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A COMPARATIVE PHYTOCHEMICAL STUDY OF CAPRIFOLIACEAE by CHARLES WILLIAM GLENNIE B. Sc. Dalhousie University, 1963 M. S. University of Rhode Island, 1966

A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctor of Philosophy in the Department of - Botany

We accept this thesis as conforming to the required standard

The University of British Columbia

November, 19&9• In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study.

I further agree tha permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.

Depa rtment

The University of British Columbia Vancouver 8, Canada Abstract

A comparative phytochemical study of the Caprifolia- ceae.was undertaken. No such study has ever been done.

Extracts of fresh leaf material from 5° taxa collected through•

out North America were examined for their content of phenolic compounds.

Acid and base hydrolysis of the extracts yielded a

large array of common phenolic acids and several unknowns.

The unknowns occurred in small amounts and were not identified.

The cyanogenetic glucoside, sambunigrin, was found to occur

only in the genus Sambucus. Chlorogenic acid isomers were

identified in the unhydrolyzed extracts of all plants exam•

ined. A chromatography system for the separation of these

isomers is described. The dicaffeoylquinic acid isomers

(isochlorogenic acid) occurred in all genera examined except;

Sambucus which contained the monocaffeoylquinic acid isomers.

Scopoletin, a 6 Me-7-OPI-coumarin, was present in hydrolyzed extracts of Meigela. This is the only report

of scopoletin in the Caprifoliaceae although other coumarins have been found in Diervilla and Symphoricarpos. A crys•

talline compound was isolated from extracts of the leaves

of Viburnum davidii and was identified as 2 ' , 4-, 4'-trihydroxydihydrochalcone. This is the first report of this compound iv

being found, in plant material and also it is the first-report

of a dihydrochaleone with the 2',V dihydroxy structure rather

than the 2',4',6'trihydroxy structure as found in phloretin.

The flavonoids identified in this study yielded either

the flavonols kaempferol or quercetin or the flavones apigenin

or luteolin on hydrolysis. These four compounds displayed

a wide variation of glycosylation and distribution throughout

the taxa examined.. The flavonols, which are considered to be the more primitive compounds, are found in Viburnum and

Sambucus instead of the flavones. This would suggest that

these genera are the. most primitive of the family. The

relatively more advanced flavones are more prominent in

Symphoricarpos and Trlosteum thus suggesting that they are

the more advanced genera of the family. The comparatively

primitive compound amentoflavone (a biflavonyl) was found

in Viburnum carlesii and V. X burkwoodii.

The occurrence of sambunigrin in Sambucus only and the

absence of the dicaffeoylquinic esters in Sambucus support the

idea that Sambucus is sufficiently different to be put into

its own family, Sambucaceae (Hoch, 1892), ACKNOWLEDGMENTS

Thv. author wishes to express his sincere apprecia• tion to Dr. B. A. Bohm, for his guidance during this study.-

The author also wishes to sincerely thank the members of his graduate committee for their advice,and encouragement,

Thanks are also due to Dr. Warren Steck, National

Research Council, Saskatoon for authentic samples of chloro• genic acid isomers, Dr. L. Horhammer, Munich for a sample of amentoflav;me and to Mr. Marcel Raymond, Montreal Botanical

Garden, for allowing the author to collect plant material.

The author wishes to express special thanks to Dr.

Roy Taylor, University of British Columbia Botanical Garden, for help during the preparation of this manuscript.

This investigation was supported by a National Resea.rc

Council Grant and carried out in the laboratories of the

.Botany'Department, University of British Columbia.

The author also wishes to thank Dr. R. D. Gibbs,

McGill.University, for consultation and encouragement during this study. TABLE OF CONTENTS

PAGE

I INTRODUCTION . 1

II LITERATURE REVIEW

(A) Early Classification Systems 3

(B) Current Classification of the

Caprifoliaceae ...... ^

(D) Other Systematic Work on the

Caprif oliaceae ...... t . 12 (E) Chemistry of the Caprif oliaceae 15

III MATERIALS AND METHODS

(A) Collection of Plant Material ...... 23

(B) Extraction of Plant Material 28

(D) Examination of Plant Material for Hydrogen Cyanide Producing Compounds 30

(E) Identification of Sambunigrin ...... 31 (F) Identification of Chlorogenic Acid Isomers ..... 31 (G) [dentification of Scopoletin'in Hydrolysates of Weigela Species ...... 32 (H) The Isolation of 2 ' , k, k'-Trihydroxydihydro- chalcone from Viburnum davidii 34 vi

PAGE

(I) Identification of the Sugar Residue .... 35

(J) Determination of the Glueose-Aglycone Ratio 36

(K) Base Cleavage of the Aglyeone . 37

(L) "^C Precursor Studies 37

(M) Identification of Flavonoids ...... 38

IV RESULTS

(A) . Distribution of Phenolic Acids ...... 4l

(B) Plants Containing a Hydrogen Cyanide

Producing Compound ...... 50

(D) Distribution of Chlorogenic Acid Isomers . 51

(E) Identification of Scopoletin in Hydrolysates or" Weigela Species 6o '(F) The Isolation and Identification of 2',4,4'-. Trihydroxydihydrochalccne from Viburnum davidli 60

(G) Identification of Flavonoids ...... 65

V DISCUSSION

(A) Distribution of Phenolic Acids ...... 91

(B) Plants Containing Hydrogen Cyanide Producing Compounds ...... 91

•(C) Distribution of Chlorogenic Acid Isomers . 92

(D) Presence of Scopoletin in Hydrolysates of Welgela Species and Hybrids . . . . 93

