Biosynthesis of Some Phenolic Acids and Lactones

BIOSYNTHESIS OF SOME PHENOLIC ACIDS AND LACTONES

IN HIGHER PLANTS USING CARBON-14

Ragai K. Ibrahim, M. Sc.

A Thesis Submitted to the Faculty of Graduate Studies and Research in Partial Fulfilment of the Requirements for the degree of Doctor of Philosophy.

Department of Botany, McGill University, Montreal. April , 19 61 . ACKNOWLEDGMENTS

I wish to express my sincere thanks and indebtedness to Dr. G. H. N.

Towers, for his continua! help and valuable criticisme in directing this work. Thanks are also tendered to Professer M.V. Roscoe, Cbairman of the Department of Botany, for making the facilities of the department available for this study; and to Dr. W. Boll, for useful criticism.

The writer is grateful to Dr. P.I. Abell, of the Chemistry Department,

University of Rhode Island, for the determination of the infra-red spectra of hydrangenol and a new coumarin derivative ; and to Dr. B.A. Bohm,

N.R.C. Post Doctoral Fellow in this laboratory, for the interpretation of the infra-red spectrum of the latter compound and the preparation of its acetyl derivative. 14 The generous gifts of cinnamic acid-ring & -3-C , from Dr. E. Conn,

University of California ; of an enoculum of Escherichia coli 83-24, from

Dr . B. Davis, Harvard Medical School,Mass.;of scopoletin and iso-ferulic acid, from Dr. V. Runeckles, Imperial Tobacco Company, Montreal ; of a sample of scopoletin, from Dr. W.A. Andreae, Canada Department of Agriculture; and of the gydrangea flowers, from Mr. A. Bisaillon, Montreal Botanical

Gardens, are also acknowledged.

I wish also to thank the World University Service of Canada for a scholarship in 1958-1959 ; the authorities in McGill University for the awa~d of a Graduate Scholarship in 1959-1960 ; and the National Research Council of

Canada for grants to Dr . Towers , for financial support of this work. TABLE OF CONTENTS Page

INTRODUCTION 1 REVIEW OF LITERATURE ON PHENOLIC ACIDS AND PHENOLIC LACTONES 3 I. Naturally occurring phenolic acids and phenolic lactones in higher plants. 3 II. Origin of benzene rings in phenolic compounds. 12 1. The shikimic acid pathway. 12 2. The acetate pathway. 15 III. Sorne biosynthetic pathways of pbenolic compounds. 17 REVIEW OF METHODS OF SEPARATION AND IDENTIFICATION OF PHENOLIC ACIDS AND PHENOLIC LACTONES 20 MATERIALS AND METHODS 27 1. Plant material. 27 2. Chemicals. 27 3· Carbon-14 compounds. 28 4. Preparation of radioactive shikimic acid. 28 5. Preparation of plant extracts. 29 6. Chromatography. 31 7. Spray reagents. 33 8. Elemental analysis. 34 9. Spectropbotometry. 34 10. Techniques for administering carbon-14. 34 11. Radioautography and radioactivity determination. 35 EXPERIMENTAL AND RESULTS 38 A. PHENOLIC ACIDS 38 I. Chromatographie separation and identification of pbenolic acids. 38 II. The phenolic acid patterns of sorne common plants. 44 III. Identification of the phenolic acids of Hydrangea macrophylla. 56 Compound No. 8. 59 IV. Isolation and identification of three dihydroxybenzoic acids from Gaultheria procumb.ens and Primula acaulis. 65 1. ~-Pyrocatechuic acid. 65 2. Gentisic acid. 66 3. 2-Hydroxy-5-methoxybenzoic acid. 66 v. Biosynthesis of C6-Cl acids from c14-labelled compounds in higher plants. 67 1. Formation from benzoic acid. 67 2. Formation from salicylic acid. 70 3. Administration of ~-pyrocatechuic acid-c14 and gentisic acid-c14 to Gaultheria leaf disks. 70 4. Formation from phenylpropanoid compounds. 73 B. PBENOLIC LACTONES 77 I. Identification of the phenolic lactones of Hydrangea. 77 1. Isolation and identification of hydrangenol glucoside. 77 2. Isolation and identification of hydrangenol. 78 TABLE OF CONTENTS (Continued) Page

3. Isolation and identification of umbelliferone 85 4. Isolation and identification of compound No. 15. 89 Alkaline hydrolysis of compound No. 15. 96 II. Biosynthesis of phenolic lactones from cl4_labelled compounds in Hydrangea. lOO 1. Biosynthesis of hydrangenol. 101 2. Degradation of hydrangenol and identification of the degradation products. 105 3. Degradation of hydrangenol-c14. 109 4. Biosynthesis of ether phenolic constituants of Hydrangea from c14-labelled compounds. 112 5. Administration of umbelliferone-c14 to §ydrangea leaf disks. 117 6. Administration of L-phenylalanine-u-cl4 to aydrangea leaf disks. 117 DISCUSSION 121 I. Phenolic acids, their identification by chromatography and their distribution in plants. 121 II. The biosynthesis of sorne hydroxybenzoic acids in higher plants. 127 III. The phenolic constituants of Hydrangea. 137 IV. Biosynthesia of the phenolic constituents of Hydrangea. 140 SUMMARY 150 CLAIM TO ORIGINALrrY OR CONTRIBUTION TO KNOWLEDGE 153 REFERENCES 154 INTRODUCTION

One characteristic feature of higher plants is their ability to synthesize phenolic compounds in great variety and quantity. Lignin, which constitutes 20-30 ~ of the dry weight of plants, is formed of phenolic polymers.

Phenolic compounds appear to be metabolically inert substances and are considered to be stable and characteristic end products in living plant tissues. No general function can be ascribed to these compounds and no explanation bas been given to their extraordinary diversity. This is possibly one of the reasons why little attention has been given to them by plant physiologiste and biochemists in the past.

With the advent of paper chromatographie techniques, it was shown that many simple phenolic compounds are widely distributed in plants.

The work of Bate-Smith (1954 a, 1956 a, b) and Williams (1955, 1956,

1957) provided a great deal of information on the distribution of certain phenolic acide (cinnamic acid derivatives). However, c6-c3 a large number of phenolic compounds which can be detected on paper chromatograms of plant extracts have not been identified.

As chromatographie methode for the separation of c -c and c -c 6 1 6 3 phenolic acids bad not been fully explored, it was a prominent part in the planning of the present work to develop a suitable method for the

1 2

separation and identification, by two-directional paper cbromatography, of the plant phenolic acide. The application of this method bas been found useful in the distribution of phenolic acide in higher plants.

The use of carbon-14 labelled compounds and the discovery of the pathvays of synthesis of the aromatic amino acide, phenylalanine and tyrosine, from carbohydrates (Davis, 1955, 1958), and of certain benzenoid compounds from acetate (Birch and Donovan, 1953), stimulated interest in the problems of biogenesis of aromatic compounds in plants.

A significant discovery was tbat of Brown and Neish (1955 a, b) who

showed that lignin, inspite of the uncertainty of its structure1 is formed of aromatic monomers of the type. c6-c3 It was also the aim of this work to include an investigation of the biosynthesis and metabolic relationships of certain phenolic acide, since early in the course of a survey of the plant phenolic acide, it was discovered that hydroxylated benzoic acide are widely distributed in plant tissues. As nothing appeared to be known of their relation ships to one another, a study of this problem was undertaken.

Later, in the co~se of these etudies, attention was given to aydrangea macrophylla Ser., a plant which was found to be a particularly rich source of phenolic compounds. Of these constituents, three were found to be phenolic lactones , and the novel structure of one of them, the phenyl iso-coumarin,hydrandenol,prompted a study of its biogenesis. 3

REVIEW OF LITERATURE ON PHENOLIC ACIDS AND PBENOLIC LACTONES

I. Naturally Occurring Phenolic Acide and Phenolic Lactones in Bigher Plants

The most common, naturally occurring, phenolic acide are the c -c (benzoic) and the c -c (cinnamic), acide. Although these 6 1 6 3 acide show a variety of bydroxylation and methoxylation patterns (see Figure 1), bydroxylation is usual in the -o- and -p-position. Table 1 is a list of some of the well known, naturally occurring benzoic acid and cinnamic acid derivatives and their distribution in bigher plants.

The development of paper partition cbromatograpby bas proved to be an excellent tool for the characterization of phenolic acids in plant extracts. Most of these acide show different fluorescence characteristics in ultraviolet light and give different colours witb diazotized pbenolic sprays.

Bate-Smitb (1954 a, 1956 a) and Williams (1955) gave an account of the identification, by cbromatography and UV-fluorescence, of the more common phenolic constituents of plants. A survey of tbeir systematic distribution was given by Bate-Smitb (1956 a).

Griffiths (1958) studied the distribution of gentisic acid among dicotyledonous families and sbowed its wide spread occurrence. Prior to his survey, this acid was only reported to occur in microorganisme."

K~ves and Varga (1959) found that benzoic acid and cinnamic acid derivatives are widely distributed among dry fruits. Figure 3 Pathways of synthesis of phenylalanine and tyrosine from carbohydrates in Escherichia coli, (see Davis, 1958). --

(*P) Stands for orthophosphate. I. ~-Keto-3-deoxy-D-arabo-heptonic acid-7-phosphate. II. Dehydroquinic acid. III. Dehydroshikimic acid. IV. Shikimic acid. V. 5-Phesphoshikimic acid. VI. Intermediate compound. VII. Prephenic acid. VIII. Phenylpyruvic acid. IX. p-Hydroxyphenylpyruvic acid. x. Phenylalanine. XI. Tyrosine. ..::t COOH COOH COOH COOH ~ OH OH ~O H

OH HO

~OH ~ Benzoic Salicylic P- Hydroxybenzoic o_Pyrocatechuic Genti sic

"0 COOH c . COOH COOH COOH a1 en +" tJ c ..-1 a1 0 .-l N p, c (]) c p •.-l OCH HO OH 3 "0 bO (]) c OH OH +" ...-! OH OH :::1 J..< +" J..< Protocatechuic Vanillic Syringic Gallic •.-l :::1 +> tJ en tJ '§ 0 en >, CH=CH-COOH .-l CH= CH-COOH CH= CH-COOH CH= CH-COOH q...., c 0 s0 (]) s H a1 0 .-l tJ :::1 s en J..< "0 0 ..-1 OH q...., tJ al .-l OH a1 tJ OH J..< ..-1 Cinnamic o- Coumaric p-Coumaric :::1 s Caffeic +> a1 tJ c :::1 c J..< •.-l +' tJ Cl) CH2CH2COOH CH= CH- COOH CH= CH-CO OH .. .-l OH (]) J..< ~ OCH CH ..-1 3 OCH3 rz..

OH OH OH Me li lotie Phloretic Feru lie Sinapic 5

Table 1

Distribution of Some Naturally Occurring Phenolic Acide in Plants

Ac id Plant 11aterial Reference

Benzoic, as :

Benzyl acetate Jasmine, byaa intbe and gardenia tlowera (Guenther, 1949)

Methyl benzoate Clove and tuberose volatile oill (Guenther, 1949)

Glucose-6-benzoate Vacainium vitia-idaea (0e1s1man & B1nreiner,l95~) o-Rydroxybenzoic -(salicylic), as: Methyl salicylate Oaultheria proaumbene (Procter, 1843)

Methyl salicylate, primeveroaide Oaultheria procumbens (Cabours, 1843)

. vic1anoside Viola cornuta (Picard, 1926)

~-Rydroxybenzoic* Catalpa ovata (Nakaok1 & Morita,l955)

2,3-Dihydroxybenzoic (2-pyrocatechuic) Populus balsamifera (Gor is & Canal, 1936 :S)

2,5-Dihydroxybenzoic* (gent1sic) W1dely d1stributed (Gr1ff1ths, 1958)

3,4-Dihydroxybenzoic (protocatecbuic) Hibiscus aubdarita (Perkin, 1909)

2-Hydroxy-4-methoxybenzoic methyl ester Primula viscosa (Gor1s & Canal, 19~5) 6

Table 1 (continued)

2-Bydroxy-5-methoxy Priulm a aur 1 cu 1 a & benzoic methyl ester E• officinalis (Goris & Cana1,1936b)

2-Hydroxy-6-methoxy ,.. &Reic4stein benzoic Colchicum autumnale (Santavy/ 1951}

4-Hydroxy-3-methoxl Berberis thunbergii & benzoic (vanillic) !!.· amurensis (Ishida & Okamura,1956) 3,4-Dimethoxybenzoic (veratric) Subadilla officina11s (Merck,l839)

3,4,5-Trihydroxy* Caesa1Einia coriaria benzoic (gallic) (~ coriaria) (Stenhouse, 1843)

3,5-Dimetho.xy-4- hydro.xybenzo,i:c (syringic) Robinia Eseudocacta (Power, 1901)

Cinnamic (free and esterified) Cinnamom essentia1 oi1s (Geissman&Hinreiner,1952) o-Hydroxycinnamic - * (2,-coumar ic) Angraecum fragrans (Zwenger, 1872) (c.f. Karrer) 1958) o-Hydroxyhydro ,, lt ~innamic(melilotic) lt tr

~-Hydroxycinnamic (p-coumaric )* Prunus serotina (Power & Moore, 1909) p-Hydroxyhydro ëinnamic(phloretic) Pyrus communia (Griseda1e&Towers,1960)

3,4-Dihydroxycinnamic tr (caffeic)* Cinchona cuErea ( Korner, 1882)

3,4-Dihydro.xyhydro cinnamic LfcoEodium clavatum (Zetsche & Bugg1er,1927) 7

Table 1 (continued)

4-Hydroxy-3-methoxy- cinnamic(ferulic)* Nepbrolepis !E.. (Kurth, 1950)

3-Hydroxy-4-œethoxy- a cinnamic(iso-ferulic) Catalpa ovata (Hiramoto&Wata~e,l940) " 3,5-Dimethoxy-4- hydroxycinnamic (sinapic)* Cruciferae ~

Malvaceae (Bate-Smith1 1954a)

Pyrus malus (Car~wright et al 1955) ~-Coumaryl qudnic --1 Chlorogenic* Pyrus malus {llulme.,l953)

Hydrangeic Hydransea opuloides {Asahina & Asano, 1930a)

* Indicates wide distribution in plants. tomaszewski (196o) recorded the occurrence of each ot ~-cocmaric,

~-hydroxyben.zoic and gentisic acids in 97 ~~ caffeic acid in 80 ~~ and

ferulic acid in 63 ~ of 122 species investigated by the use of paper

chromatography.

The substituted c6-c1 derivatives, especially ~-bydroxybenz aldehyde, vanillin and syringaldehyde are so widely distributed that they seem almost ubiquitous (Geissman and Hinreiner, 1952). The tri hydroxybenzoic (gallic) acid is a well known constituent of tannins

{gallotannins), leuco-anthocyanins and gallocatechins (Bate-Smith and

Metcalfe, 1957). Esters of quinte and gallic acide, e.g. theogallin, have been isolated from infusions of tea (Cartwright and Roberts,1955).

An important constituent of tanning materials is ellagic acid which bas been shown to be widely distributed in htgher plants (Bate-Smith,

1956 bj Hillis and Clark, 196o).

The cinnamic acid group inclides a variety of hydroxy and methoxy acide which occur mainly as esters. Ferulic and sinapic acids seem to be commonly present in monocotyledone, caffeic acid in dicotyledone, while p-coumaric acid occurs with almost equal frequenc~ in both mono cotyledons and dicotyledone (Bate-Smith, 1954 a). Ferulic and sinapic acids occur in ester form whose combination is still unknown (Bate-Smith,

1958) 0

No other dihydroxycinnamic acide, trihydroxycinna~ic acids or their derivatives, than caffeic, ferulic and sinapic acide appear to 9

bave been found in plants. However, the occurrence of 3,4-methylene-

dioxycinnamic acid (Simmons and Stevens, 1956) and 3,5-dihydroxy-

cinnamic acid (Hermann, 1958) bas been claimed in plants.

Caffeic acid occurs in the free state (Sechet-Sirot !! al,l959)

and esterified witb quinic acid in the form of the depsides which are

known as neo-, iso-, and pseudo-cblorogenic acids (Barnes, Feldman

and White, 1950 ; Corse, 1953 ; Dickinson and Gawler, 1954 a, b).

Recent interest in cblorogenic acid bas centered on its function as a for substrate polypbenolase and its relation to the darkening of injured

fruits.

The related depside, ~-coumaryl quinic acid, wbicb bas been

identified in apples (Bradfield!! !!1 1952 ; Cartwright !! !!1 1955)

is believed to be as a common a constituent of plants as cblorogenic acid (Bate-Smith, 1958). Recently, another ester, quinic acid-1,4-di-

~-coumarate bas been isolated from the pineapple plant (Sutherland and

Gortner, 1959).

One of the well known classes of c6-c3 compounds, related to the hydroxycinnamic acids, are the coumarine. Tbese are derived from cis ~-coumaric acid wbich bas undergone lactonization. Coumari~s are widely distributed, being especially abundant in the Umbelliferae and

Rutaceae. They range from coumarin itself to furano- and cbromano- coumarine. Geissman and Hinreiner (1952) gave a concise review of the occurrence of the structurally related coumarine. More recently, 10

a survey was made of the distribution of coumarine in 120 speciee

repreeenting 36 plant families (Reppel, 1954). Methode for the

identification of the most common, naturally occurring coumarine,

by the use of chromatography, have been deecribed by Swain (1953)

and Reppel (1957). Table 2 contains some examples of coumarine

known to occur in plants.

Table 2

Some Naturally Occurring Coumarine in Plants (see Reppel, 1957) :,)0

Coumarin Plant material

Coumarin Melilot us officinalis

Umbelliferone (7-hydroxy-) Hydrangea paniculata

Herniarin (7-methoxy-) Herniaria hirsuta

Esculetin (6,7-dihydroxy-) Aeeculus hippocastanum

Scopo~etin (6-methoxy-7-hydroxy-) Scopola carniolica

Daphnetin (71 8-dihydroxy-) Daphne odora

Fraxidin (6-methoxy-7,8- dihydroxy-) Fraxinus mandehurica

- 11

Another group of phenolic lactones are the phenyl iso-coumarine.

These are c6-c2-c6 compounds (like stilbenes) with one more carbon on ring A forming a lactone. Hydrangenol and phyllodulcin are two examples; the former has been isolated from the sepale of Hydrangea hortensia (Asahina and Miyake, 1916). The structural formulae of both compounds are given in figure 2.

OH*

HYDRANGENOL * Shows the position of glucose in hydrangenol glucoside.

PBYLLODULCIN

Figure 2: Structural formulae of hydrangenol and phyllodulcin. 1 ;, •.

II. Origin of Benzene Rings in Phenolic Compounds

In the past, it was believed that the aromatic nucleus arose from

a simple hexose. More recently, Geissman and Rinreiner (1952) suggested

that the link between inositols and the c (B)-c unit of flavonoids 6 3 appears to be the related pair of the cyclitol carboxylic acids, quinic

and shikimic acids. Their suggestion was based on the close structural

relationships between quinic and shikimic acide and D-glucose. Rall(l937)

placed quinic acid in the key position as the link between aliphatic and

aromatic compounds in plants, and advanced a theory for its biogenesis

from shikimic acid.

The introduction of tracer techniques utilizing carbon-14 labelled

compounds made it feasible to study the problem of the biogenesis of

benzene rings of phenolic compounds.

1. The Shikimic Acid Pathway

The outstanding work of Davis (1955, 1958) with biochemical mutants

of Escherichia coli established a pathway for the formation of the

aromatic amino acids, phenylalanine and tyrosine, from carbohydrates.

This pathway (see Figure 3) showed that dehydroquinic, dehydroshikimic

and shikimic acids were obligate intermediates in the formation of all

aromatic compounds required by ! · ~ for growth. From etudies of the

formation of carbon-14 labelled shikimic acid from glucose labelled in

different carbone, it was concluded that shikimic acid was formed from Figure 5 Early reactions leading to the formation of lignin and related compounds (see NeishJ 1960).

I. Phenylalanine. II. Tyrosine. III. Phenylpyruvic acid. IV. p-Hydroxyphenylpyruvic ac id. v. Phenyllactic acid. VI. p-Hydroxyphenyllactic ac id. VII. Cinnamic acid. VIII. Dihydrocinnamic acid. IX. p-Coumaric acid. x. Caffeic ac id. XI. Ferulic ac id. XII. Sinapic acid. 13

COOH 1 C=O 1 GOOH CHO CH2 1 1 C-OP' * H-C- OH HO-C-H 1 1 CH2" + H-C-OH ~ H-C-OH ~ CH20p* H-C-OH1 1 CH20P* Phospho- enol- 0-Erythrose- pyruvate 4- phosphate I II l

COOH +- COOH +-- COOH 1 ][

COOH

COOH \ / x

OH OH JX 14

a four-carbon sugar and a three-carbon compound, the latter being formed during glycolysis (Srinivasan et al, 1956).

Quinic and shikimic acids have been shown to be widely distributed among higher plants. Quinic acid was isolated from apple fruits (Hulme,

1951), grass (Hulme and Richardson, 1954), tobacco (Palmer, 1957) and

roses (Weinstein, Williams and Laurencot 1 1959 . ·). Shikimic ac id was identified in gymnosperme (Battori, Yoshida and Hasegawa, 1954 ; Manskaya and Kodina, 1959), grass (Richardson and Hulme, 1955), apples (Hulme,

1956, 1958) and Eucalyptus (Yoshida and Rasegawa, 1957 ; Hillis, 1959).

The shikimic pathway has not been established in higher plants with the same degree of certainty as in microorganisme... However, in shikimic acid-c14 was shown to be converted readily to ligninjTriticum and Acer spp. (Brown and Neish, 1955 a, b), the ring of caffeic acid in

Salvia splendens (McCalla and Neish, 1959 b), quercetin in Fagopyrum tatoricum (Underhill, Watkin and Neish, 1957), pungenin in Picea pungens

(Neish, 1959), ~-coumaric and melilotic acids in ~elilotus alba(Kosuge and Conn, 1959 a, b), coumarin in Hierochloë odorata (Brown, Towers and

Wright, 1960), and the aromatic amino acids phenylalanine and tyrosine in wheat and buckwheat plants (Gamborg and Neish, 1959), and ih wheat plant (McCalla and Neish, 1959 a). Conversion of quinic acid to shikimic acid , phenylalanine and tyrosine was also shown in young rose plants

(Weinstein, Porter and Laurencot, 1959 a, b). 15

2. The Acetate Pathway

The formation of aromatic nuclei from acetic acid molecules by

head-to-tail linkages was first suggested by Collie (1907). This theory

bas been further developed by Stewart (1948), Birch and Donovan (1953)

and Robinson (1955), especially after the discovery of coenzyme A.

According to Birch and Donovan, head-to-tail condensation of acetic

acid molecules produces polyacetic acid, which by means of a further

acyl condensation forma an aromatic ring (see Figure 4). The oxygen

atoms of the acetate carboxyl groups will be the phenolic oxygena on

the aromatic ring.

Birch, Massy~Westropp and Moye (1955) provided the first evidence

for this pathway by establishing the formation of 6-methylsalicylic acid 14 from acetate-1-C in Penicillium griseofulvum. The acetate pathway has since been shawn in higher plants, especially in the biosynthesis of symmetrically hydroxylated phenollc compounds of the phloroglucinol type (ring A of the c - c - c compounds). The latter was demonstrated 6 3 6 for quercetin in buckwheat (Watkin, Underhill and Neish, 1957 ; Geissman and Swain, 1957), cyanidin in red cabbage seedlings (Grisebach, 1957) and the dihydrochalkone, phloridzin in the apple plant (Hutchinson,

Taper and Towers, 1959). 16

R-COOH + 3 CH 3 -COOH >

HO OH 1 -----=.,...~ R-CO R-CO-CH 2

Figure 4 : The acetate pathway. 17

III. Seme Biosynthetic Pathways of Phenolic Compounds

In the last few years and since the establishment of the shikimic

acid pathway of biosynthesis by Davis (1955), a great deal of work

has been carried out to e1ucidate the pathways of syntheses of the groups various/of phenolics occurring naturally in higher plants. Biosynthetic

and metabolic pathways of aromatic compounds have been reviewed in

microorganisme" ( Evans, 195 8) , and in higher plants, especial1y flavonoids (Geissman and Hinreiner, 1952; Bogorad, 1958) and phenylpropanoids

(Geissman, 1958 ; Neish, 1960).

The work of Neish and his group at the Prarie Regional Laboratory,

Saskatoon, has provided a great deal of information about the simple phenolic cinnamic acids. They were shown to be precursors of lignins and of ether aromatic compounds in plants . Neish (1960) summarized these resulta in a. scheme shown in Figure 5. He showed that the phenylpropanoid intermediates in tbe scbeme were readily converted to 11gnin.

This was determined by measuring the radioactivity of vanillin and syringaldehyde obta1ned by ni trobenzene oxidation of the extractive- free plant material (Br own and Neisb, 1955 a, b , 1956 ; Wright, Brown and Neish, 1958 ; Brown, Wright and Neish, 1959). They also showed that two monocotyledonous species could readily convert tyrosine to lignin, whereas nine ether species r eadi1y uti 1ized phenylalanine as a 1ignin precursor. This scheme has been supported by the resulta of ether workers. 18

PRO TEINS / ~ 0 CH2-CHNHz-COOH HOOCH2-CHNH2 -COOH I jf CARBOrDRATE II 1 0 CH2· CO-COOH ~ SH IKINI c ----') HOO CH2-CO-COOH 1 ~ 1l ~ IV ll Q U 1 N 1 C 0 CH2· CHOH.COOH HOOCH2-CHOH.COOH v l Gramlne .. -::;" VI 0 CH·CH-COOH / Ho CH·CH·COOH VII i~ HO 0 CH= CH-COOH l CH2~CH2~COOH lX t ~ HOO CH:CH-COOH O OUERCETIN HO X VIII J

HOO CH=CH- COOH CH 0 3 XI LIGNINS 19

Geissman and Swain (1957) found that phenylalanine-~ -c14 was converted to caffeic acid, with the same arrangement of the carbon skeleton, by Nicotiana tabacum. The same result was shown for the caffeic acid moiety of chlorogenic acid in young tobacco shoots

(Reid, 1958).

