue-green 239,325 350

o-Coumaric 93 21 85 82 yellow yellow-green 227,275,325 390

p-Methoxycinnamic 95 17 87 23 dark absorbing 274,310 298

Isoferulic 89 12 67 37 mauve yellow 295,323 345

3,4,5-Trimethoxycinnamic 95 18 87 75 mauve dark 232,303 293

ItKey: BAW= n-BuOH-HOAc-H20 (4: 1:5, top layer); BN= n-BuOH-2 MNH40H(1 :1, top layer); BEW= n-BuOH-ethanol-water (4: 1:2.2).

..._~.

54 Phenolic compounds

These compounds are very easily detected, since they give characteris- tic fluorescent colours in UV light, which are intensified by further treat- ment with ammonia vapour. An advantage of PC is that these colour changes are more easily observed than on TLC plates. Cinnamic acids can be differentiated from coumarins by their less intense fluorescence and by the fact that they invariably give two spots when chromatographed in aqueous solvents, due to the separation of the cis- and trans-isomers.

Isomerization of cinnamic acids (and their derivatives) occurs during isolation on exposure of extracts to light (and particularly to UV light) so that even if the natural material in the plant is all trans, an equilibrium mixture ofcis-and trans- is normally obtained.

Identification can be confirmed by spectral measurements (Tables 2.3 and 2.4). For example, caffeic acid and its derivatives have characteristic absorption bands at 243 and 326nm, with a distinctive shoulder at 300nm to the long wave band (Fig. 2.6). Hydroxycoumarins absorb at longer wavelengths than cinnamic acids; aesculetin, the coumarin related to caffeic acid, has absorption bands at 230, 260, 303 and 351 nm. Measure- ments of the magnitudes of bathochromic shifts in spectra in the presence of alkali are also useful for distinguishing the different cinnamic acids and coumarins (see Fig. 2.6).

Separation of bound forms of cinnamic acids can be achieved using a

Table 2.4 Colour and spectral data of hydroxycoumarins R_F(xl00)in

10% Fluorescence in

Coumarin RAW Water HOAc UV light EtOH Amax

Aglycones

Coumarin" 92 67 76 none 212, 274, 282,+ 312

Umbelliferone 89 57 60 bright blue 210, 240,+ 325

Aesculetin 79 28 45 blue 230, 260, 303, 351

Scopoletin 83 29 51 blue-violet 229,253,300,346

Daphnetin 81 61 54 pale yellow 215, 263, 328

Glycosides BAW Water BN

Aesculin 53 56 13 clear blue 224, 252, 293, 338

Cichoriin 53 61 10 pale pink 228,255,290,345

Scopolin 53 64 44 mauve 227, 250, 288, 339

For key to solvents, see Table 2.3.

.. Detected by spraying paper with 5% aqueous NaOH; intense yellow-green fluorescence develops when dried paper is placed under UV light for 5-10min.

t Inflection.

Phenylpropanoids 55

0.8

/,.''\0

c

_I^{I \}_\

I \

0.6 ^{I \}

\ I

\ / V

II) //\\

0 / \

c:<II / \

.t:J₀ 0.4

'

^\

'

^\

CIl / \

.t:J

"

^\

< _{I / \}

I -/ \

\

,

" / ^\\

0.2

'

...

,---_ ..

_\ ^{.... \}_\\

\ \

\

\

\ ,

,_....'

250 300 350 400 450

Wavelength (nm)

Fig. 2.6 Ultraviolet spectra of two phenylpropanoids. Key: A, Caffeoylquinic acid (chlorogenic acid) in 95% EtOH; B, aesculetin in 95% EtOH; C, caffeoylquinic acid in EtOH-NaOH;0, aesculetin in EtOH-NaOH.