(E) Distribution of Flavonoids in the Caprif oliaceae 94

VI REFERENCES 115 LIST OF TABLES

PAGE

I Cytology of Caprifoliaceae 8

II Fluorescence, Color Reactions and Rf Values

of Phenolic Acids . . 4-5

III Distribution of Phenolic Acids 4-7

IV Rf Values of Chlorogenic Acid Isomers •. . . . . 52

V Distribution of the Isomers of Chlorogenic Acid Found in the Caprif oliaceae 54 VI Ultraviolet Spectral Characteristics of 2' , 4, 4-' -Trihydroxydihydrochale one and Related Compounds .' 62

VII Rf Values of Flavonoids ...... 68

VIII Distribution of the Flavonoids in the

Caprifoliaceae ...... 84

IX Flavonoids of Sambucus . . . 97

X Flavonoids of Viburnum 99 Xi Flavonoids of Lonicera 103 LIST OF FIGURES

PAGE

1 Chroffiatogram of Phenolic Acidi; 4-3

2 Structures>of Compounds Found in Caprifoliaceae ...... 67

3 Polygonal Representation of the Paired Affinities Indices of the Genera of the Caprif ol iaceae , , 107 INTRODUCTION

The Caprif oliaceae is a family of about 'four hundred,

species in 15 genera (Engler, 1964, p. 4-73). The family

occurs mainly in the temperate regions of the northern hem•

isphere. Members of this family are used extensively as

ornamentals. Five genera, totaling 14- species, are naturally

occurring over most of British Columbia (Taylor, 1966).

The family Caprifoliaceae. is described by shrubs

or rarely trees or herbs; leaves opposite normally without

stipules; calyx tube with 4 to 5 lobes; corolla tube with

4 to 5 lobes; stamens equal to the number of corolla lobes

and inserted on the tube; ovary inferior, 1 to 5. rarely to

8 celled, each cell with 1 to many ovules; fruit a berry,

drupe, achene or capsule.

One of the major problems in the classification of

the Caprifoliaceae has been the position of Sambucus. It was originally placed in the Caprifoliaceae (Fritsch, 1888) but in 1892 H5ch removed Sambucus from the Caprifoliaceae and placed it in a separate family. Since then most workers

have left it in the Caprifoliaceae with reservations.

The following work was undertaken to see if infor• mation obtained from phytochemical methods would support

Hoch's idea and to see how phytochemical evidence would 2

support the classification system of the Caprifoliaceae in

general.

In the literature there have been many reports of

various compounds found in members of the Caprifoliaceae but prior to this work no exhaustive phytochemical examina•

tion has been done. In this study 56 taxa in 10 of 15 genera

recognized by Engler were examined.. Included in the study were phenolic acids, the various "chlorogenic acids" and

flavonoids,, Tests for cyanogenetic compounds were also per•

formed .

A review of taxonomic research concerned with the

Caprifoliaceae is presented. In it the various categories arrived at using non-chemical evidence are described and discussed with particular emphasis upon the position occupied by Sambucus. A brief statement is also made concerning the

phytochemical position of the Caprifoliaceae with reference

to neighbouring families. LITERATURE REVIEW

(A) Early Classification Systems

Early taxonomists customarily split the Caprifolia- ceae into two tribes: the Sambuceae and the Lonicereae.

Bentham and Hooker (1873) made the separation into two tribes on corolla shape. In Tribe I, Sambuceae, they included Adoxa,

Sambucus and Viburnum with their rotate, and/or actinomorph:-.c corollas. In Tribe II, Lonicereae, they included Microsplen- ium, Triosteum, Symphoricarpos, Abelia, Linnaea, Lonlcera,

Leycesteria, Diervilla, Pen ta pyx is and Alseuosmia with their tubular, a:.id/or zygomorphic corollas.

Fritsch, in Engler and Prantl (1888), maintained that

Sambucus and Viburnum differ sufficiently from each other that they should not be put together in.the same tribe. He distinguished four tribes: Sambuceae (Sambucus), Viburneae

(Viburnum and Triosteum), Linnaeeae (Linnaea, Symphoricarpos and Dipelta) and Lonicereae (Lonicera, Diervilla, Alseuosmia and Leycesteria) . In a later paper Fritsch (1892) presentee, the same separations of the family again, stating that Sam• bucus and Viburnum are not similar enough to be put in the same tribe, Hoch (1892) agreed with Fritsch in his separation of the Sambucus into its own tribe but carried the separation 4

further and established a new family, the Sambucaceae,

(B) Current Classification of the Caprifoliaceae

Hutchinson (1948.) suspected the Caprif oliaceae to be of mixed origin. He felt that Lonicera and Linnaea. are related but that Sambucus and Viburnum are very different

from them. The last two have several separate stigmas with

short styles compared with slender styles and capitate stignas

of Lonicera and Linnaea, He also pointed out that Sambuc\is has large spreading cymes of small flowers resembling those of some members of the Hydrangeaceae. Hutchinson stated

that Viburnum is no doubt related to Sambucus and the cymes

in some viburnums have outer flowers which are very much larger and sterile like Hydrangea but he hastened to add

that this could be due to parallel evolution.