The biosynthesis of coumarin from the cis form of o-coumaric acid has been shown to proceed by the shikimic acid pathway via phenylalanine and cinnamic acid. It was shown to be readily formed from trans cinnamic, ~-coumaric, phenylalanine and shikimic aci~in

Hierochlèë odorata and Melilotus officinalis (Brown, Towers and Wright,

1960). Kosuge and Conn (1959 a, b) obtained carbon-14 labelled coumarin 14 from Melilotus alba plants which were allowed to photosynthesize C • o2 14 They showed that coumarin-c was converted to melilotyl glucoside and melilotic acid when fed back to ~· ~plants.

Scopoletin (6-methoxy-7-hydroxycoumarin) was also shown to be formed from ferulic acid in wheat, corn and sunflower plants (Reznik and Urban, 1957) and from phenylalanine in tobacco plant (Reid, 1958). 2p

REVIEW OF METHODS OF SEPARATION AND IDENTIFICATION OF PHENOLIC

ACIDS AND PHENOLIC LACTONES

This section includes a brief review of the metbods wbicb have been used for the preparation of plant extracts and the identification, by chromatograpby, of pbenolic acide and pbenolic lactones.

Extraction of plant material is usually carried out witb bot,

80 ~ methanol, ethanol or metbanolic HCl. The alcobolic extracts are evaporated to dryness and extracted witb ether after the bydrolysis of glycosides and esters. The aromatic acide are then found in the ether extract. A preliminary extraction with low boiling petroleum ether bas frequently been used to remove fats, waxes, ethereal oils,etc.

Other workers have extracted finely powdered plant material directly witb ether in Soxhlets or percolators. Aromatic carboxylic acids are much stronger acids tban phenols, and may be extracted from organic solvents by shaking with dilute solutions of carbonate or bicarbonate.

MCCalla and Neish (1959 b) described a procedure for the separat ion of phenolic acids from plant extracts. The method constituted of evaporating the etbanolic extracts of the plant material to dryness.

The residue was tben dissolved in hot water and filtered tbrough Celite.

The filtrate was hydrolyzed witb 2N NaOH at room temperature for five bours, acidified and continuously extracted with ether. The ether extract was shaken with dilute bicarbonate solution wbicb was re-extracted 21

continuously with ether after acidification. The advantage of this

method is that it can be applied for the isolation of the phenolic acide

which occur in the free state, in the form of glycosides or esters

according to the nature of hydrolysis.

Separation of individual phenolic acids bas also been carried out

by making use of special physical or chemical properties. For example,

benzoic acid and its analogues have been separated by steam distillation

from plant extracts. However, the fact that certain phenols are also

volatile with steam makes this method of limited use.

In recent years, chromatographie methode have been most frequently

applied (Brown and Hall, 1950 ; Long, Quayle and Stedman, 1951) for

the separation of phenolic compounds.

In one-directional paper chromatography, (4:1:5) toluene:acetic

acid:water (Bogorad and Granick, 1953 ; Bate-Smith, 1955, 1956 a) has

been employed, although with this solvent system, the dihydroxy acids

do not move. Moreover, it causes streaking or tailing of spots and

gives very diffuse zones and low Rf values for the ether acids. Increas

ing the amount of acetic acid in the solvent composition increases the

Rr values of the phenolic acids. The organic layer of a mixture of benzene:acetic acid:water bas 'been used in different proportions (2:2:1,

Bray, Thorpe and White, 1950 ; 6:7:3, Griffiths, 1957 ; 1:1:1, Tomas

zewski, 1960). Of these, (6:7:3) benzene:acetic acid:water gives the most useful separation of the monohydroxy and dihydroxy acids. 22

For phenolic acids wbich do not move in the benzene solvent, n-butanol:acetic acid:water, in various proportions (4:1:5, Bate-Smith,

1949, cf. Block, Durrum and Zweig, 1955 ; 4:1:2.2, Roberts, 1956 ;

6:1:2, Cruickshank and Swain, 1956) bas been found suitable. Forestal solvent, (30:3:10) acetic acid:concentrated HCl:water, bas also been found useful in the separation of ellagic acid from plant extracts

(Bate-Smith, 1956 b).

In a study of the phenolic acids of human urine, Armstrong, Shaw and Wall (1956) used the following solvent systems: (8:1:1) ~- propanol:ammonia:water , (2:2:1) benzene:propionic acid:water, and

(8:2:2) ~-butanol:acetic acid:water, all in one-directional descending paper chromatograpby. These solvent systems gave good separation of hydroxbenzoic acids and hydroxycinnamic acids.

Very recently, Grinstead (1960) reported the use of (4:1) iso- propanol:7N aqueous ammonia in the separation of monohydroxybenzoic and dihydroxybenzoic, acids. -m-Hydroxy-, -p-hydroxy-, 2,4-dihydroxy-, and 2,6-dihydroxybenzoic acids were reported to have Rf values of

0.30, 0 .16, 0 . 22 and 0.71, respectively. ~and para-hydroxybenzoic acids cannet be separated witb the other solvent systems. This applies also to the two dihydroxybenzoic acids.

In two -directional paper chromatography, 20 ~ HCl, 22 ~ KHco 3 and 20 ~ KCl have been employed to irrigate the second direction after using the benzene solvent for the first (Boscott, 1952, cf. Block et~' 23

1955). HCl causes spreading of spots which reach undesirable sizes.

KHC0 and KCl solutions precipitate on the paper chromatogram after 3 drying. This is a disadvantage since it increases self absorption when radioactivity determinations have to be made on the paper chromatogram.

The use of circular paper chromatography has been reported by van Sumere et al (1957) in the separation of coumarine and phenolic acide of uredospores of wheat stem rust. They were unable to get suitable Rf values when non-polar solvents, such as toluene:acetic acid:water, were used. In acidic systems, Rf values tended to be too high, whereas in alkaline systems, the majority of the compounds of interest did not move from the origin. They obtained satisfactory separation after the filter paper was impregnated with a mixture of

(1:1) formamide:ethanol. The solvent systems, (1:2) benzene:petroleum ether (b.p. 100-120°C), chloroform-saturated ammonia and (4:1) ~ butanol:water, were found suitable for the separation of phenolic acids and coumarine from spore extracts.

For the chromatographie separation of coumarine, (4:1:5) ~-butanol: acetic acid:water and phenol-saturated water (Swain, 1953 ; Harborne,

1960), water-saturated iso·-amyl acetate, iso-amyl alcohol and (4:1:2) iso-amyl alcohol:acetic acid:water (Nakabayashi, 1953), have been found useful.

Reppel (1957) studied the chromatographie behaviour of sorne coumarin derivatives using 12 different solvent systems and tried to correlate their 24

Rf values and chemical structure. Among these solvents, 10 ~ acetic acid1

1 (9:1) ~-propanol:25 %aqueous ammonia and ~-butanol-saturated 25 ~ aqueous

ammonia were part1cularly useful. He provided a key for the identification

of coumarins based on Rf values, fluorescence in ultraviolet light

and colour reactions. The solvent systems (50:2:48) ~-butanol:benzene:

ammonia and (7:3) ~-propanol:ammonia, have also been used in the chroma-

tography of hydrangenol and its glucoside (Asen, Cathey and Stuart,l960).

Chromatography of phenolic acids on a cellulose column bas been

reported by McCalla and Neish(l959 b) in the isolation of caffeic acid

from plant extracts. The developer was (100:100:10:3) benzene:ethyl

acetate:formic acid(9~):water. This method could be useful for the

isolation of other phenolic acids by using appropriate mixtures of

solvants as developers.

Seki, Inamori and Sano (1959) reported the use of cation exchange

resins in the separation of 14 phenolic acids with 80-90 ~ recovery. on + This was performed by adsorption Amberlite IRC-50 (H) and elution with

(6:1:2) and (9:1: 2) 0 . 2N HCl :acetone:methyl ethyl ketone. In another

+ procedure, they used Duolite C-25 (Na) as adsorbent and 0.1 M citrate

buffer in 20 %ethanol as eluent.

Gas chromatography was app1ied to the ana1ysis of phenols by

Fitzgerald (1959), but with only partial separation of the phenols which were used.

Many phenolic compounds are fluorescent in ultraviolet light. The 25

common presence of a double bond in the side chain of some phenolic acide and in the pyrone ring of coumarine is essential for the fluorogenous resonance of the molecule. In case of cinnamic acid derivatives, whereas -o-coumaric, -p-coumaric, caffeic and ferulic acids are fluorescent in UV-light, their dihydro derivatives are virtually nonfluorescent.

Goodwin and Kavanagh (1950) studied the colour and relative intensity of fluorescence of 98 coumarin derivatives. They discussed the influence on fluorescence of substitutions at different positions on the coumarin molecule. Some of the resulta reported in their investigation are summarized as follows. They found tbat coumarin, a nonfluorescent substance, when irradiated in alkaline solutions, rapidly formed a compound which exhibited a yellowish green fluorescence. Several

derivatives of coumarin (5-methyl-1 6-methyl-1 7-methyl-, 8-methyl- and

6-cblorocoumarin) also behave similarly. Wbereas umbelliferone (7-hydroxy-

coumarin) is one of the most fluorescent of all coumarins, 3-hydroxy-1

4-hydroxy-1 5-hydroxy- and 8-hydroxycoumarins are nonfluorescent. The addition of a second hydroxyl group to umbelliferone at position 5 or 6 causes at least a 15-fold reduction in the maximum fluorescence. On the

other hand 1 dapbnetin (7,8-dihydroxycoumarin) is nonfluorescent. The methoxyl is less effective tban the hydroxyl group in influencing the fluorescence of the molecule. The most fluorescent of the methoxy- coumarine appears to be 6,7-dimethoxycoumarin followed by 7-methoxycoumarin. These fluorescence characteristics reported by Goodwin and 26

Kavanagh are helpful in the identification of coumarine.

Identification of phenolic acide and phenolic lactones by their

Rf values on paper chromatograms developed in different solvent systems bas proved useful (Swain, 1953 ; Bate-Smith, 1954 a, 1956 a, b ;

Williams, 1955, 1956, 1957 ; Armstrong et !!1 1956 ; Reppel, 1957 ;

Rarborne, 1960).

Chromogenic sprays have also been employed for the detection of these compounds. D1azot1zed ~-n1troan111ne, diazotized sulfan111c acid and 1 ~ ferric chloride solution are amongst the spray reagents used.

Ferric chloride solution reacts with most of the substituted benzoic acids and also with 3,4-dihydroxycinnamic acid. 27

MATERIALS AND METHODS

l. Plant Material

Most of the plants used in the study of phenolic acids were

obtained from the greenhouse of the Botany Department, McGill University.

Gaultheria procumbens L., which was used for the isolation of two

phenolic acide, was collected from the woods, 25 miles west of Montreal.

These plants were transferred with soil to the greenhouse and served as

material for carbon-14 experimente.

Mature flowers of Hydrangea macrophylla Ser. were obtained from

Montreal Botanical Gardens for the isolation of bydrangenol and its glucoside. A number of cuttings of the same species was supplied from the same place and was allowed to root in "Vermiculite", then transferred to pots and allowed to grow in the greenhouse. Tbirty potted plants of

Hydrangea were purchased from Alcontara Co. Ltd., Montreal, and served for the isolation of certain other phenolic compounds. These plants produced flowers later, and served as material for carbon-14 experimenta.

2. Chemicals

The monometbyl ethers of the dihydroxybenzoic acide (2- hydroxy-4- methoxy-, 2-hydroxy-5-methoxy-, and 2-hydroxy-6-methoxybenzoic acids) were prepared by the method of Graebe and Martz (1905). p-Coumaric and f erulic acide wer e prepared by Doebner modification (Adams, 1942) .

Phloretic acid was obtained from the alkaline hydrolysis of phloridzin 28

(Cremer and Seuffert, 1912). Scopo1etin and 1!2-ferulic acid were

gifts from Dr. W.A. Andreae, Canada Department of Agriculture and Dr.

V. Runeckles, Imperial Tobacco Company, Montreal. Other phenolic acids

or standards referred to elsewhere were ava1lable from commercial sources.

3. Carbon-14 Compounds 14 14 14 Benzoic acid-C OOH, salicylic acid-C OOH, L-phenylalanine-U-C , 14 14 14 L-tyrosine-U-C , sodium cinnamate-2-C · , soqium acetate-l-e , sodium 14 14 acetate-2-C. and D-glucose-U-C were purchased from Merck and Company, . 14 Montreal. L-Phenylalanine-1-C was purcbased from California Corporation 14 for Biochemical Research, Los Angeles 63, and L-phenylalanine-3-C from

Research Specialities Company, Richmond, California.

Cinnamic acid labelled on the ring and the ~-carbon was a gift from 14 Dr. E. Conn, University of California. Shikimic acid-U-C was prepared in this laboratory following the method reported by Srinivasan ~al (1956).

4. Preparation of Radioactive Shikimic Acid

Twenty ml of sterile nutrient solution and 5 ml glucose (containing

3.5 mg uniformly 1abelled glucose with an activity of 500 f' diluted with a 50 mg non-labelled glucose) were added to/treshly grown culture of

Escherichia ~ 83-24. The inoculum was a gift from Dr. B.D. Davis,

Harvard Medical School, Mass.,u.s.A. The mixture was incubated with

0 continuous shaking at 37 C for 40 hours. The culture medium was acidified

0 14 to pH 2 with concentrated HCl, heated to 80 C to drive off C o , and 2 29

filtered through a bed of Celite. The filtrate was passed through a column (12.5 x 1 cm) of washed Darco-G60 charcoal. The column was washed with 300 ml distilled water until the washings were free from inorganic phosphate, and then with 100 ml of 5 ~ ethanol. Shikimic acid was eluted with 250 ml of 25 ~ ethanol and the eluate evaporated to dryness under a jet of filtered air when the residue turned blue in colour.

This was dissolved in a small volume of 85 ~ ethanol and chromatographed one-directionally using the organic layer of a mixture of (4:1:5) ~ butanol:acetic acid:water. The band corresponding to shikimic acid was marked with the aid of a co-chromatographed authentic sample, eut off the paper and eluted with 85 ~ ethanol. By this treatment, shikimic acid was purified from the coloured contaminant and was separated from phosphoshikimic acid. Atte.mpt to crystallize shikimic acid from dry glacial acetic acid after the addition of 25 mg non-labelled shikimic acid were unsuccessful. The solution was then evaporated to dryness in vacuo and dissolved in 5 ml distilled water. Two-directional chroma tography using phenol and butanol solvents showed two radioactive contaminants which amounted to about 25 ~ of the radioactivity.

5. Preparation of Plant Extracts

Fresh leaves, roots or whole plants were chopped up to small pieces and directly plunged into boiling 85 ~ ethanol and extracted by refluxing on a steam bath for four to six hours with two or three changes of ethanol until all the colouring matter was extracted. The combined ethanolic

extracts were filtered and taken to dryness either by means of a jet

of filtered air or by distillation using a rotary film evaporator at

a reduced pressure. For analysis of this et~nol soluble traction,

. the residue was dissolved in a small amount of 85 ~ ethanol and

applied directly to chromatography grade paper.

For the purpose of investigating the phenolic acids, the dry

residue of the ethanolic extracts was dissolved in a small amount of

hot water and filtered through a bed of Celite using a filter pump.

The filtrate was treated in one of the following ways:

a. Acidified with 2N HCl to pH 4, this fraction when extracted with

ether contained the free phenolic acide.

b. Hydrolyzed with 2N HCl by retluxing on a steam bath for an hour ,

this traction contained the phenolic acids which were present as

glycosides.

c. Hydrolyzed with 2N NaOH for 6 hours at room temperature in a nitrogen

atmosphere. The alkaline extract was acidified with 6N HCl and

extracted with ether. The ether extract contained the phenolic acids

originally present as esters as well as alkali-sensitive glycosides.

Each of the acidified extracts was continuously extracted with ether in a liquid-liquid extractor* for 6-12 hours. The ether extract ~s shaken

* Three different sizes of liquid-liquid extractors were used to hold 8, 100, or 500 ml of the acidified extracts (Quickfit catalogue Nos. EX8/00,

EX25 1 and EX9/53 1 respectively). 31

with amal! volumes of 2 ~ sodium carbonate solution (5-25 ml) in a

separatory funnel. The combined alkaline extracts were acidified and

re-extracted continuously with ether for 6-12 hours. The final ether

extract was taken to dryness under a jet of filtered air at room

temperature and the resid~e dissolved in a small volume of 85 ~ ethanol

for chromatographie analysis of phenolic acids.

6. Chromatography

Unless otherwise st~ted, two-directional, descending, filter

paper chromatography was used during the course of this work.

Paper: Chromatography grade Whatman No. l and No. 3 filter papers were

used without washing or previous treatment. Extracts were applied as

small circular spots at the intersection of !ines parallel to and 10 cm

from two adjacent edges of the No. l sheets, and as bands on No. 3 sheets.

The latter were chromatographed only in one direction.

Chambers: The chromatography chambers used, were made of marine ply-wood,

25 inches high and 11 inches wide, and were lined on the inside with a

coating of paraffin wax. The chamber used for the benzene solvent was

lined with Arborite. The aqueous layer of each of the organic solvants

used was placed at the bottom of the chamber in a shallow open vessel.

This was essential because of the highly volatile nature of the solvent and its tendency to be lost by absorption in the wood or paraffin. The glass troughs measured 21 x 1.5 inches. Eighty ml of solvent was used 32

to irrigate a pair of sheets which were placed opposite each other in

the trough. The sheets were draped over anti-siphon roda and anchored

in the trough with a heavy glass rod. The irrigated sheets were removed

from the chambers when the solvent front was two inches from the lower

edge and were allowed to dry in the fume bood.

Solvents: The following solvent systems were used.

a. Phenol solvant: (4:1) Mallinkrodt or Merck, phenol:water (Consden,

Gordon and Martin, 1944).

b. Butanol solvent: (4:1:5) n-butanol:acetic acid:water (v/v/v)

(Bate-Smith, 1949 c.f. Block, Durrum and Zweig, 1955).

This pair of solvents (a and b) gave good separation of the compounds in total ethanol extracts. The phenol solvent was used in the direction

parallel to the long axis of the sheets, and the butanol solvent, at

right angles to the flow of the first.

c. Benzene solvent: (6:7:3) benzene:acetic acid:water (v/v/v)

(Griffiths, 1957).

d. Formate solvent: (10:1:200) sodium formate:formic acid:water(w/v/v)

(Smith, 1958). This pair of solvents (c and d) was used for the chroma

tography of phenolic acids. The benzene solvent was used for the irrigat

ion of the first direction, and the formate for the second.

All the above-mentioned solvent systems were prepared from reagent

grade chemicals and distilled water. The butanol solvent was always

freshly prepared. 33

The period of irrigation with each solvent, without previous

equilibration of the sheets, was 30-34 hours for phenol, 14-16 hours

for butanol, 6-7 hours for benzene and 4 bours for formate, solvents.

Variations in these periode were due to fluctuations in temperature.

1. Spray Reagents

Since most of the pbenolic acide are cbaracteristically fluorescent

in ultraviolet light, they were examined under an ultraviolet lamp

0 (3660 A ) and the fluorescent spots were marked lightly witb pencil.

Paper chromatograms were then sprayed witb one of the following chromo-

genie reagents.

a. Diazotized p-nitroaniline (Bray, Thorpe and White, 1950) was

prepared as follows: 5 ml ~-nitroaniline solution (0.3 ~ in 8 ~ BCl w/v)

was mixed with one ml sodium nitrite solution (5 ~ w/v) and 15 ml sodium

acetate solution (20 ~ w/v). This mixture was always fresbly prepared before use. Paper cbromatograms were then oversprayed witb 5 ~ sodium

bydroxide solution. It was found that, if paper chromatograms to be

sprayed with this reagent were exposed to phenol or ammonia vapeurs, a dark pink background colour developed which interfered witb visualiz- ation of the spots.

b. Diazotized sulfanilic acid (Evans, Parr and Evans, 1949) consisted of a fresbly prepared mixture of two volumes sulfanilic acid ( 9 g in

90 ml concentrated HCl per liter of water), one volume sodium nitrite solution (5 ~ w/v) and two volumes sodium bydroxide solution (20 ~ w/v). 1950 c. Ferric chloride solution (Bray, Thorpe and Whit~) was used as 1 ~ aqueous solution (w/v).

8. Elemental Analysis

Carbon and bydrogen analyses of hydrangenol, its glucoside, diacetyl- hydrangenol and 2,6-cresotic acid were carried out by Schwarzkopf

Microanalytical Laboratory, New York. Blemental analysis of compound

No. 15 was done by c. Daesslé, 5757 Decelles St., Montreal.

9. Spectrophotometry

The absorption maxima of the isolated phenolic compounds in 85 ~ ethanol were determined on a Beckman model DU spectrophotometer, using l cm silica cella. For phenolic lactones, absorption maxima were also determined after the addition of two drops of 2N NaOH to the ethanolic solutions. Infra-red spectra were determined by Dr. P.I. Abell, Chemistry

Department, University of Rhode Island, using an Baird-Atomic I.R. spectre- photometer with a NaCl priam.

10. Techniques for Administering Carbon-14

Carbon-14-labelled compomtds were administered to plant tissues as aqueous solutions. These solutions were always kept in the frozen state and taken out of the freezer, sometime before carrying out the experiment, to bring them to room temper ature. Radioactive solutions were administered to leaf disks, flower sepale, root segments or whole intact plants. 35

Leaf disks 1 11 mm in diameter, were obtained from young, se1ected leaves with a Ganong leaf punch. The disks were infiltrated by placing them in a suction flask containing tap water. Suction was applied to the flask while being stoppered for 2-3 minute intervals. After infiltration the disks no longer floated in water. Batches of l0-l2 leaf disks were blotted, floated on the radioactive solutions in small Petri dishes, and the latter placed under a bank of fluorescent lights for 16-24 hours.

When radioactive solutions were administered to flower sepals, the latter were treated exactly as the leaf disks after being eut into halves.

Root segments,when used as experimental material, were washed free of soil partiales, eut into one-half inch segments, blotted, and then floated on the radioactive solutions without previous infiltration.

With whole plants, the soil was removed from the roots by washing thoroughly with tap water followed by blotting with filter paper. The roots were placed in small beakers containing the radioactive solutions and were allowed to stand under the bank of fluorescent lights for 24 hours, during which time tne solutions were absorbed by the roots and water was added to keep the roots moist. At the end of the metabolic period, the roots were thoroughly washed with water, blotted and then directly extracted with boiling, 85 ~ ethanol. ll. Radioautography and Radioactivity Determination

Radioactivity measurements of plant extracts were determined before chromatography. This was done by pipetting aliquote of the final 85 ~ ethanolic extracts, which had been made to a measured volume, on to 2 alumini~ planchettes so as to contain lesa than 200 pg/cm • The material

was therefore counted at infinite thinness, and no correction for self

absorption was ~de. All radioactivity determinations were carried out

using a thin end-window IWAAA Geiger-M61ler tube (end-window thickness

1.25 mg/cm2 ) attached to a Berkeley decimal ecaler, model 2001. The

relative efficiency of the instrument was determined to be 8.5 ~. The

radioactivity of each sample was determined in triplicate and counts were

taken to a mean error of 2 ~ or better. A background count was recorded daily.

Distribution of radioactivity in the different compound& in plant extracts was determined by direct counting on paper cbromatograms. For this purpose, developed chromatograms were eut to 17 x 14 inches in aize and placed in contact with Kodak X-Ray, No screen, safety films under a heavy weight for 1-2 weeks depending upon the radioactivity of the plant extracts. The films were developed for 6 minutes in Kodak rapid X-Ray developer (later, 4-minute developing was found to give better resulta), washed with water and then fixed in Kodak fixative solution for 7-10 minutes. The resulting radioautographs were matched with tbeir corresponding paper cbromatograms, whose corners bad been marked witb radioactive ink. The radioactive spots, corresponding to the exposed areas of the radioautograph, were marked on the paper chromatograms ligbtly witb pencil, and counted as described above. 37

During the course of this work, an instrument for determining

radioactivity of beth soluble and insoluble fractions was purchased.

Dried extracts and insoluble residues were oxidized with van Slyke (van Slyke et al, 14 reagentj~95l) to c o2 and measurements of radioactivity were made with a Nuclear Chicago model 6oO dynamic condenser electrometer

{Dynacon) incorporating an ion chamber. Samples were combusted in

duplicate with an errer of less than 4 ~. EXPERIMENTAL AND RESULTS

A. PBENOLIC ACIDS

I. Chromatographie Separation and Identification of Plant Phenolic Acide

One percent ethanolic solutions of standard c -c , c -c and 6 1 6 2

C6-c3 phenolic acids, were applied to large sheets of Whatman No. 1 chromatography paper. These were chromatographed two-directionally by the descending method using the upper layer of a mixture of (6:7:3) benzene:acetic acid:water for the first direction and a mixture of

(10:1:200) sodium formate:formic acid:water for the second. The phenolic acids which are slow moving in the benzene solvent and do not separate well, were chromatographed using a second pair of solvents consisting of the upper layer of a mixture of (4:1:5) ~-butanol:acetic acid:water for the first direction, followed by the formate solvent for the second.