Table 2.5 Separation of caffeic acid derivatives

R_F(X100) in

Caffeic derivative Structure BAW BEW Water TLC"

Chlorogenic acid caffeoylquinic acid 63 44 67,84 16 Isochlorogenic acid dicaffeoylquinic acids 79 67 10 30 Rosmarinic acid ester of caffeic and

3,4-dihydroxy-

phenyllactic acid 83 62 27 65

l-Caffeoylglucose 50 63 61,72 14

Solvent key: BAW= n-BuOH-HOAc-H20 (4: 1:5); BEW= n-BuOH-EtOH-HP (4: 1 :2.2).

ColoursinUV light, bright blue; UV+NH₃₁green.

°TLC on silica gelintoluene-ethyl formate-formic acid (2: 1: 1).

combination of PC and TLC on silica gel. Such separations are illustrated here by reference to the bound forms of caffeic acid (Table 2.5). Separation of the glycosides of hydroxycoumarins can be carried out using similar procedures as for the free coumarins (Table 2.4).

..i'·:·...·..

II

56

Phenolic compounds (b) Furanocoumarins

By contrast with the simple hydroxycoumarins, furanocoumarins are gen- erally lipid-soluble and can be isolated during extraction of dried plant material with ether or light petroleum. They occasionally occur in bound form as glycosides and have then to be released by prior acid hydrolysis.

TLC on silica gel is most commonly used for their separation. Suitable solvents include pure chlorofonn, chlorofonn containing 1.5% ethanol, ether-benzene (1: 1), and ether-benzene-lO% acetic acid (1: 1: 1); times of development vary between 1 and 2h. Typical separations (Rrs relative to bergapten) that can be obtained for five common furanocoumarins in ether-benzene (1:1) and chlorofonn, respectively, are as follows;

bergapten (100, 100), isobergapten (112, 167), pimpinellin (108, 86), isopimpinellin (97, 43) and sphondin (92, 90).

Furanocoumarins are detected by their blue, violet, brown, green or yellow colours in UV light. The colour may be intensified by spraying plates with 10% KOH in methanol, or 20% antimony chloride in chloro- form. Furanocoumarins can be further identified by their UV absorption;

unlike hydroxycoumarins, they do not, as a rule, exhibit bathochromic spectral shifts in alkaline solution. Other techniques can be applied in furanocoumarin separation. For example, Reyes and Gonzalez (1970), in isolating twelve coumarins from the roots of Ruta pin nata,used PC in water and GLC on QF-I at 174°C for distinguishing the different com- pounds. Procedures for surveying plant material for furanocoumarins are given by Crowden et al. (1969). HPLC of furanocoumarins has been achieved on normal phase silica gel columns, using hexane-ethyl acetate (4: 1) (Thompson and Brown, 1984).

(c) Phenylpropenes

These compounds are detected, together with essential oils, in ether extracts of plant tissues. Particularly rich sources are fruits of the Umbelliferae and other plants used as spices or for flavouring. They are easily separated on silica gel plates in benzene, mixtures of benzene with chloroform (10%) or light petroleum (20%) or in n-hexane-chloroform (3: 2). They give coloured spots when sprayed with vanillin-IMH2S04(2 g vanillin, 1g H2SO4 diluted to 100mI with 96% ethanol) or with Gibbs reagent (Forrest et al., 1972). Typical Rrs and colours for most of the common phenylpropenes are given in Table 2.6. It will be noted that pairs of isomeric compounds (e.g. anethole and estragole, eugenol and isoeugenol, etc.) have similarRr values but the shift in the position of the double bond in the side-chain considerably alters the colour reactions. For example, with vanillin-I^M H₂SO₄^,the compounds which have the double bond adjacent to the benzene ring give reddish colours, whereas the

Phenylpropanoids

Table 2.6 RFvalues and colours of phenylpropenes

57

,.

^'

^!~.