In his Genera of Flowering Plants, Hutchinson (1967)

suggested that Sambucus and Viburnum are the more primitive genera of the Caprifoliaceae. They have short divided styles and actinomorphic corollas. The difference between them is mainly in the leaves, those of Sambucus being compound which

is a more sdvanced type. He went on to suggest that the capsular fruit of Dlervilla made it somewhat primitive and that Triosteum is little more than a reduced Lonicera, with annual stems from a woody perennial rhizome with connate leaf bases so familiar in some species of Lonicera. 5

In discussing the geographical distribution of the family, Hutchinson pointed out that: Lohicera, Linnaea, Sam• bucus and Viburnum are found widely distributed in the north• ern hemisphere whereas only Sambucus and Viburnum are found

In the southern hemisphere. Diervllla occurs naturally only, in eastern North America, and its counterpart Weigela is native to eastern Asia,

Hutchinson divided the Caprifoliaceae into five tribes..

The Alseuosmieae was separated because it has leaves.which are alternate or crowded into false whorls while all the rest have opposite leaves. Sambuceae we,s split off because although its leaves are opposite they are pinnately compound. The other tribes have simple leaves, the Viburneae being separated because of its actinomorphic corolla, short and often three lobed style and occasional sterile enlarged outer flowers in the cyme. The Lonicereae and Triosteae both have more or less zygomorphic corollas with elongated styles; the Lon• icereae are usually trees or shrubs (woody) whereas the Trios• teae are herbs with annual stems from a perennial creeping rhizome.

Takhtajan (1969, p. 230) included the Sambucaceae in the Caprifoliaceae but expressed doubt that this should be done.

Cronquist (1968, p. 304) stated that some botanists have expressed doubt that the Caprifoliaceae should be maintained 6

as a separate family from the Rubiaceae. Hutchinson (1969, p. 130) suggested that Sambucus and Viburnum show more prim•

itive characters than are found in any. genus of Rubiaceae.

Hutchinson went on to state that comparison between the two families is scarcely possible owing to the great disparity

in size. Ferguson (1966) stated that the presence of stip• ules is probably the most useful character for distinguishing the closely related Rubiaceae. But- he went on to point out that there seems to be no single character which separates

the two families consistently. He also pointed out that •

Viburnum and Sambucus show affinities with the Cornaceae, a relationship supported by pollen morphology.

Kern and van Steenis (195D stated that the Caprifolia• ceae seems to be most related to Valerianaceae, the foliage

of Sambucus and the occurrence of valerianic acid in Viburnum adding weight to this argument. In discussing a possible mer• ger of the Caprifoliaceae into the Rubiaceae they take the

stand that this would be undesirable as the present genera

of the Caprifoliaceae would be distributed throughout the

Rubiaceae and their affinity and relationships with each

other would not be seen.

A very widely used classification of the Caprifolia-. ceae is that of Rehder (194-0, p. 827), He did not break the

family down into tribes but used the rotate corolla of Sam• bucus and Viburnum to separate them from the other genera. 7

Render's classification will be applied to the plants in

this study, His breakdown of the genera into sections will

be used in the discussion and this breakdown of the genera

will be applied to other work being discussed in the Liter•

ature Review,

Caprifoliaceae has long been of interest to taxon-

omists because of the occurrence of natural hybrids within

the genera Sambucus, Viburnum, Symphoricarpos, Lonicera and

Weigela. Table I lists the major cytogenetical work done

in the genera of Caprifoliaceae in this study. TABLE I

CYTOLOGY OF CAPRIPOLIACEAE

a Tax on n 2nl Reference

Abelii 16 Sax and Kribs (1930).

Abelia 36 Poucques (194-9)

Diervilla Sax and Kribs (1930)

Diervilla .36 Poucques (194-9) , Hardin. (.1968)

Kolkwitzia 16 Sax and Kribs (1930)

Linnaea borealis L. l6 32 Packer (1964)

Linnaea. borealis ssp. longiflora Hult." 32 Taylor and Mulligan (1968, Part 2, p. 109)

Lonicera 9, 18, Sax and Kribs .2? (1930)

Lonicera Poucques (1949), Janaki Ammai and Saunders (1952) and Green (1966)

Lonicera deflex- lea lyx Bot. Brown (1932)

Lonicera peri- clymehum L. 18 (?) Hagerup (1941) 9

TABLE I - Continued

Tax on n cn Reference

Sambucus 18 Sax and Kribs (1930)

Sambucus 36 Battaglia (194-6)

Sambucus burpceriana 38 Battaglia (1946)

Sect. Eusambucus

S. canadens is L. 36 Hounsell (1968)

S. canadensis var, maxima Sch. 36 Ourecky (1966)

S. nigra L. 36 Hounsell (1968)

Sect. Ebulus

S. ebulus 36 Hounsell (1968)

Sect. Botryosambucus

S. sieboldiana Graebn. 38 Hounsell (1968!

S. raoemosa L. var. arborescens Gray 38 Hounsell (1968)

S. raoemosa var. var, pubens Koehne 38 Hounsell (1968)

Symphoricarpos 9 Sax and Kribs (1930) 10

TABLE I - Continued

Taxon n 2n Reference

Viburnum 9 Sax and Kribs ; (1930)

Viburnum 9 Poucques (19^9)

n - chromosome counts of meiotic material

2n - chromosome counts of mitotic material 11

Using the classification of Render, Thomas (1961)

set up a table of chromosome counts. His study revealed

that the base chromosome number is nine but there were excep•

tions with eight or ten. Thomas suggested that nine is the

basic number as it is by far the dominant number in Viburnum .

as well as in the Caprifoliaceae in general. The x = 8

situation appears to have arisen by the loss of a chromosome

pair and x = 10 by the gain of a chromosome pair from species with x = 9. He found chromosome aberrations fairly common

in several species, which of course, would increase the chance

of chromosomes being gained or lost.