Identification of phenolic acids was carried out by examining paper chromatograms under an ultraviolet lamp before spraying. The fluorescence was further examined af.ter exposure to ammonia vapors. Chromatograms were then sprayed with one of the following reagents (a) diazotized p nitroaniline, (b) diazotized sulfanilic acid, or (c) 1 ~ ferric chloride solution. The colour reactions of these acids and their ultraviolet fluorescence are listed in Table 3· Figures 6 and 7 show the location of the acide on two-directional chromatograms in the benzene-formate and the butanol-formate solvents, respectively. The numbering of the spots on these maps corresponds to that in Table 3. 39

Table 3

Fluorescence and Colour Reactions of Some Phenolic Acide

Fluorescence Colour reactions -- Phenolic acid in UV-ligpt Diazotized p- Diazotized iJFerric nitroanilinë/ sulfanilic chloride Na OH ac id

1. 2-Hydroxybenzoic (aalicylic) Dark blue Pink Yellow Light brown

2. ~-Hydroxybenzoic None Pink Yellow Buff

3· 2,3-Dihydroxybenzoic Dark blue Blue fades <2-pyrocatechuic) to white Yellow Violet-blue

4. 2,4-Dihydroxybenzoic None Blue turne Yellow- ( (j -resorcylic) yellow- brown Light brown brown

5· 2-Hydroxy-4-methoxy- Light brown Yellow Light brown benzoic None

6. 2,5-Dihydroxybenzoic Bright Blue fades Buff Violet- (gentisic) blue to white blue

7· 2-Hydroxy-5-methoxy- Bright Light "blue, Light brown Blue benzoic blue fades

8. 2,6-Dihydroxybenzoic Blue fades Brown Light brown ( '1 -resorcylic) None to brown

9. 2-Hydroxy-6-methoxy- Orange- Light brown benzoic None Brown yellow

10. 3,4-Dihydroxybenzoic Light brown Olive-green Violet-blue (protocatechuic) None

11. 4-Hydroxy-3-methoxy- Purple Orange Light brown benzoic(vanillic) None

12. 3,5-Dihydroxybenzoic Orange- Brown Light brown ( o< -resorcylic) None brown 40

Table 3 (continued)

13. 3-Hydroxy-5-methoxy- Orange- Light pink Green benzoic None yellow

14. 3,4,5-Trihydroxy- None Buf:f' Reddish Blue benzoic{gallic) brown

15. 4-Hydroxy-3,5- Light dimethoxybenzoic None Blue Red violet (syringic)

16. 2-Hydroxyphenyl- None Pink- Yellow- Light violet, acetic purple orange fades

17. ~-Hydroxyphenyl- None Light Pink None acetic purple

19. 2,5-Dihydroxyphenyl None Buf:f', de- Brown- Decolorises acetic(homogentisic) colorises purple

20. 2-Hydroxycinnamic White- Purp~· Orange None (2-coumaric) yellow

21. 2-Hydroxydihydro- one Purple Orange- None cinnamic(melilotic) yellow

22. ~-Hydroxycinnamic Dark blue Blue fades Light 9l"own None (E_-coumaric) to grey

23. ~-Hydroxydihydro- None Purple- Light pink None cinnamic(phloretic) violet

24. 3,4-Dihydroxy- Bright Light blue, Bu:f':f' Dark green cinnamic(caf:f'eic) blue turne white

25. 4-Hydroxy-3-methoxy Blue-green Purple None cinnamic (:f'erulic) Blue

26. 3-Hydroxy-4-methoxy Bright Purple Salmon Buff cinnamic(l!2-ferulic) blue 41

Table 3 (continued)

27. 4-Hydroxy-3 1 5- Light Light blue, Pink None dimethoxycinnamic green 1 fades (sinapic)

28. ~-Coumaryl quinic Faint violet· None Faint brown None blue

29. Chlorogenic Strong blue Light buff Faint brown Dark green on standing

30. Ellagic None Buff :raint brown Blue-grey on standing 42

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

0 CD

.0 Q)

0 :..., ~ 0 U)

U).. \J -...1 0 (JI "' 6 ®~ m ca tB ::11 •N 0 •::11 ~ Cl n e ~ ca 0 n-· @) ~ Cl B %8 n a. ? 1[ 1\) Cl c;v@ •- "' 0 0 ? l @) ~®1 ( 10:1 :200) Sodium formate: for mie oc id: wattr

Figure 6 Location of standard phenolic acids on a two-directional paper chromatogram. Numbering of spots corresponds to that in Table 3. 0.1 0.2 0.3 0.4 0.5 0.6 o.7 o.a o.g

0 Ù)

0 G 0 èD

0 0 :...,

p U1 U1

::::J 1 CD c: 0 -::::J Q.... 0 (') !. 0 (') 0, 0 na: 0 i-.>

( 10:1: 200) Sodium formate: formic acid ~ water Figure 7 Location of standard phenolic acids, which are either slow moving or do not move in the benzene solvent, on a two directional paper chromatogram. Numbering of spots corresponds to that in Table 3. II. The Phenolic Acid Patterns of Some Common Plants

A survey of the distribution of pbenolic acids in a variety of common plants was ~arried out by the use of two-directional paper cbroma tography using the benzene and formate solvants. No attempt was made to detect the acids which did not move in the benzene solvent. The plant material consisted of stems and leaves unless otherwise stated. The methode used for preparing the plant material have been described betore.

Plant species were ehosep to represent different plant groups. Fifty three species representing 30 plant families were investigated. Identific ation of phenolic acide was confirmed by comparing their location and eolour re~ctions with those on a sprayed chromatogram of standards. In some cases, plant extraets were co-chromatographed with reference compounda to prove the identity of some phenolic acide; in others, small amounts of the acide were isolated and identified by melting points and co-chromatography with authentic samples.

The quantities of phenolic acids on paper chromatograms were estimatad after spraying, by visual comparison of the size of spots and the intensity of their colours with standards.

Table 4 contains a list of the plants investigated and the common phenolic acide identified on their chromatograms. The unidentified phenolic compounds are also included in the same table in a separate column. Il'\ ...:t- Table 4

The Pbenolic Acids of Some Commop Plants

Plant species Treat- POB Gen. Van. Syr. p-C. Caf. Fer. Sin. Otber Unidentified ment - ac ids compounds *

Moss es Cat ber in ia !!! . B, A x t t - x x x - a 7, 20, 46 Polytricbum !!!· B, A xx - xx - x x x - 5, 19, 22

Spb@gnum ~· B, A xx - xx -- - x - 7, 19, 22 Lycopodiaceae Lycopodium lucidulum Mincb. B, A x x xx - x x xx - salie. 7, 19, 30 Polypodiaceae Nepbrolepis !!!· B, A x x x - x - - - o-coum. 3, 26, 35 Selaginellaceae Selaginella !!!· A x x xxx xx t - - - 28 S. kraussiana A. Br. B, A xx x xxx t - - - - 1

~· martensii Spring. B, A x x x t x x x x 1

Gymnosperms Pinaceae Pinus excelsa Wall. A x - x - x - x - " " B xx - x - xxx xx xxx - \0 ..:t Table 4 (continued)

Plant species Treat- POB Gen. Van. Syr. E-c. Caf. Fer. Sin. Other Unidentified ment ac ids compounds * - Juniperus virsiniana A x - x - x - x - " Il Linn. B t - t - xx - xx t Taxodium disticbum Rich. A - - xxx - xx xx x - 7, 29 " " B - - x - xxx xxx x xxx 7 Ts~a canadensis Carr. A x - xx - x - x - 17 " " B t - t - xx xxx x - Taxaceae Taxus. cuspidata Sieb.& A x - x - x - x - 17 Il " Zucc. B t - x - xxx - xx - Podoearpus !E_. B, A x t x t x x t x salie. 7

Dicotyledone Casuarinaeeae protee. Oasuarina equisetifolia A 6x x t o-eoum. - - - - - Iirotoe. Linn. B x t - -- - x x o-eoum. " Salieaceae" Salix purpurea Linn. B, A - x x - x x -- salie. 1,2,25,43,44J45,49 Populus tacamahaca Mill. A x x x x x x - - protee. Moraceae Maelura pomifera Scbneid A - - t - x - x x 7 Polygonaceae Polygonum siêboldii : A x x x - t x t t protee. De Vriese t' ..:t Table 4 (continued)

Plant species Treat- POB Gen. Van. Syr. p-C. Caf. Fer. Sin. Other Unidentified ment - ac ids compounds*

Crassulaceae Sedum morganianum A x x t - t - x x 1, 27

Rosaceae phlor. Pyrus communis Linn. A x x x x x x x - protoc. phlor. Malus robusta Linn. A x x x x x x x - protoc. Leguminosae Acacia farnesianaWilld. A x xxx x x x x x - 6 a n B t x x - xx xx xx - 6, 41 Lotus arabicus Linn. A xxx x - - t - - - 1, 15

Phaseolus vulgaris Linn. B, A x t ------8,33,34,39,4~

Pisum sativum Linn. B, A x t -- - x - x x protoc. Hydrangeaceae Bydransea macroph~la B, A xx x - xx xx t - protoc. ,b r. - Euphorbiaceae Phyllanthus angustifol- A - x x x x x - - protoc. -ius Swartz Geraniaceae-- Pelargonium peltatum Ait • A x x t - - - - - Diapensiaceae Diapensia lapponica Linn • A t - :x - x - xx xx 7, 18 ·~ Table 4 (eontinued)

Plant speeies Treat- POB Gen. Van. Syr. ~-c. Caf. Fer. Sin Other Unidentified ment ac ids compounds *

Ericaceae salie. Gaultheria proeumbens A x x xx - x x - - o-Îlro. 1,7,11,21,23,37,47 n .. Linn. B - x x - xx xx x x 'B'a c. 1, 11, 37 Erica carnea Linn. A x x x x x x - - .9_-pyro. Erica vagans Linn. A x x x x x x - - .9_-pyro.(t) Rhododendron dahuricum A x x x - x x - - .9_-pyro. Linn. R. mucronulatum Turcz. A x x x - x x - - .9_-pyro. PentaEterl!ium se!Eens A - x x - x xx - - .9_-pyro.(t) 1,7,13,25,31 Kletzsch. .9_-pyro.(t) ArctostaEhllos ~-~ B, A t x x - xx x x x salic.(t) 12 Spreng.

Primulaceae Primula acaulis Hill B; A x x x x x x - - c

P. veris~Linn. B, A x x xx x x x -- d

Apocynaceae Vinca minor Linn. A x x x - x x -- .9_-pyro. v. rosea Linn. A - x x x x x -- .9_-pyro. ~ Table 4 (continued)

1' Plant species l'reat- POB Gen. Van. Syr. p-C. Caf. Fer. Sin. Other Unidentified 1 ment - ac ids compounds *

Solanaceae Solanum esculentum Nees A x x x - t x - - 1, 7 Bignoniaceae Catalpa campii(fruits) A lOx 5x 6x - x xxx 5x - lt lt B 5x xx xx x 7x 6x lOx lOx 40

Labiatae Mentba piperita Linn. B, A - x x x x x - x Salvia sp1endens Ker- A x x xx xx x x x - Ge.w1. B t t x x xx xx xx - 41 " n Rubiaceae Rubia tinctorum Linn. A t x x - x x - - 1, 7, 24 Campanulaceae Caœpanula vidalii H.C. A x x x - x x - - 7, 14, 38, 51 wats.

Monocotyledone

Gramineae Triticum vulgare var. B, A x - t - x - x x ph1or(4x) 2, 9 Thatcher

Palmae P~oenix~acty11fera Linnj. A - x xxx 6x x - - x 4, 7

LI'\ LI'\

0 0

Dracaena Dracaena

Tradescantia Tradescantia

Tulipa Tulipa

Gloriosa Gloriosa

A, A,

b, b,

~-pyro., ~-pyro.,

Fer.,-ferÛlic Fer.,-ferÛlic

x, x,

POB, POB,

a, a,

d, d,

c, c,

Plant Plant

Commelinaceae Commelinaceae

Liliaceae Liliaceae

" "

acid acid

a a

salicylic, salicylic,

indicates indicates

p-hydOxybenzoic p-hydOxybenzoic

number number

gesneriana gesneriana

hydrolysia hydrolysia

species species

superba superba

latifolia latifolia

2-pyrocatechuic 2-pyrocatechuic

r r

.. ..

~f ~f

of of

virginiana virginiana

presence presence

2-hydroxy-5-methoxybenzoic, 2-hydroxy-5-methoxybenzoic,

2-hydroxy-4-methoxybenzoic, 2-hydroxy-4-methoxybenzoic,

; ;

fluorescent fluorescent

unidentified unidentified

Sin., Sin.,

Linn. Linn.

Regel. Regel.

; ;

(~, (~,

. .

sinapic sinapic

- ;

Gen., Gen.,

-, -,

:rreat-

A A

ment ment

B B

A A

A), A),

; ;

A A

1 1

A A

phlor., phlor.,

compounda compounda

compounds compounds

indicates indicates

plant plant

gentisic gentisic

; ;

POB POB

xx xx

salie., salie.,

x x

phloretic phloretic

material material

gave gave

Gen. Gen.

was was

absence absence

; ;

x x

Van., Van.,

salicylic salicylic

3,5-dihydroxybenzoic. 3,5-dihydroxybenzoic.

found found

2-hydroxy-5-methaxybenzoic, 2-hydroxy-5-methaxybenzoic,

no no

Van. Van.

xx xx

hydrolyzed hydrolyzed

x x x x

x x

t t

colour colour

; ;

vanillic vanillic

;/ ;/

2,4-0B, 2,4-0B,

t, t,

in in

*, *,

Table Table

Syr. Syr.

xx xx

; ;

x x

t t

trace trace

roots roots

2-coum., 2-coum.,

see see

reaction. reaction.

2,4-dihydroxybenzoic. 2,4-dihydroxybenzoic.

first first

; ;

p-C. p-C.

Table Table

4 4

x x

t t

x x x x x x

Byr., Byr.,

amounts; amounts;

and and

(continued) (continued)

2-coumaric 2-coumaric

with with

flawers flawers

Caf. Caf.

5 5

x x

syringic syringic

and and

alkali alkali

Fer. Fer.

Flgure Flgure

xx xx

(see (see

3,5-dibydroxybenzoic. 3,5-dibydroxybenzoic.

; ;

then then

Sin. Sin.

protoc:, protoc:,

p-C., p-C.,

Table Table

xx xx

x x

8 8

; ;

2,4-0B 2,4-0B

with with

Phl.or. Phl.or.

ac ac

Other Other

p-coumaric; p-coumaric;

B, B,

6). 6).

ids ids

base base

protocatechuic protocatechuic

acid. acid.

hydrolysis; hydrolysis;

Unidentified Unidentified

Caf., Caf.,

1 1

1, 1,

1 1

compoundl compoundl

16, 16,

36 36

caffeic caffeic

; ;

32, 32,

42 42 ; ; 51

In this survey, many unidentified phenolic compounds were detected on paper chromatograms. Almost every plant investigated contained one or more of these compounds. Table 5 includes a list of the ultraviolet fluorescence and colour reactions of the unidentified compounds which were present in amounts detectable in UV-light or by colour reaction.

Their location on a benzene-formate paper chromatogram is shown in

Figure 8. To avoid listing the plant speoiea twice, the unidentified compounds in each plant are referred to in Table 4 by the same numbere given in Table 5 and Figure 8. A brief account is given below of some of the unidentified compounds found to occur in more than one species.

Compound No. 1 : This compound showed a white fluorescnce in UV-light but gave no colour reaction with diazotized ~-nitroaniline.

It was present in acid hydrolyzates of ethanolic extracts of seven species.

Two of these species belong to the Selaginellaceae and two to the Ericaceae.

Compound No. 7 : A nonfluorescent compound which gave blue colour with diazotized E-nitroaniline, changing to light purple after a few daye.

It was detected after acid hydrolysis and in greater amounts after alkaline hydrolysis of the plant extracts. It was found to occur in 18 species

comprising two mosses,a lycopod-1 two gymnosperme, nine dicotyledone and four monocotyledone. It was present in relatively large amounts in Tulipa and Diapensttspp.

Compound No. 19 : Showed strog yellow fluorescence in UV-light but gave no colour reaction. It bad a light green colour in visible light. It 52

Table 5

Ultraviolet Fluorescence and Colour Reactions of Some Unidentified Phenolio Compounds in Plants

Compound * Fluorescence in UV-light Colour wit~ diazotized ~- nitroaniline / NaOH

1 White None 2 None Brown 3 Bright blue None 4 Strong yellow None 5 Bright green Blue 6 None Light purple 7 None Blue turne light purple 8 White blue Pink 9 None Dark blue 10 Dark blue None 11 None Yellow 12 None Dark brown 13 None Poor colour, decolorises 14 Strong yellow None 15 None Olive green, fades 16 Violet intensifies with None exposure to ammonia fumes 17 Dark blue Light brown 18 None Purple 19 Strong yellow None 20 Strong yellow Light brown, fades 21 Bright yellow None 22 Dark blue None 23 Dark blue Bright blue, fades 24 None Blue 25 None Pur!>le-blue 26 Dark blue Light blue 27 None Po or colour, decolorises ' 28 Blue Blue-green turns blue then violet 29 Dark blue Bright blue, fades 30 None Purple 53

Table 5 (continued)

Compound * Fluorescence in UV-light Colour with diazotized E- nitroaniline / NaOH

31 None Light blue, fades 32 White orange None 33 None Pink 34 Absorba Yellow 35 Dark blue None 36 Blue Purple 37 None Blue 38 Yellow None 39 Absorba Yellow 40 Dark blue None 41 Blue Purple 42 White orange None 43 None Blue 44 None Pink 45 None Purple 46 White orange None 47 Orange None 48 Ab sorbs Yellow 49 None Blue 50 None Purple-violet 51 Orange None

* Numbering of compounds corresponds to that in Table 4 (last column) and Figure 8. , 54

0,1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

0 (D

0 àJ

0 :..,.

0 èn

8 0 G Ut

0 ~

9 1\)

( 10:1: 200) Sodium formate: formic a cid: water

Figure 8 : Location of sorne unidentified phenolic compounds in plants on a two-directional paper chromatogram • . Numbering of spots corresponds to that in Tables 4 and 5. 55

was found to occur in two moss species and in Lycopodium sp.

Compound No. 41 : A blue fluorescent compound in UV-light which

gave purple colour with diazotized alkaline E-nitroaniline. It showed

two spots on paper chromatograms in the formate solvant. The spot with

lower Rf value bad more intense UV-fluorescen~e and gave more intense

colour reaction than that with the higher Rf value. These are possibly 1

~and trans isomers, characteristic of cinnamic acid derivatives. The

tact that this compound was only detected after alkaline hydrolysis of

the leaf extracts of Salvia and Acacia spp. (see Table 4) suggests its

presence as an ester in these plants. Ester~~ication of cinnamic acid

derivatives is of common occurrence in plante. Binee monomethyl ethers

of dihydroxy acids have higher Rf values in organic solvents than

their corresponding dihydroxy acids, compound No. 41 may possibly be

a hydroxy-methoxy-cinnamic acid ether than ferulic and iso-ferulic acide.

This compound bas also been reported from Salvia splendens by Dr . A.C.

Neish, Prarie Regional Laboratory, N.R.C., Saskatoon (personal communicat-

ion to Dr. Towers). 56

III. Identification of the Phenolic Acids of Hydrangea macrophylla Ser.

On chromatographing the phenolic acid fraction of the leaves of

sydrangea1 a number of unidentified compounds appeared on paper chroma•

tograms (see Table 4). None of these compounds matched any of the

common phenolic acide. This prompted an investigation of the phenolic

constituents of the different organe of the plant.

Fresh flowers, leaves and roots were extracted separately with boiling 85 ~ ethanol. The ethanolic extracts, a~ter being reduced in volume, were each divided into three aliquote. One aliquot was worked up without hydrolysis and contained the free phenolic acids. The remain

ing two aliquote were hydrolyzed1 one with acid and the ether with alkali.

These two fractions contained the phenolic acide which were bound as glycosides and est~rs 1 respectively. Two-directional paper chromatography of the final ether extracts containing the phenolic acids was carried out using benzene and formate solvents.

Table 6 includes a list of the identified and unidentified phenolic

constituents of flowers 1 leaves and roots. Their fluorescence in UV-light1 and colour reactions with diazotized p-nitroaniline oversprayed with 5 ~

NaOH are also given. The location of these compounds on a benzene-formate chromatogram is shown in Figure 9.

Compounds No. 10 and No. 15 were identified as coumarin deriv atives, and compound No. 16 as the phenyl 1!2-coumarin, hydrangenol.

Their isolation and identification are described ~n the section on "Phenolic

Lactones" (see page 77). r U"\

Table 6

Phenolic Constituents of Flovers, Leaves and Roots of Rydrangea macrophylla

-· -- --- __; __ __-- Fluorescence Colour reaction Flowers Leaves Roots .. ~o. UV-1ight vith diazotized CaQPOUDd in F A B F A B F A B map p-ni troanilinE/ NaOH ------·-·- - ·-··------·

1 Unknown White-blue Blue-purple x x xx ------2 Caffeic Bright blue Blue,fades x x xx x xx xxx - x xx 3 Unknown Absorbs Salmon - - x ------~ x li- Protocetechuic None Light brown - x - - -- - 5 Unknown Blue Purple-red ------x - xx 6 Gentisic Blue Light blue x - - - x ~ -- - 7 Unknovn None Pink ------x - x 8 Hydrangeic 1 Blue Light brown t x xx t t x x xx 9 P-CoUID&ric Dark blue Blue-grey t x xxx xx X:i - x x 1 0 Umbelliferone Strong blue Blue,turns brown t t t !1 xx x xx 4x xx 1 1 POB None Pink t x - t xx - - - - xx l 2 Unknown None Purple - 1 ------l 3 Unknown None Deep purple - i - - x xx x x xxx ·xxx t x xx l 4. Unkno~ None Brown ------1 5 Unknovn Blue Blue turns brown t t ~ t xx x xx xxx x l 6 Hydrangenol Blue I.ight brown x 4x - x x - xxx 6x 6x l 7 Ferulic Blue ü!.ght blue t t xx - t x x - x 1 8 Melilotic None Purple - .xx - - t - t - x 1 --- F, extracts vorked up without hydrolysis ; A, acid hydrolyzed extracts ; B, base hydrolyzed extracts. x, indicates presence ; -, indicates absence J t, indicates trace amounts. POB, p-hydroxybenzoic 58

Figure 9 Location of the major phenolic constituents of flowers, leaves and roots of ~· macrophylla, on a two-directional paper chromatogram. Numbering of spots corresponds to that given in Table 6 .

..... en ~ ~ •aJ ....::Il •::Il •.. Q •n -n Q n a.

~ ..Q 8 ..,• 8

0 0

----=-,.~ (10:1: 200) Sodium formate: formic a cid: water Compound No. 8 : This compound (see Table 6 and Figure 9) sbowed a blue fluorescence in UV-ligbt whicb brigbtened afrter exposure to ammonia fumes. Its Rf values i~ benzene and formate solvents were 0.40 and 0.15, respectively, with streaking in benzene. A small amount wa~ isolated from the flowers of !· macropbylla. The etbanolic extracts of tlowers were taken to dryness in a rotary film ~aporator, and the dry residue dissolved in bot water and filtered. The filtrate was first bydrolyzed witb alkali and then with acid. The acid hydrolyzate was continuously extracted with ether for 24 hours. The ether extract was shaken with 2 ~ sodium carbonate solution, after whicb, the alkaline extract was acid~fied and re-extraated aontinuously with ether. The final ether ext~act was applied to several sheets of Wbatman

No. ' cbromatography grade paper in the fom of 'bands. The sbeets were chromatographed using the benzene solvent for 10 hours in order to g.et rid of the fast moving compounds. This gave a good separation of the compound of interest. The bands corresponding to compound No. 8 were marked on paper chromatograms by tbeir fluorescence in UV-light, eut out and eluted witb 85 ~ ethanol. The eluate was chromatographed by banding on Wbatman No. 3 paper using the formate solvent for 12 bours. Compound No. 8 was eluted from the paper witb 85 ~ ethanol, the extract reduced in volume and extracted continuously with ether after acidification witb 2 N HCl. The compound was purified by re-chromatograpby in tbe butanol solvent. It was crystallized

0 from hot 10 ~ ethanol to yellowish white needles wbich melted at 176-177 C

(uncorrected). Identification of Compound No. 8

The absorption maxima determined in 85 ~ ethanol were found to be at ~ 217 and 272 mp and at 240 and 288/when two drops of 2N NaOH were added (sèe 1 Figure 10). The identification of this compound was based on the following

observations

l. Its chromatographie behaviour was similar to that of hydrangenol; ( a

phenyl iso-coumarin with two free hydroxyl groups), in the formate solvent.

It had the same UV-fluorescence and gave the same colour reaction with

diazotized ~-nitroaniline oversprayed with NàOH,as those of hydrangenol.

2. Aecording to Karrer (1968), a compound known as "hydrangeie acid" was

reported to have been isolated by Asahina and Aaario (1930 a) from the

flowers of uydrangea opuloides. It was synthesized by the same authors

(1930 b) and its melting point reported to be l80°C

3. Compound No. 8 was hyd~olyzed with 20 ~ sodium hydroxide aolution

under reflux on a steam bath for tbree hours, and the alkaline solution

was allowed to evaporate on the steam bath to dry~ess. After cooling, the

residue was acidified and continuously extracted with ether. Chromatography

of the ether extract in benzene solvent followed by formate, gave p-hydroxy-

benzoic acid, 2,6-cresotic acid and small amounts of hydrangenol ( see

Figure 11). p-ijydraxybenzoic and 2,6-cresotic acide were reported to be

the fusion producta of hydrangenol (Asahina and Miyake, 1916}. The detailed

procedure of hydrolysis and the identification of the hydrolysis products are described in the section on "Phenolic Lactones" (see degradation of

hydrangenol, page 105) . 61 .

2 40 1·0

0·8

'217

-(/) c: A B Q) "'0

0 u 0·4

-a. 288 0

220 240 260 280 300 320 Wave length (m)J) Figure 10 Absorption spectrum of a compound believed to be hydrangeic acid in (A) 85~ ethanol, and (B) after the addition of two drops of 2N NaOH. OH

COOH OH Alkaline Hydrolysis ~ CH = CH

COOH OH + CH3 COOH

2,6- Cresotic p- Hydroxybenzoic •

Figure ll : Products of the alkaline hydrolysis of a compound believed to be hydrangeic acid.

0\ 1\) From the above mentioned evidence, compound No. 8 was believed to be hydrangeic acid. However,the possibility of it being the acid form of

the lactone, bydrangenol1 is not excluded. The determination of the nuclear magnetic resonance spectrum (NMR) of this compound would be helpful.

Attempts at preparing hydrangeic acid .trom hydrangenol were unsuccess ful. Asahina and Miyake (19i6) reported tbat boiling hydrangenol with dilute KOH solution gave hydrangeic acid after acidification. This treatment was tried using different concentrations of alkali for various periode ot time. In most cases, large amounts of hydrangenol were obtained, possibly by lactonization of the resulting bydrangeic acid during acidification.

Acidification of the reaction mixture at room temperature using 2N HCl did not give better resulta. In other cases, alkaline hydrolysis for longer periode of time, resulted in bydrolysis of ·bydrangenol with only traces of bydrangeic acid detected on paper chromatograms.