^',;

RF(X100) in Colour with

Phenylpropene* Benzene n-Hexane-eHCl_J(3 : 2) vanillin-H₂SO₄ Gibbs reagent

Safrole 74 86 grey

Estragole 72 pink

Anethole 69 rose

Myristicin 50 71 brown brown

Apiole 39 41 brown brown

Thymol 38 rose

Eugenol 20 31 brown brown

Isoeugenol 29 27 red yellow

Methyleugenol 42 red-brown

Methylisoeugenol 42 purple

Elemicin 27 yellow

• These compounds can be further identified by the spectral measurements; myristicin, for example, hasA",••278 and 285, whereas apiole has a single broad band at 280 nm.

isomers in which the double bond is farthest from the benzene ring (see Fig. 2.4) give brownish colours. Procedures for surveying plant tissues for myristicin and other phenylpropanoids in fruits of the Umbelliferae are described by Harbomeet al. (1969).

(d) Lignans

Simple mixtures of these substances may be separated by PC in butanol- acetic acid-water and 15% acetic acid. More complex mixtures can be separated using butanol-acetic acid-aqueous molybdic acid on chroma- tography paper previously impregnated with dilute molybdic acid (Pridham, 1959). Complex mixtures may also be resolved by TLC on silica gel using such solvents as ethyl acetate-methanol (19:1) or benzene- ethanol (9:1).

Unfortunately, there does not seem to be a specific spray reagent, which distinguishes lignans from other simple phenols. Lignans can be seen as dark absorbing spots on paper in short UV light or can be revealed by spraying with 10% antimony chloride in chloroform. On TLC plates, they are detected by spraying with cone. H₂SO_{4 ,} They can be further identified by spectral means; they show absorption at 280- 284 nm, this band being shifted to about 298 nm in the presence of alkali.

A survey procedure for detecting the lignan glycoside arctiin in fruits of certain members of the family Compositae is described by Hansel et al.

(1964).

58 Phenolic compounds (e) Authentic markers

Most of the common phenylpropanoids are available commercially;

e.g. the hydroxycinnamic acids, coumarin, umbelliferone, aesculin and chlorogenic acid. Others can be obtained from readily available plant materials, e.g. myristicin from nutmeg, apiole from parsley seed.

2.3.3 Alternative techniques

GLC is occasionally used for detecting phenylpropanoids, because of its greater sensitivity and value in quantitative analyses. The detection of the coumarin scopolin by GLC using electron capture has been described by Andersen and Vaughn (1972) and the same authors (Vaughn and Andersen, 1971) have used this technique for measuring cinnamic acid, formed as a product of the enzymic action of phenylalanine ammonia lyase on phenylalanine. The method is ten times more sensitive than spectral measurements. Phenylpropenes can be separated by GLC on columns of 20% Reoplex 400 on Gas Chrom Q at 80-200°C/4°Cmin (Wagner and Holzl, 1968).

HPLC, with its high resolving power, is useful when complex mixtures of phenolics are encountered. Retention times of many phenylpropanoids on a Lichrosorb RP-18 column eluted with varying proportions of water- formic acid (19: 1) and methanol have been recorded by van der Casteele et al. (1983). The application of HPLC to the determination of the many phenolics present in maize extracts is outlined by Andersen and Pedersen (1983). HPLC data for representative phenylpropanoids can be found in Harborne (1989).

2.3.4 Practical experiment

(a) Detectionof scopolin in blight-infected potato tuber

One of the responses of plant tissue to infection by micro-organisms is a large increase in the SYnthesis of particular phenolic compounds. It is presumably a protective response to invasion, although it does not neces- sarily prevent the organism establishing itself in the tissue. As an example of this, the coumarin scopolin is formed in high concentrations in infected plants of the Solanaceae and is particularly easily observed in blighted potato tubers (Hughes and Swain, 1960).

Scopolin synthesis can be observed in partly blighted potatoes collected in the autumn, soon after harvest. On cutting such tubers in half, an intense blue UV fluorescent zone can be seen in the healthy tissue adjacent to the infected zone. Scopolin SYnthesis can be induced artificially by

Phenylpropanoids

59