In 1962 Egolf published a major work on the cytology

of Viburnum. In it he discussed the taxonomy and phylogeny

of the genus along with a detailed listing of chromosome

counts. He discussed the work done by Thomas (1961) and

agreed that the basic chromosome number is nine and that

species with x = 8 and x = 10 arose by the loss or gain of

a chromosome pair. Egolf*s chromosome counts agree with

Rehder's taxonomic sections of the genus. Section Thyrososna

is composed entirely of species with a base chromosome number

of eight. Only diploids are found in sections Pseudopulus.

Lentago, Megabotinus, Opulus, and Lantana with two excep•

tions. These exceptions are Viburnum carlesii Hemsl. (2n =

18, 20, 22) and V.-lantana (2n = 2?). Tetraploids and higher

polyploids are found in sections Thyrososma, Tinus, and Odon-

totinus. 12

(D) Other Systematic Work on Caprifoliaceae

Many researchers who worked on the Caprifoliaceae used Rehder's classification system.. Zaitsur (1958) suggested that greater precision in the intraspecific taxonomy of Ion• ic era could be obtained using fruit and seed characteristics;,

A technical study on the embryology of this family by Edith

Moisse (194-1) "completed" the general picture of embryo- logical data on the Caprifoliaceae. Her study yielded no differences between genera that could be used in the present study.

In a systematic study of Kolkwitzia, Weberling (1966) noted that morphologically the genus shows a closer affinity to Abella than to Lonlcera. He went on to suggest that Kolk• witzia and Abelia should be placed with Linnaea.rather than

Lonicera, In the case of Diervilla and Weigela, Bailey (192:9) suggested that the compound group passing as Diervilla be divided into three natural parts or genera "on morphological, distributional and probably phylogenetic grounds." He stated that the genus Diervilla be restricted to three species native to eastern North America: D. rivularis Gatt., D. sessilifolla

Buckl. and D. lonicera Mill. It is interesting to note that all the We Igela spp. have been called Diervilla at different times but the reverse is not so.

In discussing the phylogeny of some genera of the

Caprifoliaceae, Sax and Kribs (1930) suggested that there 13

were several species of Symphoricarpos in China and that

they have all vanished except one while those in North Amer•

ica have flourished. In a note to Science, Davy (1930) agreed with Sax and Kribs that the origin of the family Caprifolia--

ceae is probably Asiatic but questioned the assumption that

there was at'any time more than one species of Symphoricarpos

in China. Davy suggested that to reach the present level

of development that Caprifoliaceae has reached today only

one species of Symphoricarpos was necessary.

In 194-1 Fernald reinstated the name Viburnum edule

(Michx.) Raf. using V, pauciflorum LaPylaie as a synonym;

this was based on petiole differences. He pointed out that

V. edule is very closely related to V. trilobum Marsh, and

V. opulus L, and there had been much confusion but real differ•

ences could be seen in the petioles. Svenson (1940) suggested

that V. den, ta turn L. var. pubescens Ait. and V. venosum Britt. are closely related. Wirth (1941) found that V. cassinoides

L, and V. prunifolium L. are similar from the point of view

of their pharmacognosy.

Cooper (1939) carried out a study of the pericycle

of the Caprifoliaceae. In this study he pointed out that

the only tree forms are Sambucus and Viburnum. These genera would appear to be the most primitive of the family on the basis that the arboreal habit is considered to be primitive

in the angiosperms. The greatest number of species in the 14

Caprifoliaceae are shrubs, with a few woody climbers occurr• ing in the genus Lonicera.

Cooper found that very long fibres are common in the pericycle of all the shrubs and woody climbers (except

Linnaea). It was suggested that the fibres may serve to protect the phloem against crushing in the young stems.

The restricted northern distribution and dwarf growth form of Linnaea set it apart from the rest of the family, and structurally speaking, it may be interpreted as a reduced type. In the tree forms, Viburnum shows the least develop• ment of the pericycle fibres, both in number and in individual size. In Sambucus the fibres are also very short but occur in greater numbers.

Wilkinson (1948a, 1948b, 194-9)., ' in her series of papers, did some excellent work on the floral anatomy and morphology of the Caprifoliaceae. In a paper on Triosteum

(1949), she stated that the floral anatomy and morphology of Triosteum are generalized as compared with the unusual and complex: structure of the flower of Viburnum. She sugg• ested that these differences are striking enough to prevent

Triosteum from being placed in the tribe Viburneae where

Fritsch (1888) had placed it. Sambucus and Viburnum are quite distinct from Lonicereae and I.innaeeae and Viburnum is sufficiently distinct as to be set apart from any other genus. Triosteum seems somewhat similar to Sambucus 15

anatomically, but shows some similarites to Lonioereae and

Linnaeeae, She felt that the relationship between the

Caprifoliaceae and the Cornaceae was strongest between Sam• bucus and the subfamily Cornoideae. She went on to state that the structures of Lonicereae and Linnaeeae are general-, ized compared to the Cornaceae and it is doubtful that they had their origins in Cornaceous stock. Linnaeeae shows a significant similarity to Valerianaceae which seems to be more advanced in the same line as Linnaeeae.

Jcnes (19'40), in his monograph of the genus Symphori• carpos , discussed the 16 known species in North America.

He divided the genus into two subgenera: Eusymphoricarpos with its type species S. orblculatus Moench and including the two species in this study: S. occidentalis Hook, and

S. albus Elake; and Anisanthus with its type species S. mlcro- phyllus Humboldt, Bonpland and Kunth. In his key to Symphori• carpos , Rehder includes only eight species and uses no break• down into subgenera.