Compound No. 8 was predominant in the flowers and roote, witb traces in the leaves. It was detected after acid hydrolysis and in larger amounts after alkaline hydrolysis of flower and root extracts (see Table 6). This suggests that it may be present largely combined as an ester. However, it is p~i~le that compound No. 8 occurs as a glycoside which is alkali sensitive. No attempts were made to isolate the combined form of this compound.

Among the other unidentified phenolic constituents detected on paper cbromatograms of Hydrangea extracts, the following compounds were most conspicuous {see Table 6 and Figure 9 ).

Compounq No. 1 : Showed a very bright blue fluorescence in UV-light

and gave a deep blue colour with diazotized alkaline p-nitroaniline which

turned purple-blue on standing for few hours. It was detected after acid

hydrolysis and in larger amounts aft,r alkaline hydrolysis of the flower

extracts.

Compound No. 13 : A nonfluorescent compound which gave a deep purple colour with diazotized ~-nitroaniline/NaOR. It was found to occur

free and after acid and alkaline hydrolysis of leaf and root extracts. It was detected in larger amounts after alkaline hydrolyeis especially in the

roots. Its absorption maximum determined in 85 ~ ethanol was found to be

at 223 mf1 and did not shift after the addition of two drops of 2 N NaOR.

Compound No. 14 A nonfluorescent compound which gave a brown colour with diazotized ~-nitroaniline/NaOR. It was detected after acid hydrolysis and in larger amounts after alkaline hydrolysis of root extracts. 65

IV. Isolation and Identification of Three HYdroxybenzoic Acide from

Gaultheria procumbens and Primula acaulis l. 2,3-Dihydroxybenzoic (2-Pyrocatechuic) Acid

One and a quarter kilograms fresh leaves and stems of Oe.ultberia procumbens L. were extracted witb 85 ~ ethanol under reflux on a steam bath for 12 bours with three changes of ethanol. The combined ethanolic extracts {10 litera) were reduced in volume in a Cyclone evaporator to a tbick syrup. This extract was bydrolyzed with 2N HCl on a steam bath for two bours wben a reddisb brown residue {49 g) came down. The acid bydrolyzate was filtered and continuously extracted witb ether and the ether extract sbaken witb 2 ~ sodium carbonate solution. The alkaline extract, after being acidified, was re-extracted continuously with ether.

The final ether extract containing phenolic acids was applied to several sheets of Wbatman No. 3 paper in the form of bands, and chromatographed using the benzene solvent for 10 hours. Bands corresponding to 2-pyro catecbuic acid were marked on paper chromatograms by tbeir fluorescence in

UV-ligbt and eut out. They were eluted with 85 ~ ethanol and chromato

grapbed in the formate solvent. The bands of this acid were eluted1 reduced in volume and extracted continuously with ether after acidification to remove the sodium formate . The acid was purified by re-chromatography using the benzene solvent. It was crystallized from benzene-etber and recrystallized from bot water to colourless needles {35 mg). It decomposed 66

at 204-205° C (uncorrected). Mixing with an authentic sample of 2,3-di hydroxybenzoic acid did not lower the melting point. The infra-red spectrum of the isolated acid and authentic 2-pyrocatechuic acid were identical.

Co-chromatography of the isolated acid with an authentic sample using benzene and formate solvents gave only one spot.

2. 2,5-Dihydroxybenzoic (Gentisic) Acid

Gentisic acid was isolated from the same pl~nt material as used for 2-pyrocatechuic acid. The fluorescent bands corresponding to gentisic acid were treated in exactly the same way as described above. It was crystallized from benzene-ether and recry.stallized from hot water to colourless needles which decomposed at 200-201°0 (uncorrected). The melting point was not lowered when mixed with an ~uthentic sample of

2,5-dihydroxybenzoic acid. Co-chromatography witb an authentic sample of gentisic acid gave one spot.

3· 2-Hydroxy-5-methoxybenzoic acid

This compound was isolated from the roots of Primula acaulis Hill.

While both salicylic and 2-hydroxy~5-methoxybenzoic acids were present in the shoots of f· acaulis, only the latter was found in the roots. The method of isolation by chromatography, of purification and crystallization were those used for the isolation of 2-pyrocatechuic and gentisic acids.

Hydrolysis of the root extracts was carried out first with alkali since the compound was present was present as its methyl ester. This was followed by an acid hydrolysis. 2-Hydroxy-5-methoxybenzoic acid melted 0 at 144-145 C (uncorrected). Mixing the isolated acid with a sample

synthesized from gentisic acid by the method of Graebe and Martz (1905)

did not lower the melting point. The isolated and synthesized acide

bebaved similarly on chromatography.

14 v. Biosynthesis of C6-c1 Acide fro~ C -Labelled Compounds in

Higber Plants

1. Formation from Benzoic Acid 14 One ml of a solution of carboxyl-labelled benzoic acid-C (0.1 mg witb an activity of 2 pc) was administered to each ct a set of 10 leaf disks of Gaultheria Erocumbens L., Primula acaulis Hill and leaf cuttings of Triticum vulgare var. Thatcher, for 24 hours in the light.

The methods of administration of the radioactive compounds and extraction of the plant material ~re described above. Since salicylic acid occurs in Gaultheria as a methyl ester, the plant extracts were first hydrolyzed with alkali then with acid. The final radioactive extracts containing phenoli.c acids were chromatographed two-directionally in benzene followed by formate, and theo radioautograpbed. The identity of gentisic and 2-pyrocatechuic acids was confirmed by isolation from the leaves of

Gaultberia procumbens (see page 65). The distribution of radioactivity in phenolic acide was determined by direct counting on paper chromatograms and is given in Table 7. A radioautograph of the chromatographed pbenolic 14 acide of Gaultheria leaf disks administered benzoic acid-C is shown in

Figure 12. ~

Table 7

Distribution of Radioactivity in Phenolic Acids of ~af Material of Different Plants Administered Carboxyl-Labelled Benzoic Acid-C-14 for 24 Hours in Light

Exper- Plant Date of Parcentage distribution of radioactivity iment species experiment a b c d e Benz. POB Salie. 2,3-di- 2,5-di- Unidelit- OB OB ified

Gaultheria procumbens 1 Mature leaves July 1 59 80 6 5 2 5 - 2 Old leaves Jan. / 6o 62 0- 0 0 Trace 36 3 Young leaves May 1 6o 36 17 27 13 3 - Primula acaulis lt. Mature leaves July 1 59 8o 4 6 3 3 - 5 Young leaves April/ 60 6o 8 17 7 2 - Triticum vulgare

6 Mature leaves Jan. / 6o 7 0 16 0 Trace 73

a, benzoic b, ~-hydroxybenzoic c, salicylic d, 2,3-dihydroxybenzoic e, 2,5-dihydroxybenzoic 0'1 \0 p- hydroxybenzoic

salie ylie o-pyrocatechuic gentisic

Figure 12 Print of a radioautograph of the chromatographed phenolic acid fraction of Gaultheria leaf disks, administered benzoic acid-c 14 70

2. Formation from Salicylic Acid 14 One ml of a solution of carboxyl-labelled salicylic acid-e (containing 0.06 mg with an activity of 2 re> was administered to each of a set of

10 leaf disks of Gaultheria procumbens, Rhododendron dahuricum, Primula acaulis, and leaf cuttings of Lotus arabicus and Triticum vulgare for various periode of time in the light. The plant material was extracted with boiling 85 ~ ethanol and the extracts were worked up for phenolic acids in the usual manner. The final ether extracts were chromatographed, radioautographed, and the activity determined by direct counting of spots on paper chromatograms. Identity of the radioactive phenolic acids was confirmed by spraying the chromatograms with chromogenic sprays. The distribution of radioactivity of phenolic acide is given in Table 8. A radioautograph of the chromatographed phenolic acide of Gaultheria leaf disks, administered 14 salicylic acid-e 1 is shown in Figure 13. 14 14 ;. Administration of ~-Pyrocatechuic Acid-e and Gentisic Acid-e to Gaultheria Leaf Disks

Two young shoots of Gaultheria, carrying 10 leaves, were fed two ml 14 of carboxyl-labelled salicylic acid-e (contain,ing 0.6 mg with an activity of 20 pc) in a small beaker for 24 hours in light. The leaves were then detached and extracted with boiling 85 ~ ethanol and the extracts worked up for the separation of ~-pyrocatechuic and gentisic acids as described above (see page 65). No attempt was made to crystallize either of the radioactive acids. Each acid was dissolved in two ml distilled water and used for the feeding experimenta. The radioactive acids were chromatograph- .--! t'-

Table 8

Distribution of Radioactivity in Pbenolic Acids of Leaf Material of Different Plants Administered Carboxyl-Labelled Salicylic Acid-C-14 for Various Periode of Time in Light

Exper- Plant Date of Feeding Percentage distribution of activity iment species e:xperiment period

(Brs.) Salicylic ~-P.Jro-Gentisic 2-Bydro:xy- catechuic 5-metho:xy- benzoic

1 Gaultheria procumbens April/59 i 85 7 3 - " " " " 2 80 11 3 - " " " Il 12 71 16 3 - Il 24 43 25 12 " n n - 2 n n July/59 24 50 30 3 -

3 n " May /6o 24 30 53 2 - 4 Primula acaulis * July/59 24 75 1 7 15 5 Triticum vul.gare April/59 24 69 4 24 - 6 Lotus arabicus June/59 24 70 14 14 -

7 Rhododendron dahuricum n n 24 80 9 6 -

* Salicylic and 2-hydroxy~5-methoxybenzoicacids were separated by re-chromatography using 1~BCl

(\J (\J

t--

salie salie

Figure Figure

ylie ylie

13 13

unknown unknown

fraction fraction

: :

Print Print

of of

a a

Gaultheria Gaultheria

radioautograph radioautograph

leaf leaf

disks, disks,

of of

the the

o-pyrocatechu

administered administered

chromatographed chromatographed

unknown unknown

salicylic salicylic

phenolic phenolic

gentisic gentisic

acid-C

acid acid

1 1 4 4 73

ically pure, and each acid gave one spot on radioautography. Triplicata to samples were assayed for radioactivity by plating onjaluminium planchettes 14 and the activities were found to be 576,000 cpm for 2-pyrocatechuic-C 14 and 180,000 cpm for gentisic-C • 14 14 Two ml of each of 2-pyrocatechuic acid-C and gentisic acid-C was administered to a set of 10 le$f disks of Gaultheria procumb~ns in a small Petri dish for 24 hours in light. The disks were then washed with water and extracted with boiling 85 ~ ethanol. The ethanolic extracts were worked up for the chromatography of phenolic acide in the usual manner, and r their chromatograms were radioautographed. Examination of the radioautor graphe showed tbat tbere was no conversion of either 2-pyrocatechuic or gentisic acide to otber derivatives.

4. Formation from Some Phenylpropanoid Compounds 14 14 L-Phenylalanine-U-C 1 cinnamic acid ring and-3-C and L-tyrosine- 14 U-C were. adm1nistered to sets of 10 leaf disks of Gaultheria procumbens and Primula acaulis for 24 bours in the light. The amounts administered and the distribution of activity of phenolic acids are given in Table 9.

Radioautographs of the chromatographed phenolic acid fraction of Primula 14 leaf disks,administered phenylalanine-U-C 1 and of the chromatographed 14 total ethanol extract of Gaultheria leaf disks, administered tyrosine-U-C . , are shown in Figures 14 and 15 , respectively. ...::t r--

Table 9 Distribution of Activity in Phenolic Acids of Gaultheria and Primula Leaf Disks Administered C-14 Labelled Phenylpropanoid Compounds for 24 Hours in Light

Compound Plant species Percentage distribution of radioactivity administered p-Coum- o-Coum- Sali- 2_-Pyro J Gent- aric aric cylic catechufc isic

14 L-Phenylalanine-U-C Gaultheria procumqens 32 0 0 1 1 ( 0.08 mg & 2.5 pc ) 14 L-Phenylalanine-U-C Primula acaulis 2 12 30 20 10 ( 0.08 mg & 2.5 pc )

Cin~~micacid-ring & 3-C (o.o4 mg & Gaultheria procumbens 35 0 Trace Trace Trace 0.032 pc ) 14 L-Tyrosine-U-C * Trace 0 0 Trace Trace ( 0.03 mg & 2 pc) " " 14 L-Tyros ine-U-C Trace 0 0 Trace Trace n (o.o4 mg & 2 pc) "

L____ ------~------~ ~- ----

* About 70 ~ of the activity of the ethanol soluble fraction was found in sugars and malic acid (see Figure 15). 75

Figure 14 : Print of a radioautograph of the chromatographed fraction phenolic acid of Primula leaf disks, administered 1 L-phenylalanine-u-c 4. * Identified as o-coumaric acid. 76

c ~ ~ ~ ro~ CL

~ c ~ ~ c -~ ~ ~ 0 ~ 0 ~ ~ 0 ~ ~ ro ~

3 0 n

Figure 15 Print of a radioautograph of the chromatographed total ethanol soluble fraction of Gaultheria leaf disks, administered L-tyrosine-u-c14. 77

B. PHENOLIC LACTONES

I. The Identification of the Phenolic Lactones of aydrangea macrophylla

1. Isolation and Identification of Hydrangenol Glucoside

Four hundred and forty grams of fresh flowers of aydrangea macrophylla Ser. was extracted with boiling 85 ~ ethanol under reflux for 12 hours with two changes of ethanol. The combined ethanolic extracts

(4.5 litera) were reduced in volume by distillation under reduced pressure in a Cyclone evaporator to a thick syrup, which was taken to dryness under a jet of filtered air at room temperature. The brown residue (18 g) was dissolved in 300 ml of hot water, filtered hot through a bed of Celite and continuously extracted with ethyl acetate for 48 hours. The ethyl acetete extract was reduced in volume and kept under refrigeration during which a gummy precipitate formed. The precipitate was filtered off and dissolved in absolute ethanol. The ethanolic extract was filtered, evaporated to dryness, and the residue dissolved in water, acidified with 2N HCl and continuously extracted with ether for 16 hours. The aqueous extract ·was reduced in volume by means of an air jet and kept in a refrigerator where the glucoside precipitated as a pale cream coloured powder (m.p. 185-186°C)

The precipitate ~s purified by re-extraction of an aqueous solution with ethyl acetate. The solvent was removed and the residue was recrystallized from hot water to give 261 mg of white fine needles which melted at 190-

0 0 0 190.5 C uncorrected (reported 172 C, Ueno 1937 a ; 192 C, Ueno 1937 b). 78

Hydrangenol glucoside bas a faint blue fluorescence in ultraviolet light which becomes stronger after exposure to ammonia fumes. Its absorption maximum, shown in Figurel6,was determined in 85 ~ ethanol and found to be at 315 mp. It shifted to 357 mp when two drops of 2N NaOB were added. Elemental analysis of the glucoside gave C, 60.23, 59.95 ; H, 5.50,

5.35 ; calculated C, 60.5 ; B, 5.27 • The isolated hydrangenol glucoside was chromatographically pure. On two-directional paper chromatograms using phenol followed by butanol solvents, it bad Rf va~ues of 0.60 and

0.47, respectively. It reacted with 1~ ferric chloride solution giving a light red-brown colour. It gave a pink colour with diazotized alkaline

E-nitroaniline and a light orange-yellow colour with diazotized sulfanilic acid. Bydrlolysis of hydrangenol glucoside by refluxing with 2N BCl on a steam bath for an hour, gave glucose and hydrangenol. The identity of the products was proved by co-chromatography with authentic samples. The structural formula of hydrangenol glucoside is given in Figure 2.

2. Isolation and Identification of Bydrangenol (see compound No. 16, Table 6 & Figure 9)

One and one half kilograms of air-dried flowers of ~· macrophylla was extracted with 85 ~ ethanol in large Soxhlets for 24 hours with two changes of ethanol. The combined ethanolic extracts (10 litera) were reduced in volume by distillation under reduced pressure to a thick syrup, which was suspended in water and hydrolyzed with 2N BCl for an hour on a steam bath. The acid hydrolyzate was extracted with ether in a continuous 0\ t'-

J57 1.0 31S'

0.8

~ 0.6 ·--(f) c <1> 1 / / \A \ B "0 0.4- 0 0 / " - -a. 0 / 0.2

290 310 330 350 370 390 Wave le n gth (m,AJ)

Figure 16 : Absorption spectrum of bydrangenol glucoside} isolated from the flowers of Hydrangea macrophyllaJ in (A) 85~ethanol} and (B) after the addition of two drops of ~NNaOH. 80

extractor for 24 hours. To remove the pbenolic acide, the ether extract was wasbed witb 50 ml aliquote of 2 ~ NaOH in a separatory tunnel until the alkaline extracts were colourless. Binee bydrangenol was s~ightly soluble in dilute carbonate solution, the alkaline extracts were re-

( extracted with ether. The combined ether extracts were washed twice witb water and tben extracted witb 10 ~ NaOH in a separatory tunnel. On acidification of the alkaline extract witb concentrated HCl, bydrangenol precipitated as a grey-green solid. The precipitate was allowed to stand at room temperature, filtered off and then dried ~ vacuo. It was recrystallized twice from hot 20 ~ ethanol to give 2 g of white needles

0 0 which melted at 180-181 C uncorrected (reported 181-182 C, Asabina a~

Miyake, 1916).

Hydrangenol bas a strong blue fluorescence in ultraviolet light whicb does not change on eXPosure to ammonia fumes. The absorption

maximum, determined in 85 ~ ethanol was found to be at 315.5 mf1 and 358 ~ after the addition of two drops of 2N NaOH (see Figure 17). Its infra- r.ed spectrum was deternd.ned and is shown in Figure 18. Elemental analysis of hydrangenol gave C, 70 .69, 70.93 ; H, 4.89, 4.79 ; calculated C, 70.3 ;

H, 4.6 .

The diacetyl derivative of hydrangenol was prepared by allowing a mixture of hydrangenol (0.15 g), acetic anhydride (0.7 ml) and dry pyridine

(1.5 ml) to stand under nitrogen in a refr igerator for 24 hours. The mixture was poured into ice-water and the crystalline compound was filtered 0)r-l

1.0

35"8

o.a '31S.S

>. 0.6 - -{/) c: Q) "0 - 0.4 L \A \ B 0 0 // ·- -0.. 0 0..2

290 310 330 350 370 390 W a v e 1 en gt h (m)J)

Figure 17 : Absorption spectrum of hydrangenolJ isolated from the flowers of Hydrangea rnacrophylla ; in (A) 85~ethanol, and (B) after the addition of two drops of ?N NaOH. · ··- ----··------(\J ,.;..='-• ··.;( ...... co WAVE NU!IoiBERSIN CM·' WAVE NUM1ERS IN CM-' 1. il SPECnOf'HOTO~R ------:~ .1s-- NoCI-- PRISM ~ Ji. 'i ï 4L ·','· . . ... Î Î T 100 00 NO. "ll'i .. INDU DAlt 1 ~1 "1\ ,, \ioo - .o'!o"" SAIM'LE /_ h 10 1•10 ·ï tsf\11-I-*'- ... v 1 1 7 -7 g ~ J'\ / ;! h ~ "" l60 v' 60i ~ ~\ FROt.t 'E - 1!ol.- ~ " ~ ·~-.... 1\ A ) ~ ~ -CEU'.o>l ...... _, CMS. .! llfl ! !z40 1 40~ REf.CEU -CMS . rl li? J 1 ~ v ~ t.OG. "'~ CH8< --t.IG. { . ( 1 \7 '0 SOl Y. 20 û

VQI. c.cl ~ c.J "/, u~ Hydrong j!nol F.S. SOI.J GA~MM c 0 0 IAIID-ATOI\IUC, INC. 2 4 5 6 7 1 • 10 Il 12 Il 14 15 " CAMUIDGf. MASS .• U.S.A. WAVE LENGTH IN MICRONS WAVE LENGTH IN lloiiCRONS tOI.S4-.S 1 HYDRANGENOL WAVE NUMBERS IN CM·' 1. R. SPEC.:; OPHOTOMEltR WAVE NU!IoiBERSIN CM·' 1 sooo 4000 lOOO 2500 2000 1500 1400 1300 1200 1100 1000 900 1100 700 N.Cl PRIS!Iol ' ' . 6~5 100 J ï 1 ' ' NO. 100 DATE INDEX ~~~ 1 1 SAMPI.E !r"',f/ ,. 80 1 h ~ 110

· Compound No. ~ l \ ~ z (i \r ~ J (\ If ( ~( ~ 15. !::60 1 r 60!:: :1 1 ~ ~ f--__/ a < .. -- ·- - . z FROt.t ~ - ~ lA 1 r / ( .... SAMP. CELL' MM CMS. .... 40 z . ·~ REF. CELL MM CMS. r 1/ v ti ·--- CHEMj MG. MG. ~"' 1/ s v ~1 Ir" ln 1\ SOLY. • 20 '2 JVI . -- ...... vOL.' c.e.! "!. cc] "/, -- -- -· conip4 furi

Figure 18 Infra-red spectra of hydrangenol, isolated from the flowers, and of compound No. 15, isolated from t he roots of Hydrangea macrophyl la. 8~

and dried in vacuo. It was recrystallized from boiling absolute ethanol

0 to give glistening white plates (yield 91~) which melted at 182-18~ C uncorrected (reported 181-182°0, Asahina and Miyake, 1916). Elemental analysis of diacetyl bydrangenol gave C, 67.~6, 67.62 ; H, 5.04, 5.27 ; calculated C, 67.06 ; H, 4.7 •

Rydrangenol, chromatographed in phenol followed by butanol solvents, bad Rf values _of 0.77 and 0.80, respectively. On benzene-formate cbromatograms, it bad Rf values of 0.70 and 0.15, respectively, with spreading of the spot in the formate solvent. It gave a violet colour with 1 ~ ferric chloride solution, a pink colour with diazotized alkaline

~-nitroaniline and an orange colour with diazotized sulfanilic acid.

Occurrence of Hydrangenol in Different Organe of ~· macrophylla

By means of two-directional paper cbromatograpby, it was shown that hydrangenol occurs in flowers,leaves and roots of the plant (see

Table 6). Rydrangenol was also detected on paper chromatograms of unbydrolyzed root extracts and to a lesser extent in flower and leaf extracts.

The possibility of auto-hydrolysis was avoided by direct extraction of the fresh plant material with boiling ethanol. In one determination, root material was obtained from young rooted cuttings. They were rinsed witb tap water and plunged directly into boiling 85 ~ ethanol witbout previous treatment. Tbeir unhydrolyzed extracts gave detectable amounts of bydrangenol on paper chromatograms. From quantitative determination of free hydrangenol, by isolation from unbydrolyzed root extracts, it was 84

fbund to be l mg/g fresh weight. These resulta suggested that hydrangenol was present both in the free state and bound as the glucoside. No attempt was made to determine quantitatively the amounts of free bydrangenol in either flowers or leaves.

The fact that hydrangenol was detected after alkaline hydrolysis of root extracts, suggested one of two possibilities: (a) that hydrangenol was present bound as an ester in the roots, or (b) that hydrangenol glucoside was susceptible to alkaline hydrolysis. A samll amount of the glucoside was hydrolyzed with 2N NaOH at room temperature under nitrogen.

The alkaline extract was acidified with 2N HCl and continuously extracted witb ether. Chromatography of the ether extract in benzene and formate solvents gave hydrangenol in amounts sufficient for detection by fluoresc ence and by colour reaction with phenolic sprays. This showed tbat

bydrangenol glucoside was susceptible to alkaline hydrolysis. However 1 the possibility tbat hydrangenol migbt occur as an ester is not excluded.

From a number of analyses, the average yield of hydrangenol, as determined by isolation after acid hydrolysis per 100 grams fresh weight, was 100 mg for flowers, 40 mg for leaves and 250 mg for roots. The hydrangenol isolated from the roots was relatively pure and required only one recrystallization to give a product that bad a correct melting point and was chromatographi cally pure . The hydrangenol content was determined after acid hydrolysis of mature flower extracts of three different coloured varieties of H. macropbylla and was found to be 60 mg for the · pink 85

variety, 120 mg for the white variety and 200 mg for the blue variety,

all calculated on lOO g fresh weight basie. Each value was the average

of three replicate determinations.

Leaves, root and bark of three other species of aydrangea ( H.

bretschneideri Dipp., H. arborescens Linn. and H. cinerea Small ) ~- were examined, but none was found to contain hydrangenol.

3. Isolation and Identification of Umbelliferone (see compound No. 10, Table 6 & Figure 9)

Eight hundred grams of aydrangea roots were washed with running tap

water to remove the soil particles, blotted and extracted in a Waring

blendor using boiling 85 ~ ethanol. The etbanolic extract was filtered

and the residue was further extracted with ethanol under reflux for 12

hours on a steam bath with three changes of ethanol. The combined ethan-

olic extracts were reduced in volume in a Cyclone evaporator and further

reduced to a thick syrup under an air jet. This syrup was dissolved in

300 ml hot water and filtered with suction through a bed of Celite. The

filtrate was hydrolyzed with 2N HCl under reflux on a steam bath for two hours. The acid bydrolyzate was continuously extracted with ether for 12 hours. At this stage, it was believed that umbelliferone was a phenolic acid due to the fact that it was detected on paper chromatograms after carbonate extraction of the acid hyrolyzed extracts. Therefore, the ether extract was shaken with four lots, 50 ml each, of 2 ~ sodium carbonate solution in a separatory funnel, and the combined alkaline extracts were 86

re-extracted continuously with ether for 12 hours after acidification.