(E) Chemistry of the Caprifoliaceae

Gibbs (1958) did extensive biochemical work on dico• tyledonous plants in trying to establish their relationships to each other. In this paper he discussed his tests and 12 families to which he applied them. In discussing the Capri- foliaceae he found them chemically mixed. Viburnum and Sambucus 16

gave- positive HCl/methanol tests, with a few exceptions, while the rest of the family was consistently negative.

The HCl/methanol test consists of immersing wood chips in a reagent made from 25 ml of cone HC1 in 1000 ml methanol.

A positive reaction is indicated by a magenta color while in.a negative reaction the wood chips retain their buff color.

What is being tested for is not conclusively known but it has been suggested that it is the presence of catechol tannins.

Using a test for cyanogenetic glycosides he found that Sam• bucus was positive and Viburnum although positive did not-give a test typical of those obtained, with known cyanogenetic gly• cosides. The rest of the family was negative... Gibbs (per• sonal communication) has suggested that- the positive tests from Viburnum probably indicate a different type of compound from the typical cyanogenetic compounds found in Sambucus.

The results of his tests were consistent with the view that the Rubiaceae and the Caprifoliaceae are related to each other.

An excellent review work on the chemical constituents of plants was prepared by Hegnauer (1964); it has been re• ferred to extensively during the course of this work. Chloro• genic acid was found in the leaves of Sambucus japonlca Reinw. and Lonicera sp. (Gorter, 1909). Imaseki (1959) found in

Viburnum: chlorogenic acid (Ch), isochlorogenic acid (I), neochlorogenic acid (N) and Band 510 (X): Viburnum dilataturn 17

Thunb. (Ch, I, N), V. wrightii Mlq. (Ch. I, N, X) , V. sie- boldli Miq, (Ch, I, N), V. tomentosum Thunb, (Ch, I, N, X),

V. phlebotrichum Sieb. & Zucc. (Ch, I, N, X), V. sargenti

Koehne (Ch, I, N), V. furcatum BI. (Ch, I), V. sp. (Ch, I,

N, X), V. japonicum Spreng. (Ch, I, N) , V. carles ii Hemsl. var. bitchuerse Nakai (Ch, I, N, X), V. braehyandrum Rehd.

(Ch, N, X) ,. •

Leucoanthocyanins were found in the leaves of:

Viburnum lantana L., Dipelta floribunda Maxim., Kolkwitzla amabilis Gj^aebn. , two Weigela hybrids, SambuCus ebulus L, ,

Sambucus nigra L., Abe11a X grandiflora Rehd., Diervilla decora Nakai, Leycesteria formosa Wall, Lonicera nitida VIiis h- setifera Franch., Alseuosmia quercifolia A. Cunn,, A. lineariifolla A. Cunn.. and A. banks ii A. Cunn. (Bate-Smith, 1962).

Most phenolic compounds appear to accumulate in the

Caprifoliaceae in the form of glycosides (Hegnauer, 1964).

Stoh et al (1967) found 1-caffeoyl- p-D-glucose and 1-ferulc

p -D-glucose in Sambucus nigra L.

The coumarin glucoside fraxin was present in the dried roots of Diervilla canadensis Willd. (Charaux, 1911) and D. diervilla Macm. . (McCullagh et al, 1929). Esculin was found in the roots of Symphoricarpoa occidental is Hook, and esculetin was found in the wood of S, rivularis Suksdorf.

On the basis of the fluorescence of the extract, Plouvier 18

(1951) presumed that esculetin or a similar glucoside was present in selected species of Dipelta, Leycesteria, Lonicera and Welgela. The conversion of caffeic acid to esculetin

is relatively simple. Bubbling oxygen through a methanol

or acetic acid solution of cis-caffeic acid produces esculetin

in high yield. Trans-caffeic acid can be converted to the cis-isomer by irradiation with ultraviolet light (Butler and Siegelman, 1959; Kagan, 1966). Therefore any plant ex• tract exposed to both air and light could possibly contain esculetin which had been formed from caffeic acid.

Coniferin and syringin were isolated from a large number of Lonicera species (Plouvier, 1951). The glucoside, salicin, was found in Viburnum prunifollum (Evans et al,

1945. iwamoto et al, 19^5).

Hattori and Imaseki (1959) found a new glycoside with a phenolic aglycone in Viburnum furcatum Blume and called

it furcatin. Imaseki and Yamamoto (1961) described a glyco- sidase from V. furcatum which hydrolyzed furcatin into the aglycone p-vinyl phenol and apiosyl-1,6-glucose. Furcatin was not. found in 11 other species of Viburnum.examined.

King and Schwarting (194-9) isolated rutin (quercetin-

3-rhamnoglucoside) from the leaves and flowers of Sambucus canadensis L. Stroh (1958) observed that flowers of Sambucus nigra contained large amounts of rutin, quercetin-3-glucoside, chlorogenic acid and caffeic acid. Lonicera japonica Thunb. 19

contains lonicerin, the 3 rhamnoglucoside of luteolin.

Bobbitt and Rao (1965) checked the fruit of Viburnum opulus

for the bitter principle but found 310 glycosides responsible

for this bitter taste. From the flowers of V. opulus, Egger

(1962) isolated and described astragalin (kaempferol-3-

glucoside) and paeonoside (kaempf erol-3, 7-d.iglucoside) as

the principal glucosides. Traces cf quercetin glycosides were also found. In 1966 Plouvier reported finding acacetin

7-rutinoside in Kolkwitzia amabills Graebn. leaves. ,Iso-

fraxoside (fraxetol-8-glucoside) was reported from Diervilla

lonicera (Plouvier, 1969).