The ether was removed by evaporation under reduced pressure and the residue

was dissolved in 85 ~ ethanol. This ethanolic extract was applied to

several sheets of Whatman No. 3 paper in the form of bands and chroma-

tographed using the ben~ene solvent. The bands corresponding to umbelli-

ferone, marked by their strong blue fluorescence in UV-light, were eut

out the paper chromatograms, eluted with 85 ~ ethanol and chromatographed

using the formate solvant. Since eluates from the formate chromatograms

contained appreciable amounts of sodium formate, umbelliferone was purified

by continuous extraction with ether. The ether extract was reduced in

volume and transferred to a semi-micro sublimation apparatus and sublimed

0 twice to white needles (25 mg) which melted at 225-226 C uncorrected

(reported 223-224°C, Mangini and Passerini, 1957). The melting point was

not lowered when the product was mixed with an authentic sample. On two-

directional chromatography using two pairs of solvants, benzene-formate

and butanol-formate, a mixture of the isolated umbelliferone and an authentic sample gave one spot in each pair of solvents. However, this

is not the proper procedure for the isolation of umbelliferone, since it was discovered that the bulk of it was left in the first ether extract of

the acid hydrolyzate. It is recommended that the first ether extract should be chromatographed directly without carbonate extraction, since umbelliferone is slightly soluble in dilute carbonate solution. 87

Umbelliferone bad a strong blue fluorescence in UV-light which did not change in acid condition but brightened after exposure to ammonia fumes. It bad Rf values of 0.25 and 0.55 in benzene and formate solvents respectively. Spraying with diazotized alkaline E-nitroaniline, gave a dark blue colour which cbanged to dark brown and then faded to light brown. Diazotized sulfanilic acid gave a pink colour which turned deep red on standing for a few minutes. No colour reaction was produced after spraying with 1 ~ ferric chloride solution. The absorption maximum, shown in Figure 19, was found to be at 325 mf in 85 ~ ethanol and shifted to

377 IDf when two drops of 2N NaOH were added (reported 325 and 371-373 mp in

95 ~ ethanol and in 5 ~ ethanolic NaOH, respectively, Mangini and

Passerini, 1957).

Umbelliferone (7-hydroxycoumarin) is rather common in higher plants.

In a survey of the distribution of coumarin derivatives in 120 species representing 36 plant families, Reppel (1954) reported its occurrence in

16 species, two of which were Bydrangea ;arborescens L. and!!· paniculata

Sieb. Umbelliferone bas also been given the names "skimmetin" and

"hydrangin" (Karrer, 1958). It was isolated and purified by sublimation by Sommer (1859) cf. Karrer (1958) from Feru1a ga1baniflua Boisa. et Buhre.

Its glucoside, skimmin, was iso1ated from Skimmia japonica Thunb.(Eykmann,

1884 cf. Karrer, 1958). More recently, Nakahara (1956) isolated the diglucoside of umbelliferone, neohydrangin, from the inner bark of H. paniculata. On acid hydrolysis, neohydrangin gave one molecule of

~ ~

"0 "0

·-

0 0

>. >.

c c

Q) Q)

(/) (/)

CL CL

0 0

Figure Figure

0·6 0·6

0·5 0·5

0·3 0·3

0 ·4 ·4 0

0-2 0-2

o.J o.J

19 19

: :

Absorp

28.0 28.0

in in

(A) (A)

ion ion

85~ 85~

spectrum spectrum

300 300

ethanol, ethanol,

Wave Wave

of of

320 320

and and

umbelliferone, umbelliferone,

325 325

(B) (B)

length length

after after

340 340

the the

(m)J) (m)J)

isolated isolated

360 360

addition addition

from from

377 377

of of

'380 '380

the the

two two

roots roots

drops drops

400 400

of of

Hydrangea Hydrangea

?N ?N

NaOH. NaOH.

420 420 macrophylla, macrophylla, umbelliferone and two molecules of D-glucose.

Umbelliferone was found to occur in all the organs of ~· macrophylla.

Large amounts were detected on paper chromatograms of acid bydrolyzed root extracts. Smaller amounts were detected in the free state and after alkaline hydrolysis, especially in leaves and roots. This suggested that umbelliferone was most likely present as a glycoside.

4. Isolation and Identification of Compound No. 15 (see Table 6 and Figure 9)

Two kilograms of wasbed roots were cbopped to small pieces and extracted witb boiling 85 ~ ethanol in a Waring blendor. The residue was filtered off and re-extracted with ethanol under reflux for 12 bours on a steam bath. The combined ethanolic extracts (10 litera} were reduced to 500 ml by distillation under reduced pressure and tben blown down to dryness under a jet of filtered air. The residue was dissolved in 300 ml hot water and filtered througb a bed of Celite using suction. The filtrate was bydrolyzed witb 2N NaOH at room temperature for six hours in a nitrogen atmosphere, acidified, and furtber hydrolyzed using 2N

HCl under reflux on a steam bath for an hour. The acid hydrolyzate was continuously extracted with ether for 24 bours. Since this compound was slightly soluble in dilute carbonate solution, therefore the ether extract was taken to dryness, and the residue dissolved in 85 ~ ethanol and directly applied to severa! sheets of Whatman No. 3 paper and chroma- tograpbed using the benzene solvent. The bands of compound No. 15 were marked by fluorescence in UV-light before and after exposure to ammonia

fumes. Tbe bands were eut out of the paper, eluted, concentrated and

rechromatographed using the fQrmate solvent. Compound No. 15 was

eluted and purified from the sodium formate by continuous extraction with re ether after acidification. The ether extract was)cbromatographed7 using

the benzene solvent and co'-Pound No. 15 was eluted again with 85 ~

ethanol, reduced in volume and transferred to a semi-micro sublimator

and sublimed twice to fine white needles (90 mg) which melted at 157-

0 157.5 C (uncorrected). Tb+s compound bad a pleasant smell of coumarin.

Elemental analysis ot two dupli~ate aamples gave C, 63.09 1 62.72, ·, '

The infra-red spectrum, shown in Figure 18, was consistent with

a molecule containing the following functional groups: (a) a lactone,

(b) a carbonyl group, (c) a bydroxyl group, (d) a metbyl group and (e)

an ether linkage. The two latter functional groups were possibly present

as a methoxyl substitution. The possibility of the presence of a

functional carboxyl group was excluded by the fact that when this compound was chromatographed using the formate solvent, it gave no colour reaction when sprayed with an acid-base indicator, e.g. brome-cresol green. Its solutions, as determined with a Beckman model G pH meter, were found to be nearly neutral.

The absorption spectrum, determined in 85 ~ ethanol, showed a maximum at 325 ~~ shifting to 377 mF after the addition of two drops of 2N NaOH ) 91

Optical density

0 0 1\)

1\) Ot 0

Ul 0 - 0

().1 N =E 0 I.>J 0 1\J < Vj C1>

().1 ~ ('1) 0 ::J \0

~ - ().1

~ 0 0

J:. 1\) 0

Figure 20: Absorption spectrum of compound No. 15 (see Table 6 and Figure 9), isolated from the roots of Hydrangea macrophylla, in (A) 85~ ethanol, and (B) after the addition of two drops of 2N NaOH. 92

and is shown in Figure 20. These maxima are exactly the same as tbose

found for umbelliferone (see Figure 19). Mangini and Passerini (1957)

reported that substituted coumarin derivatives give absorption maxima

in this range.

Compound No. 15 had a blue fluorescence in UV-light wbich cbang~'d

to a greenisb yellow colour after exposure to ammonia fumes. It also acquired a pale yellow colour in visible light after exposure to ammonia.

It gave a dark blue colour with diazotized ~-nitroaniline oversprayed with

NaOR, wbich cbanged to dull brown then faded to a light brown colour.

With diazotized sulfanilic acid, it showed a pink colour which turned

red on standing for a few minutes. It gave no colour with l ~ ferric chloride solution. These colour reactions were similar to those given by umbelliferone, but they were visible when the compound was present in relatively large amounts.

The acetyl derivative w~s prepared by allowing a mixture of compound

No . 15 (10 mg), aceti c anhydride (0.1 ml) and dry pyridine (1 ml) to stand under nitrogen in a refrigerator for 18 bours. The mixture was poured into ice-water and the crystalline compound was filtered and dried in va cuo. It me1ted at 134-134.5°C (uncorrected). It was recrystallized from hot 50 ~ ethanol to colourless needles (yield 50 ~) which gave the same melting point.

Ther ef ore, from the accumulated evidence cited above, this compound was considered to be a coumarin derivative with hydroxyl and methoxyl 93

substitutions. From elemental analysis, the formula c10 Ha 04 was suggested. The fact tbat it bad the same UV-absorption maxima and gave the same colour reactions with diazotized sprays as those of umbellifer one, suggests a relationship to the latter. However, the only mono hydroxy-monomethoxy coumarin derivative known to occur naturally in plants is scopoletin (6-methoxy-7-hydroxycoumarin).

Binee the characteristics of compound No. 15 did not match any of the common coumarin derivatives, a comparison of its chromatographie bebaviour with seme coumarin derivatives was found necessary. Tbeir fluorescence in UV-light and colour reactions with diazotized phenolic sprays are given in Table 10. Their location on a benzene-formate paper chromatogram is shown in Figure 21. Binee an authentic sample of

daphnetin (7,8-dihydrox~coumarin) was not available for chromatography1 its Rf values were compared with those of esculetin (6,7-dibydroxy coumarin) in 12 different organic and aqueous solvent systems reported by Reppel (1957).

Although the number of coumarin derivatives available for this comparison was insufficien't to permit any definite conclusions, the following interesting observations can be drawn from Figure 21.

(a) Dihydroxycoumarins are slower moving tban 7-hydroxy:coumarin

(umbelliferone) in benzene solvent and much slower tban coumarin

itself.

(b) Hydroxy-methoxy coumarine are faster moving tban dihydroxycoumarins; ~ Table 10

UV-Fluorescence and Colour Reactions of Some Coumarin Derivatives

Fluorescence in UV-light Colour reactions

Coumarin Neutral After exposure Diazotiz:? p-nitro- Diazotized sulf- 1 to NH3 fumes aniline -NaoH anilic acid 1NaOH

a 1. Coumarin None Yellow Light buff Orange-yellow

2. 3,4-Dihydro- None Absorba Purple Yellow

3- 7-Hydroxy- Blue, changes Pink, turns 1 (umbelliferone) Deep blue Strong blue to brown deep red

4. 6,7-Dihydroxy- Yellowish Greenish yellow (esculetin) Blue pink * Khaki turns brown ' 5. 6-Metho:xy-7 -hydroxy- Blue turns Green turns (scopoletin) Deep blue Blue-green * light violet ** light brown**

6. Compound No. 15 Blue Greenish Blue turns Pink turns 1 yellow * brown** deep red **

1. 7,8-Dihydro:xy- None(?)or Bright (daphnetin) faint yellow yellow *" Brown - (??)

a, Yellow colour after spraying with lN NaOH. *, Yellow in visible light. **, No colour reaction when present in small amounts. (?), See Goodwin and Kavanagh (1950). (??), See Swain (1953). 95

0 n -Cil 0 n o.

----, 1 7 ' 1 8 ...... __ _,/ r ) (10:1•200) Sodium formate:formic acld~water

Figur e 21 : Location of sorne coumarin de ~ ivatives on a two - di rect ional paper chromatogram. 96

therefore, scopoletin bas a higher Rf value tban tbat of esculetin in the

benzene solvent.

more soluble than coumarin in the benzene solvent.

These chromatographie characteristics of coumarine in this solvent pair are similar to those of cinnamic acid derivatives.

Alkaline HYdrolysis of Compound No. 15

A small quantity of this compound was bydrolyzed with 10 ~ NaOH under reflux for 10 minutes on a steam bath. The alkaline solution was allowed to cool to 1°C, then carefully acidified with 2N HCl and extracted continuously with ether for six hours. Two-directional. chromatography of the ether extract gave two major compounds and a small arnount of the unreacted starting material

(compound No. 15). Beth compounds bad the sarne Rf value of 0.3 in benzene

&elvent and 0.50 and 0.75 in formate. The spot with a lower Rf value bad a bright blue fluorescence in UV-light which becarne brigbter after exposure t~ arnmonia furnes. Tbat with a higher Rf value, bad the same fluorescence characteristics of compound No. 15 (see Table 10). These spots were possibly

trans and cis isomers of a substituted cinnamic acid1 respectively.

On spraying with diazotized R-nitroaniline, the trans isomer gave an intense salmon colour, which changed to dark brown-green when oversprayed with 5 ~ NaOH. The colour changed to a deep olive green on standing for a

few minutes. With diazotized alkaline sulfanilic acid1 it gave an intense purple colour which turned violet-blue on standing. This compound will be 97

referred to as compound No. 15 - A.

The presence of a functional carboxy1 group on the trans isomer

(compound No. 15-A) was confirmed by spraying a formate chromatogram with bromo-cresol green indicator when this compound gave a yellow colour on a light blue background whereas the cis isomer gave a negative reaction.

The absorption maximum of compound No. 15-A, determined in 85 ~ ethanol was found to be at ~19-~22 mf which shifted to ~70 mf after the addition of two drops of 2N NaOH. This compound was therefore the free acid of that compound No. 15. The fac1/it has two hydrox~and a methoxyl substitutions makes it slightly faster moving than caffeic acid and slower moving than ferulic or sinapic acide in the benzene solvent. Isomerization in aqueous solvents is a common feature of hydroxycinnamic acid derivatives and lactonization is characteristic of their 2-hydroxyderivatives.

A small quantity of compound No. 15-A was reduced using palladium catalyst (5 ~ palladium on charcoal) in a slightly alkaline medium at 40 lh./in.2 • The hydrogenolysis product was acidified and continuously extracted with ether. Chromatography of the ether extract using benzene and formate solvents gave a nonfluorescent compound with Rf values of

0.68 and 0.80, respectively. It gave an orange-pink colour with diazotized sulfanilic acid and a brown-green colour with diazotized alkaline p-nitroaniline, which changed to olive green on standing for few minutes. This compound,referred to as compound No. 15 - B, is considered to be the di- hydroderivative of compound No. 15-A. The fact that dihydrocinnamic acid derivatives are nonfluorescent in UV-light and f~ster moving th~n their dehydroderivatives in the benzene solvent supports this tentative identifi-

cation. The chromatographie behaviour 1 UV-fluorescence and absorption

maxima of compounds No. 15 1 15-A, and 15-B are summarized in Table 11 1 and their suggested formulae are included. Compound No. 15 was detected in large amounts on paper chromatograms

of the acid-hydrolyzed root extracts 1 in smaller amounts in leaf extracts 1 and only in traces in flower extracts. The tact that small amounts were

found to occur in the free state and after alkaline hydrolysis (see Table 6) 1 suggests that this coumarin derivative is likely present as a glycoside.

No attempts were made to determine its quantities on paper chromatograms of the different parts of the plant due to the tact that the small amounts isolated from the plant material were insufficiept for the preparation of a calibration curve. 99

Table 11

Ultraviolet Characteristics and Chromatographie Behaviour of Compound No. 15 and Derivatives

Behaviour Compound No. 15 Compound No. 15-A Compound No. 15-B

Fluorescence in UV-light Neutral Blue Blue None Alkaline Green-yellow Yellow-green None

Absorption maximum(mjl) in 85~ et hanol 325 319-322 210 plus 2N NaOH 377 370 212

Rf values in Benzene solvent 0.30 0.68 Formate solvent 0.50 0.80

Colour reactions Diazotized p-nitro- Blue turns Brown-green Brown-green turns aniline/NaOH brown turns olive green olive green

Diazotized sulf- Pink turns Purple turns Orange-pink anilic acid / deep red · violet-blue changes to Na OH pink

Suggested formulae

0 COOH

No. 15 No. 15-A No. 15-B

COOH lOO

II. A Study of the Biosynthesis of Phenolic Lactones from c14-Labelled Compounds in aydrangea macrophylla

Whereas the c6-c3 (Bi) moiety of the flavonoids has been shown to be formed as a unit from C6-c3 precursors, such as phenylalanine or cinnamic acid, the formation of that of iso-flavones is different.

Flav one Iso-flavone

Phenyl ~-coumarin

Geissman, Mason and Rowe (1959), in studying, the biosynthesis of the iso- flavone~ biochanin A, showed that the loss of the terminal carbon of the

C6-c precursor is most probable, and that carbon 2 of the heterocyclic 3 ring is derived from a one-carbon compound. On the other hand, Grisebach and Doerr (1959) suggested that a phenyl migration from carbon 2 to carbon 3 occurs in the course of biogenesis of formononetin (7-hydroxy-4'-methoxy- 101

14 !!2-flavone). Therefore, the activity of phenylalanine-l-e would be

expected in carbon 4 of the iso-flavone• There is general agreement that

ring A of both flavones and iso-flavones is formed from acetate according

to the Birch and Donovan hypothesis (1953).

An inspection of the structural formula of phenyl iso-coumarine,

suggests that the origin of the carbon skeleton is different from that

of the flavones or iso-flavones. Therefore, a study of the biogenesis of

the phenyl !!2-coumarin1 hydrangenol1 was considered to be particularly

interesting.

l. Biosynthesis of Hydrangenol

The following experimenta are concerned with the biosynthesis of 14 hydrangenol from C -labelled compounds. The methode of preparation of

the plant material1 administration of radioactive compounds and isolation of hydrangenol are described above. Since the roots of ~· macrophylla

contain appreciable amounts of hydrangenol, and since the flowers were not available at the time, roots were used as experimental material. 14 14 Experiment l .0 Sodium cinnamate-2-C , L-phenylalanine-U-C 14 14 L-tyrosine-U-C and sodium acetate-2-C were administered to 4-month- old rooted cuttings, for 24 hours in the light. At the end of this period the root systems were eut off, wasbed with water and extracted with boiling 85 ~ ethanol. The ethanolic extracts were worked up for the isolation of hydrangenol. The isolated hydrangenol was recrystallized and its melting point determined. Yields ranged from 0.8-1.7 mg/ g fresh weight. An 102

accurately weighed amount was dissolved in 95~ ethanol and triplicate samples were plated on aluminium planchettes and assayed for radioactivity.

The resulta are given in Table 12. 14 Experiment 2 : The low specifie activities of hydrangenol-C ,

isolated from the first experiment, could have been the result of the transport of the administered radioactive compounds to the shoots, or the slight degree of synthesis of hydrangenol. In order to avoid losses due to transport, a second experiment was carried out using small samples of 14 4-month-old root segments. In this experiment, sodium cinnamate-2-C , 14 14 14 L-phenylalanine-U-C , sodium acetate-l-e and sodium acetate-2-C were administered to lt gram-samples of root segments in small Petri dishes for 24 hours in the light. The methode of isolation and activity determination of hydrangenol were the same as in the first experiment. The amounts of hydrangenol recovered from these samples ranged from 2 to 5 mg/g fresh weight. The resulta are given in Table 13.

Remarks on Tables 12 and 13

(a) The resulta in both experimenta are expressed in terms of dilution.

This was calculated by dividing the specifie activity of the labelled compound

(pc/mmole) by the specifie activity of hydrangenol 0uc/mmole).

(b) In calculating these dilutions, no corrections were made for the amounts of hydrangenol isolated. (c) The dilution values of the acetate feedings in both experimenta were calculated on the basie of three molecules of acetate being incorporated into one molecule of hydrangenol.

1("\ 1("\

0 0

r-4 r-4

Sodium Sodium

L-Pbenylalanine-U-C L-Pbenylalanine-U-C

L-Tyrosine-U-C L-Tyrosine-U-C

Syntbesis Syntbesis

Compound Compound

acetate-2-C acetate-2-C

cinnamate-2-C cinnamate-2-C

of of

14 14

HYdrangenol HYdrangenol

14 14

! !

i i

1 1

i i

Compound Compound

0.0062 0.0062

0.0009 0.0009

0.0005 0.0005

Amount Amount

from from

(mmole) (mmole)

C C

14 14

administered administered

-Labelled -Labelled

(p.c/mmole) (p.c/mmole)

13,200 13,200

12,500 12,500

Specifie Specifie

activity activity

1,150 1,150

960 960

Table Table

Compounds Compounds

12 12

0.130 0.130

o.o43 o.o43

0.057 0.057

. 0

Amount Amount

isolated isolated

(mmole) (mmole)

058 058

in in

HYdrangenol HYdrangenol

aydrangea aydrangea

activity activity

Specifie Specifie

(p.c/mmole) (p.c/mmole)

0.85 0.85

0.72 0.72

1.14 1.14

0.04 0.04

macropbylla macropbylla

Dilution Dilution

320,500 320,500

11,600 11,600

4,050 4,050

1,330 1,330

Rooted Rooted

cuttings cuttings

i i

1 1

i i

1 1 1 1 -g r-1

Table 13

Synthesis of HYdrangenol from C14 -Labelled Compounds in aydrangea Root Segments

Compound administered HYdrangenol Compound Amount Specifie Amount Specifie Dilution (mmole) activity isolated activity (pc/mmole) (mmole) (pc/mmole)

14 Sodium cinnamate-2-C 0.0071 989 0.0099 2.10 470

14 L-Phenylalanine-U-C 0.0007 10,500 0.015 3.81 2,760

14 Sodium acetate-l-e 0.0020 1,550 0.029 0.73 6,390 14 Sodium acetate-2-C 0.0012 1,150 0.011 0.56 6,150 105

2. Degradation of Hydrangenol and Identification of the Degradation Products

A method for the chemical degradation of hydrangenol was essential

for the study of its biogenesis. Considerable difficulties were encountered

due to {a) the low yield of the degradation products and (b) the low 14 specifie activities of hydrangenol-C isolated from the tracer experimenta.

Hydrangenol was fused with sodium hydroxide pellets in a stainless

steel bomb with a steel cap for 20 minutes using a Wood's metal bath main-

tained at 300-3500 C. The hydrolyzed mass was allowed to cool, dissolved in

a small amount of water and carefully acidified with concentrated HCl.

The acidified hydrolyzate was continuously extracted with ether for six

hours. The ether extract was shaken with 2 ~ sodium carbonate solution,

and the alkaline solution was re-extracted continuously with ether after

acidification. Two-directional chromatography of the final ether extract

in benzene followed by formate solvents, gave two main compounds, the Rf

values and colour reactions of which are given in Table 14.

Table 14

Rf Values and Colour Reactions of the Degradation Products of Eydrangenol

Fluorescence values Colour reaction Compound R! in UV-light BenzenE' Formate Diazotized p-nitro'- Diazotized sulfan iline/-riaOH anille acid/NaOH

A None 0.28 0.75 Pink Yellow

B Blue 0.83 o.Bo Deep purple Orange-yellow 106

Compound A was identified as E-hydroxybenzoic acid by co-chromate-

graphy with an authentic sample. Compound B was isolated in a small quantity

by chromatographie separation following the above-mentioned procedure. It was recrystallized twice from hot water and gave colourless needles which decomposed at 167-168°C uncorrected (reported melting point of 2,6-cresotic

0 acid 168 C, Hodgman, 1947). Elemental analysis gave C, 62.73,62.84 ; 1 H, 5.63, 5.56 ; calculated for 2,6-cresotic (2-hydroxy-6-methylbenzoic) acid C, 63.1 ; H, 5.3 • Both R-hydroxybenzoic and 2,6-cresotic (3-hydroxy-

2-toluic) acide were report.ed ta be the fusion products of hydrangenol

(Asahina and Miyake, 1916).

Since the yield of·both ~-hydroxybenzoic and 2,6-cresotic acide was relatively small (5 ~ of theoretical), further attempts were made to obtain better yields. Alkali fusion was carried out for shorter periods, i.e. 5,

10, and 15 minutes, following the same procedure mentioned above. None of these treatments gave better yields. Examination of paper chromatograms of the first ether extract (before carbonate extraction), showed at least

10 unidentified phenolic compounds which gave different colours with phenolic sprays. Seme of these compounds were thought to be phenols, intermediates in the degradation of hydrangenol. Co-chromatography of these extracts with authentic samples of phenol, catechol, resorcinol and orcinol, showed that these compounds were not degradation products.

Attempts to degrade hydrangenol using 1 ~ potassium permanganate at room temperature resulted in relatively large amounts of unchanged hydrangenol 107

and a number of unidentified compounds. Refluxing bydrangenol witb differ ent concentrations of bydrogen peroxide in the presence or absence of aqueous sodium hydroxide on tbe steam bath for various periode of time was tried but witbout success.

Sin~e degradation of bydrangenol involved opening of tbe lactone, removal of the elements of water and finally breaking the double bond as sbown in Figure 22, the use of alkaline hydrolysis for longer period was tried. Alkaline hydrolysis of hydrangenol was carried out using 20 ~ aqueous sodium bydroxide under reflux for three hours on a steam bath. At tbe end of that period, the condenser was removed, and the flask contain ing the alkaline solution was allowed to stand on the steam bath for six bours until the contents were reduced to a dry residue. The alkaline residue was allowed to cool, dissolved in a small volume of water, acid ified witb concentrated HCl and continuously extracted with ether. Chroma tography of the ether extract gave g-hydroxybenzoic acid, trace of a compound believed to be hydrangeic acid (see compound No. 8, Table 6),

2,6-cresotic acid and unchanged hydrangenol. The products of hydrolysis,

except for hydrangeic acid1 were almost equal in amounts as determined by visual comparison of the size of spots ·and the colour developed witb phenolic sprays. No otber intermediates were detected on paper chromatograms of the ether extracts.

Degradation of milligram-samples of hydrangenol, following the above mentioned procedure gave easily detectable quantities of both ~-hydroxy- co 0 .-f

+fl:?O ,. - H 0 OH 2 CH2 - CHOH OH ')

OH OH COOH COOH ~ 0 + COOH 8 OH CH CHfCH00H 3 2,6-Cresotic p-Hydroxybenzoic

Figure 22 : Steps of degradation of hydrangenol. 109

benzoic and 2,6-cresotic acids on paper chromatograms when 20 pl of the final ether extract (equivalent to 160 rs of hydrangenol) were chromatographed. 14 ;. Degradation of Hydrangenol-C 14 Unfortunately, hydrangenol-C isolated from the tracer experimenta reported in Tables 12 and 13 was degraded by fusion with solid sodium hydroxide and. the products of hydrolysis could not be detected by radioautography.

After many attempts, three samples of hydrangenol-C14 with appreciable radioactivity were obtained for degradation etudies. Degradation of these samples was carried out using 20 ~ aqueous sodium hydroxide as described above. The final ether extracts were chromatographed one-directionally using the benzene solvent. Standard hydrangenol, f-hydroxybenzoic and

2,6-cresotic acids were used as reference markers. The paper chromatograms were radioautographed and their radioautographs are shawn in Figure 23. 14 The source and specifie activities of the degraded hydrangenol-C and the moiety found to be radioactive are given in Table 15.

0 0

r-1 r-1

administered administered

Sodium Sodium

L-Phenylal~~ine L-Phenylal~~ine

. .

Compound Compound

-3-C -3-C

-2-C -2-C

-2-cl4 -2-cl4

acetate-

cin~tmat~ cin~tmat~

Wbole Wbole

Young Young

Root Root

Plant Plant

* *

segments segments

root root

infloresc-

The The

material material

en en

to to

system! system!

ce ce

specifie specifie

Results Results

the the

1 1

low low

weight weight

Fresh Fresh

12.4 12.4

12.2 12.2

activities activities

2.0 2.0

(g) (g)

of of

yield yield

Degradation Degradation

obtained obtained

1 1

Amount Amount

14.7 14.7

12.0 12.0

(mg) (mg)

2.8 2.8

of of

the the

Table Table

of of

isolated isolated

on on

1 1

0.057 0.057

degradation degradation

0.047 0.047

0.011 0.011

(mmole) (mmole)

Hydrangenol-C Hydrangenol-C

·-·--

hydrolysis. hydrolysis.

Hydrangenol Hydrangenol

15 15

!_Specifie !_Specifie

products products

~cpmfmg) ~cpmfmg)

14 14

700 700

400 400

500 500

Obtained Obtained

were were

activity activity

1 1

(cpmfmmole) (cpmfmmole)

181,000 181,000

106,000 106,000

130,000 130,000

in in

not not

Various Various

determined determined

Radioactive Radioactive

p-Hydroxybenzoic p-Hydroxybenzoic

2,6-Cresotic 2,6-Cresotic

Experimenta Experimenta

due due

acid acid

moiety moiety

acid acid

acid acid * * Figure 23 Prints of radioautographs of the chromatographed 14 products of degradation of hydrangenol-c ,

isolated from the following organs of Hydrangea.

LEFT: A root system administered sodium cinnamate-2-c14 .

MIDDLE :A young inflorescence administered L-phenylalanine-3-c14.

RIGHT : Root segments administered sodium acetate-2-c14. lll

0 c cu 0'1 c 0...... "0 > .!:.

u (5 Nc cu .0 > >< 0 \- "'0 > .!:. a.1 112

4. Biosynthesis of Other Phenolic Constituents of Hydrangea macrophylla From cl4_ Labelled Compounds

Carbon-14-labelled compounds were administered to sepal strips, intact inflorescences, leaf disks, eut shoots, root segments, and intact root systems, for 24 hour periode in the light. The methods of administration of the radioactive compounds, and preparation of the plant extracts were described before. The total ethanol extracts were evaporated to dryness, the residues dissolved in a small amount of water, hydrolyzed with alkali, then with acid, and worked up for the phenolic acids. No carbonate extraction was carried out, the first ether extract being applied directly to chromatography paper. This extract contained the phenols, phenolic acids and phenolic lactones. Extraction with dilute carbonate solution was omitted as it did not aeparate the acids from the lactones. i The extracts were chromatographed two-directionally using benzene and formate solvents. Developed chromatograms were radioautographed and radioactivity determination was carried out by direct counting on the paper,

The C14 -labelled compounds administered to different organs of

Hydrangea, and the distribution of activity in the ether soluble compounds are given in Table 16. Radioautographs of the cbromatograpbed ether extracts of three feeding experimenta are shown in Figures 24, 25, and

26 . These are marked with an asterisk in Table 16. rltc"\ rl

Table 16

Distribution of Radioactivity in Ether Soluble Fraction of Flowers, Leaves and Roots of aydrangea macrophylla Administered Carbon-14-Labelled Compounds for 24 Hours in Light

Amount Plan t 1 Percentage distribution of rad ioactivity Compound administered materia1 p-Coum- o-Coum Caff Fer- Hydrang Umbe1l- No.l5 ~aric -aric eic ul ic eno1 if erone

2 pc , 0.032 mg Infloresc~nce 14 0 0 0 0 .44 14 ll L-Pheny1alanine-U-C 2 pc , 0 . 0 32 mg Sepal strips 5 3 10 0 0 l 1 { 5 fc , o.o82 mg Root srstem * 2 4 0 1 2 20 30

14 Leaf disks 12 0 0 0 0 18 L-Phenylalanine-1-C { 4 pc , 0. 084 mg 65 2.5 pc, 0.052 mg Root segments 1 0 7 0 Trace 64 16 14 2 pc , 0.018 mg Inflorescence 0 0 0 2 L-Pheny1a1anine-3-C 5 30 5 1

3.05 0.5 mg Sepa1 strips 12 0 1 0 1 1 14 pc, * 3 Sodium cinnamate-2-C 6 .1 pc1 1 mg Shoot system * 30 3 10 5 0 25 18 Trace ~ 6 . 5 pc ' 1. o6mg Root system 11 3 4 1 54 17 14 Shikimic acid-U-C 1.8 pc, 10 mg Root system 0 0 0 0 4 25 67

* The radioautographs of these feeding experimenta are shown in Figures 24, 25 a nd 26 . 114

(b- c:""

(')

0 1 (') c:0 3 , 0 1 n (')"" g 3 0... o·

Figure 24: Print of a radioautograph of the chromatographed ether extract of Hydrangea roots, administered L-phenylalanine-U-c14 * Traces of activity did not show on print. n D ~ 2 ~ u ~ n' n ~ 2 3 3 Q c* n ~ 3 n cr ~ ~ ~ 0 ~ ~

Figure 25: Print of a radiociutograph of the chromatographed ether extract of Hydrangea sepal strips, administered sodium cinnamate-?-Cl4 * Traces of activity did not show on print . ll6

"'0 1 0 0 c 3 0 ~ . 0

Figure ?.6: Print of a r adioautograph of the chroma t ographed ether e x tr~ct of Hydr a ~gea shoots , administered sodium cinnamat e- ? -C1 . 117

14 5. Administration of Umbelliferone-C to Hydrangea Leaf Disks

F'our 'radioactive spots of umbelliferone were eut out of two-directional paper chromatograms obtained in the above experimenta and eluted with 85 ~ ethanol in the cold. The eluate was evaporated to dryness under an air jet. Since eluates of paper chromatograms, run in the formate solvent, contain appreciable quantities of sodium formate, it was necessary to separate this from umbelliferone. This was accomplished by continuous extraction with ether after slight acidification. The ether extract which contained the umbelliferone was evaporated and the residue dissolved in about ! ml of distilled water.

Four disks,obtained from young Hydrangea leave~were then floated on the radioactive umbelliferone in a small Petri dish, for 16 hours in the light. At the end of this period, the leaf disks were riosed with tap water and directly extracted with boiling 85 ~ ethanol. The ethanolic extract was worked up in the usual manner and the final ether extract was chromatographed using the benzene and formate solvents. A radioautograph showing the incorporation of the label of umbelliferone into ether phenolic compounds is given in Figure 27.

14 6. Administration of L-Phenylalanine -U-C to Hydrangea Leaf Disks

As both umbelliferone and compound No. 15 were readily formed from 14 C -labell ed phenylalanine, shikimic and cinnamic acids (s ee Table 16), and the label of umbelliferone was incorporated into compound No. 15 (see Figure

27), an investigation of the biosynthetic relationship of these two compounds 118 Î 0

Figure 27 Print of a radioautograph of the chromatographed ether extract of Hydrangea leaf d~sks, administered umbelliferone-c1 . 119

was carried out. 14 L-Phenylalanine-U-C (5 pc ) 0.08 mg) was administered to 48 young leaf disks in a Petri dish, 5 inch in diameter, in the light. Samples of eight leaf disks were taken after three, six and 12 hour periods, for the analysis of phenolic acids and phenolic lactones. The remaining 24 disks were thoroughly rinsed with tap water and transferred to another Petri dish where they were floated on 3 ml of water and allowed to photosynthesize in the light. Eight leaf disks were taken after three, six and 12 hour periods for extraction with boiling 85 ~ ethanol. The ethanolic extracts of each of the six samples were evaporated to dryness and the residues hydrolyzed first with alkali and then with acid. The acid hydrolyzates were continuously extracted with ether for six hours and the ether extracts evaporated to dryness. The residues were dissolved in 85 ~ ethanol to measured volume and duplicate samples were assayed for radioactivity using the combustion method.

Aliquots of these extracts were chromatographed two-directionally using the benzene and formate solvents. The developed chromatograms were radioautographed and the activity of the spots was determined by direct counting on paper. The total activities of the ether soluble fraction and the distribution of activity in phenolic compounds are given in Table 17.

~ ~

r-:1 r-:1

0 0

Total Total

~ ~

Activity Activity

~ ~

Activity Activity

activity activity

* *

** **

Activities Activities

in in

Absolute Absolute

Total Total

of of

umbelliferone(fc umbelliferone(fc

compound compound

umbelliferone umbelliferone

unidentified unidentified

compound compound

of of

extra extra

Phenylal~ine-u-c Phenylal~ine-u-c

activity activity

ether ether

activities activities

of of

ct ct

No. No.

a a

(pc)* (pc)*

Ether Ether

No. No.

0 0

15(tc) 15(tc)

determination determination

1~ 1~

' '

Administered Administered

Soluble Soluble

-·

obtained obtained

-· -·

0.001 0.001

0.032 0.032

3 3

1~ 1~

0.001 0.001

-· -·

3 3

Hours Hours

60 60

3 3

for for

Fraction Fraction

by by

Various Various

of of

phenylalanine-U-c

0.156 0.156

0.052 0.052

0.023 0.023

6 6

the the

combustion combustion

Table Table

Hours Hours

~1 ~1

27 27

15 15

of of

12 12

Periods Periods

Hydrangea Hydrangea

hour hour

17 17

12 12

. 0

0.111 0.111

0.0~9 0.0~9

of of

period period

285 285

Hours Hours

39 39

29 29

17 19 19 17

in in

duplicate duplicate

Light Light

Leaf Leaf

14 14

in in

Disks Disks

wate~ wate~

0.068 0.068 0.355 0.355

3 3

0.160 0.160

~5 ~5

Hours Hours

25 25

samples samples

Transferred Transferred

Administered Administered

was was

0.158 0.158

0.376 0.376

0.087 0.087

6 6

using using

unavailable. unavailable.

~2 ~2

Hours Hours

20 20

23 23

to to

the'Dynacon'. the'Dynacon'.

water water

12 12

26 26

30 30

21 21

Hours Hours

- ** ** 121

DISCUSSION

PHENOLIC ACIDS

I . Their Identification, by Chromatography, and Their Distribution in Plants

The need for a method of identifying the C6-c and C6-c phenolic 1 3 acids on two-directional paper chromatograms became evident in the initial stages in the investigation of the plant phenolic acids.

Of the solvent systems reported before (see Review of Methods), two were found particularly us~ful for two directional chromatograpby. These were (6:7:3) benzene:acetic acid:water (Griffiths, 1957) and(l0:1:200) sodium formate:formic acid:water {Smith, 1958). However, 2,4-dihydroxybenzoic and

2,6-dihydroxybenzoic, acids had exactly the same Rf values in both solvent systems. Similarly, salicylic acid and 2-hydroxy-5-methoxybenzoic acid were not well resolved. The advantages of the solvent system (4:1) iso-propanol:

7N aqueous ammonia (Grinstead, 1960) have been pointed out. But, the behav not iour of the cinnamic acid derivatives in this solvent wasjsatisfactory since it gave poor resolution of these acids with tendency of spots to tail.

It should be noticed that each of, p-coumaric, caffeic, ferulic, iso- ferulic and chlorogenic acids, gave two spots in the formate solvent due to

cis-trans isomerization. These are represented by the numbers 22 1 22' ;

24,24' ; 25 1 25' ; 26, 26' (see Figure 6) and 29, 29' (see Figure 7). The cis form of o-coumaric acid lactonizes spontaneously and is detected as isomerization coumarin. Cis-transjof hydroxycinnamic acids was first reported by Williams

(1955). 122

The methods of preparation of plant extracts have been reviewed. The procedure reported by McCalla and Neish (1~59 b) was adopted after slight modification, and was found applicable for the separation of phenolic acids which occur free, combined as glycosides, or as esters.

Since benzene and formate solvants were only used in the course of this study; no reference will be given to the phenolic acide, such as gallic, ellagic or chlorogenic acids which do not separate with the benzene solvent.

The following observations may be drawn from the investigation of the phenolic acids of a variety of plants (see Table 4).

1. 3,5-Dihydroxybenzoic acid which was detected in Primula acaulis and P. veris, and 2,4-dihydroxybenzoic acid which was found in Gloriosa superba have not previously been reported as naturally occurring phenolic acids in higher plants. Their identity was confirmed by co-chromatography with authentic samples in different solvent systems.

2. 2-Hydroxy-4-methoxybenzoic acid which occurred in P. acaulis and P. veris 1 and 2-hydroxy-5-methoxybenzoic acid which occurred in ~· acaulis, were not found in any of the other species investigated. The methyl ester of the former acid has been isolated from f· viscosa (Goris and Canal, 1945) and the methyl ester of the latter, from P. auricula and P. viscosa by the same authors (1936b). A study of the distribution of both these monomethyl ethers of dihydroxypenzoic acids should be extended to include other genera of the

Primulaceae and perhaps closely related families. 123

3. Another phenolic acid of restricted distribution, 2,3-dihydroxybenzoic

(2-pyrocatechuic), was found to occur in 7 species (of five genera) of

the Ericaceae. The detection of 2-pyrocatechuic acid in five genera of

the Ericaceae suggests that it may be of Wide spread occurrence in this

fam11y. It was a1so detected in two species of Vinca, y. minor and V.

rosea. The identity of 2-pyrocatechuic acid was confirmed by isolation

of a sma11 quantity from the 1eaves of Gaultheria procumbens. It bas been iso1ated from Popu1us ba1samifera (Goris and Canal, 1936 a), and from

the 1eaves of Vinca minor where it occurs as 3-~-D-g1ucosyloxy-2-hydroxybenzoic acid (King, Gi1ks and Partri4ge, 1955).

4. Whi1e 2-pyrocatechuic acid was detected in . 7 ericaceous species, the

c~osely re1ated 2-hydroxybenzoic (salicy1ic) acid was on1y found in

Gaultheria procumbens and a trace in Arctostaphylos uva-~. Salicylic acid was a1so found to occur in tycopodium, Podocarpus, Salix and Primula spp.

5. Among the common pheno1ic acids found to occur in the different species investigated, ~-hydroxybenzoic acid was recorded in 85 ~' gentisic acid in

95 ~' each of p-coumaric and vani111c acids in 90 ~' caffeic acid in 65 ~ and feru1ic acid in 50~ of the total number of species. Simi1ar resulta have been reported by ether workers studying the distribution of phenolic acids in vascular plants ( Bate-Smith, 195 4 a ; Griffiths, 1958 ; Koves" and

Varg~, 1959 ; Tomaszewski, 1960).

6. Whi1e gentisic acid was found to occur in almost every plant investigated, including moss and fern species, it was absent in gymnosperme. The only 124

hydroxybenzoic acid detected in gymnosperme was p-hydroxybenzoic acid.

Griffiths (1959), in a study of the distribution of gentisic acid in green

plants, by the use of paper chrornatography, reported its absence in all the

fern species investigated and in two cycads (Cycas and Zarnia). He also

reported that the occurrence of gentisic acid appears to be associated

with the woody habit of plants, and suggested that it is concerned in

lignification or sorne associated process. However, degradation etudies

of lignin (Brown and Neish, 1955 a, b) have not yielded any fragment with

the 2,5-hydroxylation pattern. Moreover, 21 5-dihydroxycinnarnic acid or any substituted derivative has not yet been isolated from plant material.

7. The widespread occurrence of vanillic acid has not been reported before. It was found in all groups of plants tested, i~cluding masses and

ferns. Tomaszewski, in a persona! communication to Dr. Towers 1 reported

the frequent occurrence of vanillic acid in plants.

8. The fact that p-coumaric, caffeic, ferulic and sinapic acids were detect-

-ed in larger amounts after a l kaline hydrolysis of plant extracts, suggests that these acids occur in the ester form. It was found that1 when alkaline hydrolysis of plant extracts was followed by acid hydrolysis,there was some destruction of bath ferulic and sinapic acids. Caffeic acid commonly occurs as an ester of quinic acid, known as chlorogenic acid (Fischer and Dangschat,

1932). Bate-Smith (1954 a) has suggested that ferulic and sinapic acids occur esterified, but the nature of the compound or compounds involved in ac id esterification is still unknown. ~-CoumaricJoccurs esterified with quinic 125

acid as ~-coumaryl quinic (Bradfield et al, 1952 ; Cartwright et al, 1955).

Recently, it has been reported that ~-coumaric acid occurs esterified with glucose in the berries of all cultivated potato varieties (Corner and

Harborne, 1960). However, small amounts of p-coqmaric and caffeic acids were detected after acid hydrolysis of plant extracts. This suggests that either both acids are also present as glycosides, or that their esterified forms are susceptible to acid hydrolysis.

9. Syringic acid was detected in 35 ~ of the species investigated, with more frequency in monocotyledons than in dicotyledons. It was absent in mosses, ferns and gymnosperms, although traces were detected in Selaginella and Podocarpus spp. Bate-Smith (1958) has suggested that the oxidation state of phenolic compounds found in the lesa woody plants differa from those in the woody ones in that the latter types are more highly oxidized.

Creighton, Gibbs and Hibbert (1944) reported that the lignine of angio- sperme and the Gnetales gave syringaldehyde and vanillin on oxidation with alkaline ni trobenzene, while the Coniferales and Ginkgo biloba as well as the ferns gave only vanillin on oxidation. Dr. Towers (1951) extended this st udy and obtained syri ngaldehyde from nitrobenzene oxidation of the lignine. of Sel aginella and speci es of Podocarpus . However, the ability of Selaginella and certai n gymnosperms to synthesize the syringyl nucleus of lignins is r emarkable and should be considered in evolutionary s tudies.

10 . Whereas mos t of the dihydroxybenzoi c and trihydroxybenzoic acids a nd their monomethyl ethers are known to occur naturally in plants, little is 126

known of the cinnamic acid analogues. Acids auch as ~,3-dihydroxycinnamic,

2,4·-dihydroxycinnamic (umbellic), 2,5-dihydroxycinnamic, 3,4,5-trihydroxy

cinnamic and their monomethyl ethers, bave not been reported as naturally occurring phenolic acids in plants. The lack of knowledge of their chroma tographie bebaviour and their colour reactions with spray reagents precluded their identification on paper chromatograms of plant extracts. Bate-Smith

(1956 c) remarked on the absence of 3,4,5-trihydroxycinnamic acid in nature and suggested that if it bad been found anywhere, it would have been in such plants as tea where trihydroxy compounds and cinnamic acids are found.

He also suggested that its absence may be due to its instability.

As a result of studying the phenolic acids of 53 species, a large number of unidentified compounds (see Table. 5) were detected. Most of these compounds were fluorescent in ultraviolet light and gave colour reaction with diazotized p-nitroaniline. Sorne of them could possibly be phenolic aldehydes, ket0nes or alcohols. The possibility of their being cournarins or other phenolic lactones is not excluded, since a number of these compounds are s lightly soluble in dilute carbonate solution and would appear in the phenolic acid fraction. Reference to sorne of these compounds is given in the section of resulta. 127

II. Discussion of the Biosynthesis of Sorne Hydroxybenzoic Acids in Higher Plants

Whereas the pathways of synthesis of the c -c acids have been 6 3 outlined in the work of McCalla and Neish (1959 b), little is known of

the formation of the c -c phenolic acids in higher plants. It bas 6 1 been suggested that they are possibly formed from phenylpropanoid

compounds, by the removal of a two-crbon fragment from the three-carbon

side chain (Geissman and Hinreiner, 1952 ; Geissman, 1958 ; Neish, 1960).

However, protocatechuic acid bas been shown to be formed directly from

non-aromatic precursors in micro~rganisms. The biosynthesis of of 6-methyi- 14 salicylic acid from C -carboxyl-labelled acetate bas also been demonstated

in Penicillium griseofulvum (Brich et ~~ 1955).

Among the plants used in the present study, Gaultheria procumbens,

and Primula acaulis were particularly interesting. The former contained

~-hydroxybenzoic (salicylic), ~-hydroxybenzoic, 2,3-dihydroxybenzoic (~-

pyrocatechuic), 2,5-dihydroxybenzoic (gentisic) acids, and the latter,

salicylic, ~-hydroxybenzoic, gentisic and 2-hydroxy-5-methoxybenzoic acids.

The resulta given in Table 7 and Figure 12, show that carboxyl- 14 labelled benzoic acid-C was readily converted to ~-hydroxybenzoic, 14 salicylic, ~-pyrocatechuic and gentisic acids, all labelled witb C , in

Gaultheria and Primula leaf disks. These conversions possibly took place

through the corresponding glycosides, since these phenols are rapidly converted to glycosides when administered to plant tissues and rarely occur 128

free. Methy1 salicylate primeveroside {Cahours, 1843), 2-pyrocatechuic

acid glucoside {King~ !1, 1955) and a gentisic acid-containing compound

(Griffiths, 1959) are known to occur naturally in plants.

The hydroxylation of benzoic acid was more remarkable in young leaf

disks of Gaultheria and Primula {see experimenta 3 and 5, Table 7) than

in mature leaves of the same species. In these two experimenta, there was

more incorporation of the label of benzoic acid into 2-monohydroxy- and -o-dihydroxybenzoic acids than in the corresponding -p-hydroxy acids. Mature leaves of Gaultheria and Triticum on the ether band, could not convert

benzoic to ether hydroxybenzoic acids, except that a small amount of

salicylic acid was formed in the latter. Most of the activity of the

phenolic acid fractions was found in unidentified compounds shawn in

Figures 28 and 29, respectively. One of these compounds which appeared

on the radioautographs of bath plants bad Rf values of 0.28 and 0.85 in

benzene and formate solvents, respectively. This compound was not fluoresc-

ent in UV-light and gave no co1our with diazotized sprays.

This remarkable diffe~ence between young and mature leaves is not

understood. It may be the result of changes in the enzyme systems which

occur with maturation of these tissues. This phenomenon was demonstrated

by Mrs. S. Lawson, who was working in this laboratory in 1960. In ber

studies of arbutin biogenesis in Pyrus, she found that only young leaves

could synthesize arbutin (hydroquinone-~-D-glucoside)from administered phenylalanine or shikimic acid. In mature leaves on the ether band, the 129

benzoic

unknown

Figure 28 Print of a radioautograph of the chromatographed phenolic acid fraction of Gaultheria leaf disksJ administered benzoic acid-cl4. c ::::1 ':l' ::::1 0 ~

Figure 29 Print of a radioautograph of the chromatographed phenolic acid fraction of wheat leaves, administered benzoic acid-c14. 131

14 activity of phenylalanine-C was incorporated into chlorogenic acid and

other esters of cinnamic acid derivatives.

Andreae and Good (1957) demonstrated the formation of labelled benzoyl 14 aspartic and benzamide in pea sections, administered benzoic acid-C with

either inactive aspartic acid or ammonium salta. However, none of the

unidentified compounds of Gaultheria or Triticum extracts could have been an

ester, since the extracts were subjected to hydrolysis. 14 Salicylic acid-C was found to be readily converted to ~-pyrocatechuic

acid-C14 and gentisic acid-C14 by leaves of Gaultheria, Primula, Rhododendron,

Lotus and Triticum (see Table 8 and Figure 13). There was more incorporat- 14 ion of the label from salicylic acid-e into ~-pyrocatechuic acid than

into gentisic acid in Gaultheria leaves (see experimenta 1-3, Table 8),

which suggests that further hydroxylation, ortho to the hydroxyl function

of salicylic acid, is predominant in this plant. In Primula on the other

hand, there was more activity in gentisic and 2-hydroxy-5-methoxybenzoic

acids than in ~-pyrocatechuic acid (see experiment 4, Table 8).

There was no evidence of loss of hydroxyl groups, since no radioactive 14 benzoic acid was formed from salicylic acid-e administered to leaves of

different plants. Similarly, ~-pyrocatechuic and gentisic acide were not

interconvertible. This was demonstrated when each of the radioactive

acids was administered to Gaultheria leaf disks.

Conversion of salicylate to gentisate bas been shawn to occur in animals (Lutwak-Mann, 1943 ; Schayer, 1950 ; Alpen et al, 1951), and in 132

human subjects (Alpen et al, 1951). On the other hand, Benati and Ciceri

(1951) found that when gentisic acid was administered to rabbits, it was

rapidly absorbed from the intestine and excreted as the sodium salt without

transformation. Recently, Inmori et al (1958) showed tbat a rabbit liver

enzyme preparation, in the presence of triphosphopyridine nucleotide,

converted benzoic to m-hydroxybenzoic, p-hydroxybenzoic to protocatechuic - - 1 salicylic to gentisic, ~-hydroxybenzoic to protocatechuic and gentisic,

phenylacetic to -o- and·- P-hydroxyphenylacetic, -o-bydroxyphenylacetic to

homogentisic and ~-hydroxyphenylacetic to homoprotocatechuic acids.

The oxidation of hydroxybenzoic acids bas been studied in micro-

organisme by the use of simultaneous adaptation techniques. Walker (1952)

showed that Pseudomonas fluorescens grown on salicylate was adapted to

catechol, but to none of the possible dihydroxybenzoic acids or phenols.

When the organism was grown on m- or p-hydroxybenzoate, it was adapted to

gentisic and protocatechuic acids respectively. Oxidation by bacteria,

of salicylate to catechol, was also reported by Roof, Lanon and Turner(l953).

They found that bacteria grown on gentisate or 2,3-dihydroxybenzoate, on

the other hand, showed no adaptation to any ether intermediates. However,

the oxidation of gentisate was suggested to be analogous to that of homo-

gentisate in micro3rganisms with the formation of a straight chain dicarbo:-

xylic acid (Roof et al, 1953 ; Evans, 1958 ; Lack, 1959).

From the accumulated evidence cited above, it seems that the hydroxylation of benzoic acid to R_-hydroxybenzoic and salicylic acids, and further 133

hydroxylation of the latter to ether dihydroxybenzoic acids, resembles the patterns obtained with rabbit liver enzyme by Inmori et !h (1958). However, higher plants differ from animale in their ability to convert both benzoic and salicylic acids to 2-pyrocatechuic acid. This was shown in five species including a monocotyledon (see Tables 7 and 8). It is an interesting fact, that the wheat plant, which contains none of these acids in a free or bound form except p-hydroxybenzoic acid (see Table 4), is capable of converting salicylic to gentisic and 2-pyrocatechuic acids.