Amentoflavone was found in the bark of Viburnum pruni-

folium by Horhammer et al (1965). This biflavonyl had been

reported only in the gymnosperms until- Horhammer et al re•

ported its occurrence in this species of advanced angiosperms.

Horhammer et al (196?) also reported scopoletin and esculetin

in the bark of V_;_ prun if olium and DL-catechin in the bark

of V. opulus.

In discussing the anthocyanins it is Interesting to

note that Novell! (1953) reported a new nuclear staining dye

extracted from the ripe fruit of Sambucus nigra. This stair

showed a pH-dependerit color change from red to blue and was

probably the anthocyanin, sambucyanin. Reichel et al (1957,

i960) reported four anthocyanins in the fruit of Sambucus

nigra: chrysanthemin, cyanidin-3-Q sambubioslde and two 20

unidentified pigments. The new sugar sambubiose was identi• fied as xylose-lj?-^2-glucose.

Yoimgken (1931) and Youngken and Munch (194-0) studied the pharmacognosy of Viburnum alnifolium (which contains an unknown toxic principle) and V. lentago' and discussed their history, structure and pharmacological effects.

Plouvier (19^4) reported the presence of ursolic acid in the flowers of Viburnum opulus L. var. sterile DC.

Valeric acid was reported (Kolodynska and Praezko, 1966) in the dried bark of Sambucus nigra. Heyl and Barkenbus (1920) reported valeric acid present in Viburnum tinus L., V. lantana

L., and V. prunifolium.

Iii a study of the carotenoids of the berries of

Lonicera japonica, Goodwin (1952) found phytofluene,p -caro• tene,^ -carotene, ^ -carotene, lycopene, crjrptoxanthin, zea- xanthin, auroxanthin and ^ -carotene. Huneck e_t al (1965) found betalic acid, betalin and amyrin, £2 -sitosterol, ceryj.- alcohol, and tj -nonacosane in the non saponifiable fraction from the light petroleum extract of the bark of Sambucus racemosa. Using the same extraction procedure on the bark of S. nigra, Laurie et al (1964) found ^-amyrenone, c< -amyrin, betulin, oleanolic acids and ^-sitosterol.

Horhammer e_t al (1967) reported the presence of sitosterin,

at Kew Gardens, found saponin in the leaves of Abelia unl- flora R. Br., Diervilla japonica DC., Lonicera caprifolium

L. , L. japonica Thunb., L. ledebourii Esch. , L. morrow ii

Gray, L. standishii Hook., L. tartarica L., L. somentella

Hook, et Thomps. , L. xylosteum L. , Symphoricarpos mollis

Nutt., S. racemosa Michx. and Viburnum macrophyllum Thunb.

He failed zo find any cyanogenetic glycosides in these plants.

Bourquelot and Danjou (1909) reported the isolation

of the cyanogenetic glucoside sambunigrin from Sambucus nigra and its absence in Sambucus ebulus L, and S. racemosa L,

Mannitol was found in Viburnum rhytidophyllum Hemsl. but not in 25 other species of the family (Plouvier, 1951).

A sugar alcohol, viburnit, was isolated from Viburnum tinus

L. (Hegnauer, 1964).

Hegnauer (1964) suggested that the occurrence of alkaloids in the Caprifoliaceae was doubtful. The -hydro• chloride of a base having chemistry and pharmacological activ•

ity similar to coniine was isolated from Sambucus nigra (De

Santis, 1894). Another crystalline hydrochloride of a base was isolated from the leaves and bark.of S. nigra; the base was named sambucin (Malmejic, 1901) ,. Hegnauer hastened to point out that later investigations did not reveal either

of these compounds.

In a phytoserological study, Hillebrand and Pairbrothers

(1966) found that the three tribes Lonicereae, Linnaeeae, 22

and Diervilleae form a close serological unit, while the

Viburneae and Sambuceae are distinct from the other tribes and from each other. They also pointed out that there is greater serological correspondence between Cornus (Cornaceae) and the Caprifoliaceae than between representatives of the

Rubiaceae and the Caprifoliaceae. MATERIALS AND METHODS

(A) Collection of Plant Material

The plants studied in this work are listed below.

Voucher specimens have been deposited in the University of

British Columbia Herbarium, .

Accession Collection . No. No. Plant Source

125335 1 Viburnum rhytidophyllum U.B.C.a

Hemsl.

125366 2 Viburnum lantana L. U.B.C.

3 Viburnum davidii Franch. U.B.C.

125365 4 Viburnum opulus L. var. U.B.C.

roseum L.

125364 5 Viburnum carlesii Hemsl. U.B.C.

12534-1 6 Symphoricarpos albus U.B.C. Blake 125337 7 Viburnum sargenti U.B.C.

Koehne

125843 8 Viburnum dentatum L. U.B.C.

125361 9 Viburnum tinus L. U.B.C.

125362 10 Lonicera pileata Oliv. . U.B.C. 125330 11 Linnaea borealis L. C.C.P. ssp. longiflora Torr. 125367 12 Viburnum trilobum U.B.C. Marsh. 24

Accession Collection No No. Plant S ource

13 Lonicera ciliosa Poir. U.B.C.

125342 14 Abe 11a X grandlflora U.B.C. (Andre) Rend. = (A. chinensis R. Br. X A. uniflora R. Br.)

125372 !5 Viburnum edule (Michx.) U.B.C. Raf. = (V. pauciflorum LaPylaie)

125339 16 Weigela "Bristol Ruby" U.B.C. X "Abel Ci?.rriere" hybrida Hort.