It is of interest to metion the close similarity between the oxidation processes which proceed in plant cella and that of the model peroxidase system in vitro, reported by Grinstead (1960). He showed that, in the presence of ethylenediaminetetraacetato-iron (III) and ascorbic acid, sa1icy1ic acid was oxidized by oxygen and hydrogen peroxide to 2-pyrocatechuic and gentisic acids. Benzoic acid was oxidized to a mixture of o-, m-, and p-hydroxybenzoic acids. - - - A suggested scheme of hydroxylation of the c -c acids in higher 6 1 plants is shown in Figure 30. The fa11ure of ~-hydroxybenzoic acid to be further converted to ether dihydroxybenzoic acids by 1eaf disks might be exp1ained by the low activity incorporated into it from the administered benzoic acid-c14• Whereas protocatechuic (3,4-dihydroxybenzoic)acid has been s hown to be formed from p-hydroxybenzoic acid in anima1s (Inmori, 1958) and certain micro3rganisms (Walker, 1952), Gross ,(1958) demonstrated the enzymic conversion of 5-dehydroshikimic acid to protocatechuic acid by 134 0

0 (") :::r:: 0 0 :::r::

0 :::r:: ~ () ::0 () 0 0 0 0 ::0 :::r:: G') l> 0 z (j) ~ (.)) 1\

l> z

~ 0 n 0 z 0 :::r:: () (") 0 l> 0 0 :::r:: r 0 0 (J) :::r:: :r 00 :::r:: 0 !? :::r: :::r:: /\ 0

0 () () (") :::I: 0 0 0 0 0 0 :::r:: :::r: :::r: 00 0 0 00 () :::r: 0 z :::r:: :::r:: ()j 1 () :::r: ()jo

(") 0 0 :::r:: 0 0 :::r:

Figure 30 Suggested scherne of formation of sorne hydroxybenzoic acids in higher plants. 135

Pseudomonas fluorescens. Similar resulta were also shown with P. ovalis

{Hattori, Yoshida and Hasegawa, 1958}. However, the formation of proto-

catechuic acid in higher plants, by further hydroxylation of ~-hydroxy-

benzoic acid,in a similar manner as 1with R-coumaric and caffeic acids

(McCalla and Neish, 1959 b), may be a possibility. 14 The resulta given in Table 9 show tbat phenylalanine-U-C was

converted by Primula leaf disks to 2-coumaric, salicylic, 2-pyrocatechuic

and gentisic acids {see Figure 14). On the other band, when phenylalanine- 14 14 U-C and cinnamic acid labelled on the ring and the 1 -carbon with C

were administered to Gaultheria leaf disks, most of the activity was

incorporated into ~-coumaric acid and only traces were detected in the

C6-Cl acids. These resulta suggest that C6-c1 acids are possibly formed from shikimic acid via phenylpropanoid compounds. However, the fate of

phenylalanine is quite different in different plants. Ortho-hydroxylation

is predominant in sorne plants auch as Primula, where phenylalanine is

converted into o-coumaric and salicylic acids. The latter has been shown

to be a good precursor of gentisic and 2-pyrocatechuic acids. In other

plants, p-hydroxylation appears to be the predominant type where phenyl-

alanine is largely converted to p-coumaric acid as was shown with Gaultheria

leaf disks (see Table 9). Apple and pear leaves are other examplesof plant material which convert administered phenylalanine or cinnamic acid to

~-hydroxy compounds, sucp as E-coumaric and E-coumaryl quinic acids

(Grisedale and Towers, 19p0; Avadhani and Towers, unpublished resulta). 136

There was almost no synthesis of C6-c3 or C6-c1 acide from 14 tyrosine-U-C in Gaultheria leaf disks (see Table 9). About 70 ~of the activity of the ethanol soluble fraction, was found in sugars and malic acid (see Figure 15). It has been reported by many workers that tyrosine is a poor precursor of aromatic compounds in plants (Underhill,

Watkin and Neish, 1957 ; Hutchinson, Taper and Towers, 1959 ; Grisedale and Towers, 1960).

Douglass and Hogan (1958) demonstrated the formation of some hydroxybenzoic acids from flavonols in vitro. They found tbat quercetin, morin, and 3,4-dihydroxyflavone, when subjected to the action of rat kidney homogenates, gave 3,4-dihydroxybenzoic, 2,4-dihydroxybenzoic and ~-hydroxybenzoic acids, respectively. Also, Booth, Jones and DeEds (1958) showed tbat when naringin, phloridzin and their aglycones were administered to rats, all gave ~-hydroxyphenylpropionic acid as well as small amounts of p-coumaric and ~-hydroxybenzoic acids. It has also been shown with microorganisme, tbat one of the degradation products of rutin (quercetin rhamnoglucoside), is the C6-c1 acid, protocatechuic acid (Hattori and Noguchi, 1959 ; Westlake et al, 1959). The possibility exista therefore of the formation of c -c acids from the breakdown of flavonoids in higher 6 1 plants. This bas never been investigated.

Three ether hydroxybe~oic acids, 2,4-dihydroxy-, 2,6-dihydroxy~, and 3,5-dihydroxybenzoic acids are known to occur natu~ally in plants.

Their pattern of hydroxylation is unusual and their biogenesis in higher plants is worthy of study. 137

Although vanillic and syringic acids were easily detectable on many of the chromatograms in the tracer experimenta reported in the present work, these acids were never found to be radioactive. The origin of these ubiquitous acids in plants is not known. They are homologues of ferulic and sinapic acids and could conceivably be formed from c -c acids by 6 3 removal of two carbone of the three-carbon aide chain. However,

Geissman (1958) has suggested that gallic (3,4,5-trihydroxybenzoic) acid might be directly formed from shikimic or quinic acids rather tban through prephenic and phenylpyruvic acids. This needs further investigation.

III. Discussion of the Phenolic Constituents of Hydrangea macrophylla

Phenolic Acids

Flowers, leaves and roots of ~· macrophylla were analyzed for phenolic acids which were present in the free state and combined as glycosides or esters (see Table 6 and Figure 9). Among the phenolic acids detected on paper chromatograms, ~-hydroxybenzoic, protocatechuic, gentisic, ~-coumaric, caffeic, melilotic, ferulic and a compound believed to be hydrangeic acid were present. Vanillic, syringic and sinapic acids wh'ich were shown to be of common occurrence in plants, were absent. Identification of these , acids was based on their Rf values, fluorescence in ultraviolet light and colour reaction with chromogenic sprays.

P-Hydroxybenzoic, protocat echuic and gentisic acids were only detected after aci d hydrolysis of flower and leaf extracts, they were absent in the roots. ~-Hydroxybenzoic and gentisic acids were found to occur in small quantitiee in the free state in flowers and leaves. p-Coumaric and

caffeic acide on the other band, were present in all the organs of the plant. They were detected in the free etate in flowere and leaves after acid hydrolyeie, and in larger amounts after alkaline hydrolysis of flower,

leaf and root extracts. Ferulic acid was present in all the organs of the plant, possibly combined as an ester with traces in the free state.

Melilotic acid was detected after acid hydrolysis of flower extracts, and after alkaline hydrolysis ofroot extracts.

T~ pattern of distribution of phenolic acide in the different organe of the plant is of interest. From Table 6, it can be seen that, whereas both c -c and c -c acids were found to occur to~ether in flowers and 6 1 6 3 leaves, roots contained only C6-c acids. Moreover, the other major phenolic 3 constituents of the roots were three lactones,two of which were the coumarin derivatives, umbelliferone and compound No. 15, and the third, the phenyl ac id ~-coumar in , hydrangenol. While melilotic (dihydro-~-coumaric)jwas found to occur in flowers and roots, o-coumaric acid was absent in all organe of the plant.

Hydrangenol and its Glucoside

The phenyl iso-coumarin, hydrangenol {see Figure 2) , was first isolated by Asahina and Miyake (1916) from the sepals of Hydrangea hortens~

Dipp. Later,its identity was confirmed by synthesis from 3-methoxyphtbalic anhydride and E-methoxyphenylacetic acid (Asahina and Asano, 1930 b). Further investigation by Ueno (1937 a, b) resulted in the isolation, from the same 139

source, of hydrangenol glucoside. In more recent etudies of the phenolic constituants of H. macrophylla, Asen, Siegelman and Stuart (1957) and

Asen and Siegelman (1957), made no reference of hydrangenol or its glucoside in this species.

Hydrangenol glucoside and its aglucone were isolated from the flowers of H. macrophylla. The procedure for the isolation of hydrangenol was found applicable in the recovery of milligram quantities from one to two gram fresh weight of roots after acid hydrolysis of their extracts. The discovery that hydrangenol occurs in the roots of aydrangea has not previous ly been reported.

Very recently, Asen, Cathey and Stuart (1960) isolated hydrangenol and its glucoside from the leaves of~· macrophylla. They reported that both the glucoside and the aglucone melted at 179°C (uncorrected), reported for the glucoside 172°C (Ueno, 1937 a} and 192°C (Ueno, 1937 b). HYdrangenol glucoside isolated in this laboratory melted at 190-19Q.5°C (uncorrected).

It should be mentioned that the reference compounds usen by Asen et al were samples of hydrangenol and its glucoside isolated in this laboratory from the flowers of the same species.

The fact that hydrangenol was detected in larger amounts in flowers and roots than in leaves suggests that it is synthesized in these two organs without being translocated to the leaves. Analysis of leaf extracts showed that leaves synthesi~e hydrangenol at an advanced stage of growth while it was completely absent in buds and young leaves. 140

IV. Discussion of the Biosynthesis of the Phenolic Constituants of Hydrangea macrophylla

Considerable interest is being shawn in the biosyntbesis of aromatic

compounds in plants, especially those with c -c or c -c -c carbon skeleton. 6 3 6 3 6 The pathways of syntheses of benzenoid compounds, namely the shikimic acid and the acetate pathways, have been reviewed. Evidence of the applicability

of these pathways bas been shawn from the resulta of different workers.

The carbon skeleton of hydrangenol and the orientation of hydroxyl groups (see Figure 2) suggest that it may be formed exc1usive1y from acetate unite as proposed by Robinson (1955) for the stilbenes, which type of compounds hydrangeno1 resemb1es. On the ether band, it may be visua1ized

that ring B and carbons 3, 4, and 5 of hydrangeno1 are formed from a phenylpropanoid precursor and carbons 1, 6, 1, 8, 9, and 10 are derived from acetate according to the hypothesis of Birch and Donovan (1953).

From the r~ults of bath tracer experimenta given in Tables 12 and 13, ~ it appears that cinnamic acid is a relatively good precursor of hydrangenol 14 compared to the other compounds used. L-Pheny1alanine-U-C was also shawn to be incorporated into hydrangenol. Acetate-1-C14 and acetate-2-C14 were 14 found to be equally effective in forming hydrangenol-C . 14 The resulta of hydrangenol-C degradation1 given in Table 14 and 14 Figure 22, show that the label of each of sodium cinnamate-2-C and sodium 14 acetate-2-C was incorporated into ring A which corresponds to 2,6- cresotic acid, whereas the activity of phenylalanine-3-C14 was found in ring

B of hydrangenol represented by E-hydroxybenzoic acid. Therefore, the 141

incorporation of the label of beth cinnamic acid and phenylalanine on the

one band, and of acetate on the other band, suggests that hydrangenol may

be formed by a pathway shown in Figure 31. However, these resulta do not

show whether the e6-e unit is incorporated intact or not. The possibility 3 of the loss of the terminal carbon of the phenylpropanoid precursor and

the introduction of a one-carbon fragment in the l-position of the

hydrangenol molecule is not excluded.

Further etudies with specifically labelled compounds auch as phenyl

alanine-l-e14 are essential in order to resolve this problem. With phenyl- 14 alanine-l-e , ring A (2,6-cresotic acid) should be the only radioactive

moiety, and more precisely, all the activity from phenylalanine-l-e14 should be found in carbon 5 of hydrangenol. Further degradation etudies of 2,6-

cresotic acid are also required to determine the distribution of activity 14 14 from acetate-l-e and acetate-2-e in ring A.

The fact that compound No. 8 (believed to be hydrangeic acid, see

Table 6 and Figure 9) never became radioactive in the tracer experimenta given in Table 16, suggests that it is not an intermediate in the pathway of hydrangenol biosynthesis. It is possible that this compound . is formed at a later stage of hydrangenol synthesis. Its absence from paper chromate- grams of young root and young flower extracts supports this view. However, it is possible that it may .be synthesized by a different route.

Whereas phenylalanine and cinnamic acid were shown to be incorporated

into hydrnagenol, another phenylpropanoid compound) L-tyrosine, in spite of 142

3 ACETATE+ l

OH 0

Figure 31 A suggested pathway of hydrangenol biosynthesis. its close structural relationship to phenylalanine, was relatively ineffect- ive as a precursor of hydrangenol. The failure of tyrosine to be incorporated is remarkable, since it has the same hydroxylation pattern as that of rin~ B of the hydrangenol molec~le (see Figure 2). Underhill, Watkin and Neish (1957) showed that tyrosine was a very poor precursor in the biosynthesis of quercetin in buckwheat. Similarly, Hutchinson, Taper and

Towers (1959) reported that tyrosine was very poorly incorporated into phloridzin in Malus tissues, unlike phenylalanine which was a good precursor.

They found that tyrosine was readily metabolized to amine acids and even to sugars. Another example of the behaviour of tyrosine was demonstrated in the biosynthesis of arbutin in Py!us leaves (Grisedale and Towers, 1960).

They showed that tyrosine-u-c14 was converted to labelled sugars, organic acids and amine acids, while arbutin never had more than a trace of activity.

The law activities incorporated into hydrangenol from the c14-labelled compounds, administered to different organs of Hydrangea (see Table 16), might bave been due to the fact that the radioactive compounds were metabolized by ether routes. The resulta given in Table 16 show that most of the activity, from the administered phenylalanine-C ~ and cinnamic acid-C. ~ was largely incorporated into umbelliferone and compound No. 15, and to a lesser extent into p-coumaric and caffeic acids. In most of these experimenta, hydrangenol was not radioactive . Calculation of the ratio of ortho to para hydroxylation among the identified phenolic compounds in tbese feeding experimenta, showed that ~-bydroxylation was predominant especially in the 144

roots. In this respect, it should be noticed that ring B of hydrangenol bas a p-hydroxylation pattern. Radioactive hydrangenol (see Tables 12, and 13) was obtained with 4-month-old root material, whereas with young roots, hydrangenol never had more than a trace of activity. This suggests that in young roots, umbelliferone and compound No. 15 are synthesized continuously, while hydrangenol is only formed at a later stage of root maturity. This also appeared to be the case in the leaves where no hydrangenol was detected on paper chromatograms of young leaf extraçts, and it was found to occur in the mature leaves.

Another important point is the type of phenolic compounds which accumulate in the different organe of Hydrangea. From examination of paper chromatograms, hydrangenol was found to be the major phenolic constituent in both flowers and roots (see Table 6). Roots, in ad~ition, contained about as much umbelliferone as hydrangenol, and smaller amo~ts of compound No. 15 , whereas both coumarin derivatives were present in relatively small amounts in the flowers. In the leaves, on the other hand, there was a predominance of umbelliferone and compound No. 15, wh il-e hydrangenol was only present at a later stage of leaf maturity. Therefore, it appears thàt 2-hydroxy compounds are predominant in roots and young leaves, whereas E-hydroxy compounds are more abundant in sepals. Beth p-coumaric and caffeic acids were readily formed from phenylalanine-U-C14 14 and sodium cinnamate-2-C in sepals (see Table 16). In contrast, these labelled compounds were converted to 2-hydroxy compounds (umbelliferone and 145

compound No. 15) in roots and 1eaves. from Both umbe1liferone and compound No .. 15 were readily formedjshikimic 14 14 acid-U-C , cinnamic acid-2-C and phenylalanine labelled in different 14 carbons with C , in flowers, leaves and roots {see Table 16). In most of these experimenta, there was more activity in umbelliferone than in compound No. 15. The biosyntbetic relationsbip of botb coumarin deriv atives was investigated by administering umbelliferone-c14 to sydrangea leaf disks. Compound No. 15 was found to be the only radioactive spot on the paper cbromatogram of leaf extracts (see Figure 27) which suggests tbat the presence of the hydroxyl group of compound No. 15 in the 7-position, is most likely.

This correlation was studied further wben sydrangea leaf disks were 14 administered phenylalanine-U-a for 12 bours in the light, tben transferred to water and were allowed to pbotosyntbesize for various periods of time. The resulta given in Table 17 show tbat the total activities of the ether extracts of the leaf disks, administered phenylalanine-u-c14, increased with increase in time of administration. Wben the disks were transferred to water, the activities of their ether extracts sbowed a further increase up to six bours after removal of phenylalanine. This may possibly be due to a fraction of phenylalanine in the cells of the leaf disks wbich was further metabolized to phenolic compounds. The failure to obtain quantitative resulta for the 12-hour treatment, after removal of phenylalanine, was due to partial loss of the extract of this sample. 146

Both the percentage and total activities of umbelliferone and compound

No. 15 increased after a lag period of three hours. Six hours after the 14 removal of phenylalanine-C 1 the activity of compound No. 15 continued to increase, while that of umbelliferone decreased.

In this experiment, umbelliferone bad always higher activities than that of compound No. 15, which suggests that the latter is formed from the former. There were only traces of radioactivity found in 2-coumaric,

~-coumaric, caffeic and ferulic acids. These resulta suggest that phenylalanine is converted to cinnamic acid which then undergoes hydroxylation in the ortho position and forma these phenolic lactones.

If it is true that umbelliferone is a pre~ursor of compound No. 15, and the latter bas its hydroxyl group in the 7-position, then the only

positions possible for a methoxyl substitution are either the 5- 1 or 8- position, since the 6-position characteristic of scopoletin is excluded.

In any case, the synthesis of hydroxy-methoxy-coumarins from monohydroxy- coumarins requires the formation of dihydroxycoumarins as intermediates.

A suggested pathway of synthesis of umbelliferone and compound No. 15 1

from phenylalanine, is given in Figure 32. However 1 neither coumarin nor any of the dihydroxycoumarins were found radioactive in these tracer experimenta. This might possibly have been due to the fact that the reactions leading to the formation of these intermediates are rapid and do not result in their accumulation. Small amounts of activity were found in cinnamic and o-coumaric acids in these experimenta. Furthermore, it is not 1:' ..:t r-1

tl) c ..... ~ as !3 0 t> »1 >< 0 ~ .d Phenylanalase Hydroxylation +' Q) COOH COOH B »1 >< 0 ~ ~~,~ R 0 0 .d !:! 'H 0

Ol ..... Ol Q) .d +' Hydroxylation »c Ol 'H 0 » .d~ Hydroxylation +' HO as ~ ~ Pl

Methylation C\J 1? 1? l'(\

CH30 Q) ~ ;:j bO ..... ~

HO HO 148

known whether the conversions, shown in Figure 32, take place through

coumarin derivatives, i.e. coumarin, umbelliferone, esculetin (or daphnetin) 1

scopoletin (or 7-hydroxy-8-methoxycoumarin) etc., or through the glycosides

of their corresponding acids. It is worth mentioning that the resulta

reported in this work were obtained with hydrolyzed extracts.

About 20 ~ to 60 ~ of the radioactivity of the ether extracts of

the phenylalanine feeding1 were found in four unidentified compounds (see

Table 17). The activity of these compounds decreased steadily with increased

synthesis of umbelliferone and compound No. 15. They were fast moving in

the formate solvent and very slow moving in benzene~which resulted in their

poor separation. However, their position on paper chromatograms did not

correspond to any known phenolic acids or coumarine. Their nature was not further investigated.

The formation of coumarin, from shikimic acid, phenylalanine, cinnamic, and o-coumaric aci&has been demonstrated in Hieochloe" odorata (Brown, Towers and Wright, 1960),of scopolin, from ferulic acid in the leaves of wheat, corn and sunflower plants (Reznik and Urban, 1957) and from phenylalanine in tobacco leaves (Reid, 1958).

Finally, the fate of phenylpropanoid compounds as precursors of the major phenolic constituents of ijydrangea may be summarized as follows:

Phenylalanine was converted to cinnamic acid which was predominantly hydroxylated in the ortho position, especially in the roots and young leaves. In mature leaves and flower sepals, on the ether hand, ~ hydroxylation was 149

predominant when phenylalanine and cinnamic acids were administered, they were readily converted to p-coumaric and caffeic acids.

The need for a study of the period of active synthesis of hydrangenol

in the different organe of Hydrangea is essential for a more complete study

of its biogenesis. 150

SUMMARY

l. A method for the identification, by two-directional paper chroma-

tography, of phenolic acids was developed . This method was used in

studying the phenolic acids in 53 species of higher plants belonging

to 30 plant families. A number of unidentified phenolic compounds was

shown to occur in plants. Their chromatographie behaviour, fluoresce-

nee, and colour reactions were described.

2. Two hitherto unreported phenolic acids were found to occur in plants.

2,4-Dihydroxybenzoic acid was present in Gloriosa superba, and 3,5-

dihydroxybenzoic acid, in Primula acaulis and P. veria.

3· ~-Pyrocatechuic, gentisic, and 2-hydroxy-5-methoxybenzoic, acids were

isolated from plant material and identified .

4. By means of tracer experimenta with leaf disks, the hydraxylation of 14 certain C6-c1 acids was demonstrated. Benzoic acid-C was converted t o salicylic, -p -hydr oxybenzoic, -o-pyrocatechuic and gentisic, acide, all labelled with C14 . Salicylic acid-e14 was also shawn to be converted

in vivo to ~-pyrocatechuic, gentisic, and 2-hydroxy-5-methoxybenzoic

a cids in a numb er of plants . There was no evidence of los a of hydroxyl

groups of hydroxybenzoic acids in these experimenta . A scheme, showing

the relationships and possibl e routes of synthesis of t hese acids ,

was presented .

5. Sali cylic, ~-py r ocatechuic, and gentisic, acids were shown to be formed 151

14 from phenylalanine-U-C in Primula leaf disks. Tyrosine, on the other

hand, was largely converted to non-aromatic compounds by Gaultheria

leaf disks.

6. Flowers, leaves and roots of, Hydrangea macrophylla were analyzed, by

the use of paper chromatography, for phenolic acids and phenolic lactones,

and their relative amounts determined.

7· The phenyl iso-coumarin, hydrangenol, and its glucoside were isolated

from the flowers of the same species, and methods for their isolation

and identification were described. The occurrence of large amounts of

hydrangenol and its glucoside in the roots of Bydrangea had not previous-

ly been reported.

8. Hydrangenol was degraded and the degradation products identified as

~-hydroxybenzoic and 2,6-cresotic acids. A semi-micro method for the

degradation of milligram quantities of hydrangenol was developed. 14 . 14 14 9. Phenylal anine-U-C , sodium c~nnamate-2-C 1 sodium acetate-1-C 1 and

sodium acetate-2-C 14 were shawn to be incorporated into hydrangenol.

Hydrangenol-c14 was degraded and the products of degradation identified

by chromatography and radioautography. On the basis of these resulta,

a scheme f or the biogenesis of hydrangenol was suggested.

10. Umbelliferone (7-hydroxycoumarin) was isolated from Hydrangea roots.

The methode of isolation and identification were described and its bio-

-synthesis from c14-labelled phenylpropanoid compounds was demonstrated. 152

A hitherto unreported coumarin derivative was isolated from the roots of the same species, and identified as a methoxy-7-hydroxycoumarin. It was shown to be readily formed from C14 -labelled phenylalanine, cinnamic acid, and umbelliferone . A pathway of synthesis of methoxy-hydroxy-coumarins was suggested and discussed. 153

CLAIM TO ORIGINALrTY OR CONTRIBUTION TO KNOWLEDGE

1. A method for the separation and identification of phenolic acids, by

two-directional paper chromatography, was developed and applied to a

study of the plant phenolic acids.

2. 2,4-Dihydroxybenzoic, and 3,5-dihydroxybenzoic, acids which were not

known to occur naturally in plants, have been detected in Primula spp.

3· As far as the writer is aware, gentisic acid was isolated for the

first time from plant material (Leaves of Gaultheria procumbens).

4. It was shawn that 2-pyrocatechuic (2,3-dihydroxybenzoic) acid is of

common occurrence in the Bricaceae.

5. The conversion of benzoic acid-c14 to mono- and dihydroxybenzoic acide, 14 all labelled with C , was demonstrated in the leaves of higher plants,

and the nature of the precursors of these acide was discussed.

6. The presence of umbelliferone, hydrangenol, and hydrangenol glucoside

in relatively large quantities in Hydrangea roots, was not reported before.

7. A semi-micro method for the degradation, by alkaline hydrolysis, of

milligram quantities of hydrangenol was devised.

8. A scheme for the biogenesis of the pbenyl iso-coumarin, hydrangenol,

was proposed, based on the resulta of tracer experimenta.

9. A new phenolic lactone was isolated from the roots of !· macrophylla

and identified as a methoxy-7-hydroxycoumarin. Its biosynthesis from

phenylpropanoid compounds, and its close relationship to umbelliferone

were demonstrated. 154

REFERENCES

Adams, R., Bachmann, w., Fieser, L., Johnson, J. and Snyder, H. (1942)

Organic Reactions, vol. I, pp. 248-249. John Wiley & Sons, Inc. N.Y.

Alpen, E.L., Mandel, H.G., Rowdell, v.w. and Smith, P.K. (1951). Metabolism

of carbon-14 carboxyl salicylic acid in the dog and in man.

J. Pbarmacol. Exptl. Therap. !Qg : 150-155·

Andreae, W.A. and Good, N.E. (1957). Studies on 3-indoleacetic acid

metabolism. IV. Conjugation with aspartic acid and ammonia as processes

in the metabolism of carboxylic acids. Plant Pbysiol. ~ : 566-572.

Armstrong, M.D., Shaw, K.N.F. and Wall, P.E. (1956). Phenolic acide of

buman urine. Paper cbromatograpby of phenolic acids.

J. Biol. Chem. 218 : 293-303.

Asahina, Y. and Asano, J, (1930 a). Uber" die Konstitution von Hydrangenol

und Phyllodulcin (II. Mitteil.) Ber. 63 : 429.