17 Sambucus racemosa L. U.B.C, ssp. racemosa 18 Lonicera utahensis M.P. S. Wats.

19 Lonicera involucrata M.P. Banks.

20 Lonicera tartarica U.B.C. L. var. alba Lorsel.

21 Sambucus racemosa L. U.B.C. ssp, pub ens' (Michx. ) House

125369 22 Sambucus racemosa L. U.B.C. ssp. racemosa = T§_. callicarpa Greene)

125368 23 Lonicera nltida Wils. U.B.C.

125338 24 Weigela floribunda U.B.C. (SlebT-and Zucc.) • Mey.

125348 25 Weigela florida (Sieb. U.B.C. and Zucc.) A, DC. 25

Accession Collection No. No. Plant S oure e

125359 26 Viburnum X burkwoodii U.B.C. Burkw. = (V. carlesii Hemsl. X V. utile Hemsl.)

125370 27 Viburnum opulus L. U.B.C. var. nanum David.

125336 28 Kolkwitzia amabilis U.B.C. Graebn.

125340 29 Viburnum dentatum L. U.B.C. var. venosum Britt.

30 Sambucus coerulea Raf. Montana

31 Lonicera xylosteum L. Rhode Island •

32 Lonicera sempervlrens Rhode L. . Island

33 Viburnum recognition Rhode Fern, Island

34 Sambucus canadensis Rhode L. Island

125329 35 Linnaea boreal is L. Williams ssp. americana Lake,B.C. (Forbes) Halten.

125344 36 Symphoricarpos 00c i- Williams dentalis Hook. Lake,B.C.

125345 37 Lonicera involucrata Williams Banks. Lake,B.C.

38 Viburnum opulus L. Nova Scotia var. roseuia L.

39 Viburnum trilobum Nova Scotia Marsh.

4o Lonicera oblongifolia Nova Scotia Hook. 26

Accession Collection No. No. Plant Source

41 Sambucus canadensis L. Nova Scotia 125371 42 Sambucus nigra L. Nova Scotia 125349 45 Triosteum sinuatum M.B.G. Maxim. b 46 M.B.G. Lonicera sp. 125331 47 Weigela hortensis M.B.G. (Sieb. and. Zucc . ) Mey. 125353 48 Diervilla rivularis M.B.G. Gatt.

125347 49 Abelia X grandiflora M.B.G. (Andre) Rehd. = (A. chinensis R. Br. X A« tin if lora R. Br.) 125350 50 Lonicera maackll M.B.G. Maxim. 125351 51 Weigela hybrida Hort. M.B.G. var. nivea Bonard.

125346 52 Abelia X grandiflora M.B.G. (Andre) Rehd. = (A. chinensis R. Br. X A. uniflora R. Br.) 125358 53 Lonicera myrtillus M.B.G. Hook. var. depressa (Royle) Rehd.

125332 54 Triosteum fargesii M.B.G. Franch,

125333 55 Triosteum pinnati- M.B.G. fidum Maxim.

125352 56 Weigela florida (Sieb. M.B.G. and Zucc.) A. DC, forma variagata Sie- bold. 27

Accession Collection No. No. • • Plant Source

125354 57 Lonicera ledebourii M.B.G. Esch. 125334 58 Lonicera syringantha M.B.G. Maxim, var. grandiflora Maxim.

125355 59 Viburnum urceolatum M.B.G. (Sieb. and Zucc.)

125356 60 Weigela praecox M.B.G. TLemoine) Bailey 125357 6l Viburnum.prunlfolium M.B.G. L.

a U. B. C. - University of British Columbia, Vancouver, C. C. P. - Cypress Creek Park, B. C; M. G. B. - Montreal Botanical Garden; M.P. - Manning Provincial Park, B, C.

b ~ Number 46 was not identified beyond the genus name. 28

(B) Extraction of Plant Material

The plant material was killed toy blending in boil•

ing 95 per cent ethanol in a Waring blender to which a small

amount of CaCO^ was added to keep the extracts neutral.

After exhaustive extraction (3 changes of ethanol) the plant,

material was discarded and the combined ethanol extracts were evaporated to dryness under an air jet. The resulting

residue was taken up in a small amount.of hot water and filtered

through a bed of Gelite using suction. The filter pad was

exhaustively washed with hot water after which the Celite

•containing the water insoluble fraction was discarded. The water soluble fraction was then divided into two aliquots: a small one for the study of phenolic acids and a larger

one for ethyl acetate extraction. The latter was extracted

exhaustively with ethyl acetate in a continuous liquid-liquid

extractor, after which the ethyl acetate fraction was. saved and the water soluble fraction discarded.

The smaller aliquot from above was divided into three parts, (a), (b), and (c) which were treated in the following

manner: (a'; was made 2N with HC1; (b) was made 2N with

NaOH; and (c) was left neutral. Samples (a) and (b) were heated at 80°C for 3.0 minutes. After cooling (b) was made 29

slightly acid by adding HC1. All three samples were then extracted separately with ethyl ether using a separatory funnel. Five portions of ether were used for each sample, the extracts combined and taken to dryness under an air jet.

The water residues were discarded.

After being blown to dryness the ether-soluble resi• dues were each taken up in a small amount of 95 per cent ethanol and spotted on sheets of Whatman No. 1 chromatography paper. The chromatograms were developed in two dimensions using the organic layer of benzene:acetic acid:water (10:7:3) for the first direction and 2 per cent formic acid for the second. The chromatograms were air dried and inspected under ultraviolet light, using both long wave (366nm) and short wave . (?53.?nm). The chromatograms were exposed to ammonia fumes and re-examined with ultraviolet light of both wave lengths. The ultraviolet lamps used were Mineralights manu• factured by Ultraviolet Products Inc., San Gabriel, California.