Asahina, Y. and Asano, J, (1930 b). Uber" die Konstitution von Bydrangenol

und Phyllodulcin, III. Mitteil.: Synthese des Hydrangenols.

Ber. §.2 : 2059.

Asahina, Y. and Miyake, K. (1916). Hydrangenol, a chemical constituent of

Hydrangea hortensia. Jour. Pharm. Soc. Japan No. 408 : 121-126.

Chem. Abstr. gQ : 1523.

Asen, S., Cathey, H.M. and Stuart, N.W. (1960). Enhancement of gibberellin

growth-promoting activity by hydrangenol isolated from leaves of

Hydrangea macrophylla. Plant Physiol. 22 : 816-819. 155

Asen, S. and Siegelman, H.W. (1957). Effect of aluminium on absorption

spectra of the anthocyanin and flavonols from the sepale of Bydrangea

macropbylla var. Merveille. Proc.Amer.Soc.Hort.Sci. 1Q: 478-481.

Asen, s., Siegelman, B.w. and Stuart, N.W. (1957). Antbocyanin and

phenolic compounds in red and blue sepale of Hydrangea macrophylla

var.Merveille. Proc.Amer.Soc.Hort.Sci. 22 : 561-569.

Barnes, H.M., Feldman, J.R. and White, w.v. (1950). Iso-chlorogenic acid.

Isolation from coffee and structure etudies.

J, Am. Chem. Soc. 72 : 4178-4182.

Bate-Smith, E.C. (1954 a). Ferulic, sinapic and related acid~ in leaves.

Chem. & Ind. 1457-1458.

Bate-Smith, E.C. (1954 b). Leuco-anthocyanidins. 1. Detection and

distribution of anthocyanidins formed from leuco-anthocyanidins in

plant tissues. Biochem. J. ~ : 122-125.

Bate-Smith, E.C. (1955). The commoner phenolic constituants of plants and

their systematic distribution. Unpublished resulta, Low Temperature

Research Station Record Memorandum No. 271.

Bate-Smith, E.C. (1956 a). The commoner pbenolic constituants of plants

and their systematic distribution. Sei. Proc.Roy. Dubl. Soc. 27 :

165-176. s Bate-Smith, E.C. (1956 b). Chromatography and sy~ematic distribution of

ellagic acid. Chem. & Ind. R 32-33·

Bate-Smith, E.C. (1956 c). Symposium on vegetable tannins. Chem. & Ind.

478-479. 156

Bate-Smith, E.C. (1958). Symposium on systernatic distribution. Plant

phenolicsas taxonomie guides. Proc.Linn.Soc.Lond. 169 : 198-211.

Bate-Smith, E.C. and Metcalfe, C.R. (1957). Leuco-anthocyanins. 3. The

nature and distribution of tannins in dicotyledonous plants.

J. Linn. Soc. 22: 669-705.

Benati,O. and Ciceri, C. (1951). Metabolisrn of gentisic acid.

Arch. ital. sei. farrnacol. 2 : 178-181.

Birch, A.J. and Donovan, F.W. (1953). Studies in relation to biosynthesis.

r. Sorne possible routes to derivatives of orcinol and phloroglucinol.

Austr. J. Chem. 2 : 360-368.

Birch, A.J., Massy-Westropp, R.A. and Moye, C.J. (1955). Studies in relation

to biosyntbesis. VII. 2-Hydroxy-6-rnetbylbenzoic acid in Penicillium

griseofulvum Dierckx. Austr. J. Cbern. 8 : 539-544.

Black, R.J., Durrum, E.L. and Zweig, G. (1955). A Manual of Paper Cbroma

tography and Paper Electrophoresis. Academie Press Inc., New York.

Bogorad, L. (1958). The biogenesis of flavonoids. Ann. Rev. Plant Pbysiol.

2 : 417-448.

Bogorad, L. and Granick, S. (1953). Protoporpbyrin precursors produced by

Chlorella mutant. J. Biol. Chern. 202 : 793-800.

Booth, A.N., Jones, F.T. and DeEds, F. (1958). Metabolic and glucosuria

etudies on naringin and pbloridzin. J. Biol. Chem. ~ : 280-282.

Bradfield, A.E., Flood, A.E., Hulme, A.C. and Williams, A.H. (1952).

Chlorogenic acid in fruit trees. Nature 170 : 168-169. 157

Bray, H.G., Thorpe, W.V. and White, K. (1950). The fate of certain organic

acids and amides in the rabbit. 10. The application of paper chroma

tography to metabolic etudies of hydroxy acide and amides.

Biochem. J. 46 : 271-275.

Brown, F. and Hall, L.P. (1950). Separation of carboxylate ions on the

paper chromatogram. Nature 166 : 66-67.

Brown, S.A. and Neish, A.C. (1955 a). Shikimic acid as a precursor in

lignin biosynthesis. Nature 112 : 688-689.

Brown, S.A. and Neish, A.C. (1955 b). Studies of lignin biosynthesis using

isotopie carbon. IV. Formation from sorne aromatic monomers.

Can. J. Biochem. Physiol. ~ : 948-962.

Brown, S.A. and Neish, A.C. (1956). Studies of lignin biosynthesis using

isotopie carbon. v. Comparative etudies on different plant species.

Can. J. Biochem. Physiol. ~ : 769-778.

Brown, S.A., Towers, G.H.N., and Wright, D. (1960). Biosynthesis of coumarine.

Tracer etudies on coumarin formation in> Hierochlo~ odorata and Melilotus

officinalis. Can. J. Biochem. Physiol. 2§ : 143-156.

Brown, S.A., Wright, D. and Neish, A.C. (1959). Studies of lignin biosynthesis

using isotopie carbon. VII. The role of ~-hydroxyphenylpyruvic acid.

Can. J. Biochem. Physiol. 21 : 25-34.

Cahours, A. (1843). Untersuchungen ~ber das Oel der Gaultheria procumbens.

Ann. 48 : 60-65.

Cartwright, R.A. and Roberts, E.A.H. (1955). Theogallin, as galloyl ester of

quinic acid. Chem. & Ind. 230-231. 158

Cartwright, R.A., Roberts, E.A.H., Flood, A.E. and Williams, A.H. (1955).

The suspected presence of ~-coumaryl quinic acid in tea, apple and

pear. Chem. & Ind. 1062-1063.

Collie, J.N. (1907). Derivatives of the multiple keten group.

J. Chem. Soc. 91 : 1806.

Consden, R., Gordon, A.H. and Martin, A.J.P. (1944). Qualitative analysis

of proteine : a partition chromatographie method using paper.

Bichem. J. 38 224-232.

Corner, J.J. and Harborne, J.B. (1960). Cinnamic acid derivatives of

potato berries. Chem. & !nd. P 76.

Corse, J.W. (1953). A new isomer of chlorogenic acid from peacbes.

Nature 172 : 771-772.

Creighton, R.H.J., Gibbs, R.D. and Hibbert, H. (1944). Studies on lignin

and related compounds. LXXV. Alkaline nitrobenzene oxidation of plant

materials and application to taxonomie classification.

J. Am. Chem. Soc. 66 : 32-37·

t~ Phlorin Cremer, M. and Seuffert, R.W. (1912). uber ein Spaltungsprodukt des

Phloridzins. Ber. 45 : 2565-2571.

Cruickshank, I.M.T. and Swain, T. (1956). Study of phenolic constituants of oil flax. J. Exptl. Bot. I : 410-415.

Davis, B.D. (1955). Intermediates in amine acid biosynthesis.

Advances in Enzymol. 16 :247-312.

Davis, B.D. (1958). On the importance of being ionized.

Arch. Biochem. Biophys. 78 : 497-509. 159

Dickinson, D. and Gawler, J.H. (1954 a). Chemica1 constituents of Victoria

pluma. J. Sei. Food Agricu1t. 2 : 525.

Dickinson, D. and Gawler, J.H. (1954 b). Isomers of chlorogenic acid.

Chem. & Ind. 1583.

Douglass, C.D. and Hogan, R. (1958). Formation of hydroxybenzoic acids

from flavonols in vitro. Fed. Proc. 17 : 213.

Evans, C. (1958). Metabolism of aromatic compounds by lower plants. Phenols,

quinones, and the problem of biogenesis of benzene nuclei. Encycl.Plant

Physiol.,vol. X,pp. 454-506, by w. Ruhland, Springer-Verlag.

Evans, R.A., Parr,W.H. and Evans, W.C. (1949). Paper partition chromatography

of phenolic substances. Nature 164 : 674-675·

Fischer, H.O.L. and Dangschat, G. (1932). Konstit ution der Chlorogensaure"

( 3. Mitteil. "uber Chinsaure" und Derivate ) • Ber. ~ : 1037-104 O.

Fitzgerald, J.S. (1959). Gas chromatography applied to analysis of phenols.

Austr. Jour. Appl. Sei. !Q : 169-189.

Gafney, G.W., Schreier, K. , Di Ferrante, N. and Altman, K.I. (1954). The

quantitative determination of hippuric acid. J.Biol.Chem. 206: 695-698.

Gamborg, O. L. and Neish, A.C. (1959). Biosynthesis of phenylalanine and

tyrosine in young wheat and buckwheat plants. Can. J. Biochem.

Physiol. 37 : 1277-1285.

Geissman, T.A. (1958). The matabolism of phenylpropane derivatives in plants.

Encycl. Plant Physiol. vol. X, pp. 543-559, by W. Ruhland,Springer-Verlag.

Geissman, T.A. and Hinreiner,E. (1952). Theories of the biogenesis of phenolic

compounds (Parts I and II). Bot. Rev . 18 77-244. 160

Geissman, T.A., Mason, J.W. and Rowe, J.R (1959). Biosynthesis of iso-

flavones. Chem. & Ind. 1577-1578.

Geissman, T.A. and Swain, T. (1957). Biosynthesis of flavonoid compounds in

higher plants. Chem. & Ind. 984.

Goodwin 1 R.H. and Kavanagh 1 F. (1950). Fluorescence of coumarin derivatives

as a function of pH. Arch. Biochem. 27 : 152-173·

Goris, A. and Canal, H. (1936 a). Etude sur la composition chimique des

bourgeons du Populus balsamifera. Bull.Soc.Chim. de France 2:1982-2009.

Goris 1 A. and Canal, H. (1936 b). Sur la composition de l'essence de Primula

auricula. Comp. Rend. 202 : 1351-1352.

Goris, A. and Canal, H. (1945). Composition chimique de l'essence de

racines de Primula viscosa. Comp. Rend. 220 : 790-792.

Graebe, C. and Martz, E. (1905). Ueber die Methyl~ethers~uren von Hydro-

chinoncarbonsaure,n Protocatechusauren und Gallusaure." Ann. ~:4 213-221.

Griffiths, L.A. (1957). Separation and identification of aromatic acids

in plant tissues by paper chromatography. Nature 180 : 286-287.

Griffiths, L.A. (1958). Occurrence of gentisic acid in plant tissues.

Nature 182 : 733-734.

Griffiths, L.A. (1959). On the distribution of gentisic acid in green

plants. J . Exptl. Bot. 10 : 437-442.

Grinstead, R.R. (1960). Oxidation of salicylate by the model peroxidase

catalyst iron-ethylenediaminetetraacetato-iron(III) acid.

J. Am. Chem. Soc. 82 : 3472-3476. 161

Grisebach, H. (1957). Zur Biogenese des Cyanidins. I. Mitt. Versuche mit 14 14 Acetat-1-C und Acetat-2-C • z. Naturforsch. 12,b : 227-231.

Grisebach, H. and Doerr, N. (1959). Zur Biogenese des 7-Hydroxy-4'-methoxy-

isoflavons. Naturwissenschaften 46 : 514-515.

Grisedale, S. and Towers, G.H.N. (196o). Biosynthesis of arbutin from

some phenylpropanoid compounds in PyrUS communia. Nature 188:1130-1131.

Gross, S.R. (1958). Enzymatic conversion of dehydroshikimic acid to

protocatechuic acid. J. Biol. Chem. 233 : 1146-1151.

Guenther, E. (1949). The Essential Oils. vol. II. D. Van Nostrad Co.

Hall, A.J. (1937). A system of structural relationships in phytochemistry.

Chem. Rev. 20 : 305-344.

Harborne, J.B. (1960). Plant polyphenols. 2. The coumarins of Solanum

pinnatisectum. Biochem. J. 74 : 270-273·

Harborne, J.B. and Corner, J.J. (1960). The metabolism of cinnamic a~ids

and coumarins in higher plants. Biochem. J. 76 : 53 P.

Hattori, S. and NogCuchi, r. (1959). Microbial degradation of rutin.

Nature 184 : 1145.

Hattori, s., Yoshida, S. and Hasegawa, M. (1954). Occurrence of shikimic

acid in the leaves of gymnosperms. Physiol. Plantarum 1 : 283-289.

Hattori? S., Yoshida~ S. and Hasegawa, M. (1958). Biological conversion of

shikimic acid. Arch. Biochem. Biophys. 74 84o-842.

Hermann, K. (1958). Hydroxycinnamic acids in leaves of grain plants.

Naturwissenschaften ~ : 111-112. 162

Hillis, W.E. (1959). Shikimic acid in the leaves of Eucalyptus

sieberiana F.Muell. J. Exptl. Bot. !Q : 87-89.

Hillis, W.E. and Clark, A. (1960). Formation of phenolic substances in

Eucalyptus gigantea and!· sieberiana. Biochem. J, ~ : 607-615.

Hiramoto, M. and Watanabe, K. (1940). Uber" die Bestandteile von Catalpa

ovata. Chem. Zentr. 11! : 1515.

Hodgman, C.D. (1947). Bandbook of Chemistry and Physics. Chemical Rubber

Publishing Co., Ohio.

Hulme, A.C. (1951). The isolation of 1-quinic acid from the apple fruit.

J, Exptl. Bot. g : 298-315.

Hulme, A.C. (1953). The isolation of chlorogenic acid from the apple fruit.

Biochem. J. 22 : 337-340.

Hulme, A.C. (1956). Shikimic acid in apple fruits. Nature 178 991-992.

Hulme, A.C. (1958). Quinic and shikimic acidsin fruits.

Qualit. Plant. Mater. Veget. 2 : 468-473·

Hulme, A.C. and Richardson, A. (1954). Isolation of quinte acid fro~ grass.

J. Sei. Food Agric. 2 : 221.

Hutchinson, A., Taper, C.D. and Towers, G.H.N. (1959). Studies of phloridzin

in Malus. Can. J. Biochem. Physiol. 21 : 901-910.

Inmori, K., Nasu, H., Sakamato, Y. and Ishihara, K. (1958). Enzymatic

hydroxylation of benzoic and phenylacetic acids. New pathway to homo-

gentisic acid. Proc. Japan. Acad. ~ : 645-648.

Chem. Abstr. 53 : 11457 (1959). Ishida, Y. and Okamura, T. (1956). Blue luminescent substance from Berberis

thunbergii va. maximowizii and~· amurensis var. japonica forma

Bretschneideri. J. Pharm. Soc. Japan 76 : 223-224, Chem.Abstr.50:7957·

Karrer, W. (1958). Konstitution und Verkommen der organischen Pflanzen-

etoffe. Birkn:user Verlag, (Basel und Stuttgart).

King, F.E., Gilks, J.H. and Partridge, M.W. (1955). A glycosidic

constituent of Vinca minor, and its identification as 3-~-D-glucosyloxy-

2-hydroxybenzoic acid. J. Chem. Soc. 4206-4215.

Korner," G. (1882). Ueber die Kaffeesaure" aus Cinchona cuprea. Ber. !2:2624.

Kosuge, T. and Conn, E.E. (1959 a). The metabolism of coumarin and o-

coumaric acid in sweet clover. Fed. Proc. 18 : 265, 1046.

Kosuge, T. and Conn, E.E. (1959 b). The metabolism of aromatic compounds

in higher plants. I. Coumarin and o-coumaric acid.

J. Biol. Chem. 234 : 2133-2137.

Koves," E. and Varga, M. (1959). Comparative examination of water-, and

ether-soluble substances in dry fruits. Phyton 12 : 93-99.

Kurth, E.F. (1950). The composition of the wax of Douglas-fir bark.

J. Am. Chem. Soc. 72 : 1685-1886.

Lack, L. (19B9) . Enzymic oxidation of gentisic acid.

Biochim. Biophys. Acta ~ : 117-123.

Long, A.G., Quayle, J.R. and Stedman, R.J. (1951). The separation of acids

by paper partition chromatography. J. Chem. Soc. 2197-2201. 164

Lutwak-Mann (1943). The excretion of a metabolic product of salicylic

acid. Biochem. J. 21 : 246-248.

Mangini, A. and Passerini, R. (1957). Ultraviolet absorption spectra and

chromophoric properties of cyclic ethers. Gazz. chim. ital. §I :

243-292. Chem. Abstr. ïg : 10716 (1958).

Manskaya, S.M. and Kodina, L.A. {1958). Quinic and shikimic acids in

plants. Doklady Akad. Nauk. SSSR. 123 : 733-736.

Chem. Abstr. 22 : 5422 (1959).

McCalla, D.R. and Neish, A.C. (1959 a). Metabolism of phenylpropanoid

compounds in Salvia. I. Biosynthesis of phenylalanine and tyrosine

in Salvia. Can. J. Biochem. Physiol. 21 : 531-536.

McCalla, D.R. and Neish, A.C. (1959 b). Metabolism of phenylpropanoid

compounds in Salvia. II. Biosynthesis of phenolic cinnamic acids.

Can. J. Biochem. Physiol. 21 : 537-547.

Merck, E. (1839). Ueber eine neue in dem Sabadillsamen enthaltene sKure.

Ann. 29 : 188-190 .

Nakabayashi, T. (1953). Coumarin derivatives. III. Paper chromatography

of simple coumarin derivatives. J. Pharm. Soc. Japan ~ : 831-833.

Chem. Abstr. 47 : 12757 (1953).

Nakahara, H. (1955). The constituents of the inner bark of Hydransea

paniculata. I. A new glycoside, neohydrangin. J. Fac. Sei., Niigata

Univ. Ser . I : 2, 63-66. Chem. Ab s tr. 2Q : 4926 (1956). 165

Nakaoki, T. and Morita, N. (1955). Medicinal resources. I. Organic acids

in the leaves of Catalpa ovata and~· bignonioides. J. Pharm. Soc.

Japan 75 : 171-172. Chem. Abstr. 49 : 8562 (1955).

Neish, A.C. (1959). Biosynthesis of pungenin from c14-labelled compounds

by Colorado spruce. Can. J. Bot. 37 : 1085-1100.

Neish, A.C. (1960). Biosynthetic pathways of aromatic compounds.

Ann. Rev. Plant Physiol. 11 : 55-80.

Palmer, J.K. (1957). Occurrence of quinic acid in tobacco leaves.

Science 126 : 504-505.

Perkin, A.G. {1909). The colouring matters of the flowers of Hibiscus

subdariffa and Thespasia lampas. J. Chem. Soc. 95 : 1855-1860.

Picard, P. (1926). Le violutoside, nouveau glucoside à salicylate de

méthyle retiré du Viola cornuta. Comp. Rend. 182 : 1167-1169.

Power, F. B. (1901). Chimie der Rinde von Robinia pseudoacacia.

Chem. Zentr. 52 : 725-726.

Power, F.B. and Moore, Ch .W. (1909}. The constituents of the bark of

Prunus se ~ otina. I. Isolation of l-mandelonitrile glucoside.

J. Chem . Soc. 95 : 243-261.

Procter, W. (1843). Ob servations on the volatile oil of Betula lenta and

on gaultherin, a substance which by decomposition yields that oil.

Amer. J. Pharm. 15 : 241.

Reid, W.W. (1958) . Biosynthesis of scopoletin and caffei c acid in Ni cotiana

tabacum. Chem. & Ind. 1439-1440. 166

Reppel, L. (1954). Ober nat~rliche Cumarine.

Die Pharmazie 2 : 278-300.

Reppel, L. (1957). Beitr&ge" zur Kenntis der Cumarine.

Die Pharmazie ~ : 6?4-662.

Reznik, H. and Urban, R. (1957). Uber" den Metabolismus 14C-markierter

FerulsRure im Pflanzenversuch. Naturwissenschaften 44 : 13.

Richardson, H. and Hulme, A.C. (1955). Shikimic acid in grass.

Nature 175 : 43-44.

Roberts, E.H.A. (1956) . The chlorogenic acids in tea and maté.

Chem. & Ind. 985-986.

Robinson, R. (1955). The Structural Relations of Natural Products.

Oxford University Press.

Roof, B.S., Lannon, T.J. and Turner, J.C. (1953). Bacteria1 oxidation of

sa1icy1ic acid and re1ated antirheumatic phenolic acids.

Pree. Soc. Exper . Biol. and Med. 84 : 38-41.

Santav~, F. and Reichstein, T. (1950). Isolation of new compounds from

Co1chicum autumna1e. XII. Compounds of Co1chicum autumnale and

their derivatives. He1v. Chim. Acta 21 : 1606-1627. Chem.Abstr.45:2151.

Sapadnjuk, J.P. (1950). Futterm~ige und toxico1ogische Bewertunge der

we~en Kaspischen Me1i1otuspf1anze. Chem. Zentr. 121 : 2453·

Schayer, R.W. (1950). The metabo1ism of radioactive sa1icylic acid.

Arch. Biochem. 28 371-376. Sechet-Sirat, J., Masquelier, J. and Tayeau, F. (1959). Pigments i~

sunflower seeds. II. Caffeic acid. Bull. soc. chim. bioe. 41:1060-1070.

Seki, T. Inamori, K. and Sano, K. (1959). Chromatographie separation of

phenolic acids. J. Biochem. (Tokyo) 46 : 1653-1657.

Simmons, N.W. and Stevens, R. (1956). Occurrence of the methylene-

dioxy bridge in the phenolic components of plants.

Nature 178 : 752-753·

Smith, I. (1958). Chromatographie Techniques.

Interscience Publications, New York.

Srinivasan, P.R., Shigeura, H.T., Sprecher, M., Sprinson, D.B. and Davis,

B.D. (1956). The biosynthesis of shikimic acid from D-glucose.

J. Biol. Chem. 220 : 477-497·

Stenhouse, J. (1843). Ueber pyrogallusaure" und einige der adstringirenden

Substanzen, welche sie liefern. Ann. 45 : l-19.

Stewart, w.s. (1948). Recent Advances in Organic Chemistry. Vol. I.

Longmans, Green and Co .

Sutherland, G.K. and Gortner, W.A. (1959). Studies of a quinyl-~-coumarate

in the pineapple plant (Ananas comosus var. coyenne).

Austr. J. Chem. 12 : 240-246.

Swain, T. (1953). The identification of coumarins and related compounds

by filter paper chromatography. Biochem. J, 53 : 200-208.

Tomaszewski, M. (1960). The occurrence of E-hydroxybenzoic acid and some

ether simple phenols in vascular plants. Bull. Acad. Polon. Sei.,

ser. sei. biol. 8 61-65. Towers, G.H.N. (1951). M.Sc. Thesis. Comparative chemistry and taxonomy

of plants. The separation and estimation of phenolic aldehydes from

the alkaline nitrobenzene oxidation mixtures of plant materials.

Ueno, Y. (1937 a). Uber" das Hydrangenolglucosid.

Chem. Zentr. 108 : 4o4o.

Ueno, Y. (1937 b). Uber" das Hydrangenolglucosid. J. Pharm. Soc. Japan

57 602-605. Chem. Abstr. 2l : 6245.

Underhill, E.W., WatkinJ J.E. and Neish, A.C. (1957). Biosynthesis of

quercetin in buckwheat plant. Part I. Can.J.Biochem.Physiol. 22

219-228.

Van Slyke, D.D., Plazin, J. and Weisiger, J.R. (1951). Reagents for the

van Slyke-Folch wet carbon combustion. J. Biol. Chem. 191: 299-304.

Van Sumere, C.F., Van Sumere-de Preter, c., Vining, L.C. and Ledingham, G.A.

(1957). Coumarine and phenolic acids in the uredospores of wheat

stem rust. Can. J. Microbiol. 2 : 847-862.

Walker, N. (1952). Pathways in metabolism of monohydroxybenzoic acids by

soil bacteria. Biochem. J, ~ : 23-24.

Watkin, J.E., Underhill, E.W. and Neish, A.C. (1957). Biosynthesis of

quercetin in buckwheat. Part II. Can. J. Biochem. Physiol. 35:229-237·

Weinstein, L.H., Williams, R.S. and Laurencot Jr., H.J. (1959 ).

Senescence of r oses. III. Isolation and identif ication of 1-quinic

acid from Better Times roses. Contr. Boyce Thompson Inst. 19

341-348. 169

Weinstein, L.H., Porter, C.A. and Laurencot Jr., H.J. (1959 a). Evidence

for the conversion of quinic acid to shikimic acid in roses.

Nature 183 : 326.

Weinstein, L.H., Porter, C.A. and Laurencot Jr., H.J. (1959 b). Quinic

acid as a precursor in aromatic biosynthesis in the rose.

Contrib. Boyce Thompson Inst. 20 : 121-134.

Westlake, D.W.S., Talbot, G., Blackley, E.R. and Simpson, F.J. (1959).

Microbial decomposition of rutin. Can. J. Microbiol. 2 : 621-629.

Willia~s, A.H. (1955). Paper chromatography of cinnamic acid derivatives.

Chem. & Ind. 120-121.

Williams, A.H. (1956). ijydroxy-aromatic acids in plants.

Chem. & Ind. 478-479·

Williams, A.H. (1957). The simpler phenolic substances of plants.

J. Sei. Food Agric. 1 : 385-389.

Wright, D., Brown, S.A. and Neish, A.C. (1958). Studies of lignin bio~

synthesis using isotopie carbon. VI. Formation of the side chain

of the phenylpropanoid monomer. Can. J. Biochem. Physiol. ~ :

1037-1045.

Yoshida, S. and Hasegawa, M. (1957). A microcolorimetric method for the

determination of shikimic acid. Arch.Biochem.Biophys. 70: 377-381.

Zetsche, F. and Huggler, K. (1927). ijydrokaffeesaure," ein Bestandteil der

Sporen von tycopodium clavatum. Helv. Chim. Acta 10 : 472-474.