Any observed changes were recorded. After examination under ultraviolet light the chromatograms were sprayed with diazotized p-nitroaniline which was prepared by mixing:

5 nl 1% p-nitroaniline in 8% HC1

1 ml 5% sodium nitrite (NaN-C^),

15 nl 20$ sodium acetate.

The chromatograms were then oversprayed with 5 per cent NaOH solution which develops the color characteristic of each 30

phenolic acid. The presence or absence of the common phen•

olic acids was then recorded.

(D) Examination of Plant Material for Hydrogen Cyanide Producing Compounds

Small samples of each unhydrolyzed plant extract were placed in 125 ml round bottom flasks fitted with glass

stoppers. The samples were dissolved in water and a small amount of emulsin added. The emulsin was obtained commercially

from Nutritional Biochemicals Corp., Cleveland, Ohio. A narrow strip of picric acid paper was suspended over the

solutions by inserting it between the ground glass neck of

the flask and the ground glass stopper. The picric acid

paper was prepared-by soaking Whatman No. 1 paper in 1% picric acid solution and air drying. This paper was then cut into

strips and stored in an air tight jar. Each strip was moistened with 5% Na^CO^ solution prior to insertion. into the flasks.

After the insertion of the picric acid paper the flasks were left overnight on the laboratory bench. In a positive re• action the picric acid paper turned dark brown while in a negative reaction the picric acid paper remained bright yellow.

The above test was carried out on herbarium specimens with the following modification. Instead of an extract being prepared, a leaf from the dried herbarium specimen was crumbled directly into a small amount of water in the round bottom flask. 31

(E) Identification of Sambunigrin

An isolate of impure sambunigrin was made from the ethyl acetate soluble fraction in the following manner.

A small portion of the ethyl acetate soluble fraction was streaked on sheets of Whatman No. 3 MM chromatography paper and run in butanol:acetic acid:water (4:1:2.2). The proper band was located by cutting a strip off the air dried chromatogram and spraying it with a solution of emulsin in water.

After incubation of this strip in a warm, moist, glass chamber the correct band could be located by the odor of the released

HCN and benzaldehyde. This band was cut out and eluted.

The amount of solid obtained in this manner was insufficient for identification and was hydrolyzed by brief warming in

2N HC1 followed by extraction with ether. The ether soluble fraction was spotted on Whatman No. 1 paper together with separate spots of benzaldehyde and p-hydroxybenzaldehyde.

These chromatograms were run one dimensionally in the organic layer of benzene:acetic acid:water (10:7:3) then sprayed with

2,4-dinitrophenylhydrazine to locate the aldehydes.

(P) Identification of Chlorogenic Acid Isomers

The chlorogenic acid isomers were identified using the chromatographic system of Grodzinska - Zachwieja ejb al

(1967). Glass plates (18 cm x 56 cm) were spread with a 32

slurry of Silica Gel G from E. Merck A. G., Darmstadt, Germany, prepared by shaking 20 gm Silica Gel G and 50 ml of 2.5$ aqueous KHSO^ per plate. The plates were activated at 100°C in a drying oven for one hour before spotting.

Standard samples of 4-caffeoylquinic acid, 5-caffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid were generously provided by Dr. Warren Steck, National Research Council. Chlorogenic acid (3-caffeoylquinic acid) plus a sample of each of the above isomers were spotted- on each plate along with the unhydrolyzed plant extracts. These chromatograms were then developed in acetophenone:methylethyl ketone (3:1) for 10 hours and air dried in a fume hood overnight. The chlorogenic isomers appeared as dull gray spots after spraying or fuming with cone NH^OH„ A small sample of each of the standards was subjected to the isolation procedure so that it was treated as were the plant extracts. This would determine if the extraction procedure caused a migration of the caffeoyl moiety from one position to another on the quinic acid.

(G) Identification of Scopoletin in Hydrolysates of Weigela Species

Fresh leaf material was collected and exhaustively extracted with boiling 95$ ethanol after which the combined extracts were evaporated to dryness. The residue was extracted 33

with hot water and the extract filtered through Celite, made

2N with cone HG1 and hydrolyzed by heating for 30 minutes.

The hydrolysate was continuously extracted with ether for

12 hours. Evaporation of the ether left a syrup which was taken up in a small volume of ethanol and applied as spots to sheets of Whatman No. 3 MM paper and chromatographed two-- dimensionally using the organic layer of benzene:acetic acid: water (10:7:3) and then 2%- formic acid in water.

The spots were located by their fluorescence under ultraviolet light and were cut out and eluted with ether.

After removal of the ether the residue was sublimed at 100°/

0.15 mm. The sublimate was recrystallized from a methanol and water mixture to yield colorless crystals, with a small amount of brown residue. This residue was removed by brief washings with cold ether. Authentic scopoletin was recrys• tallized from methanol and water to yield colorless crystals: for the following tests. Melting points of both isolated and authentic material were taken along with a mixed melting point. Ultraviolet and infrared spectra were also taken of both the isolated and authentic scopoletin. Ultraviolet spectra were run on a Unicam SP - 800 ultraviolet spectro• photometer and the infrared spectra were run on a Unicam SP -

200 G Grating Spectrophotometer (Glennie and Bohm, 1968). 34

(H) The Isolation of 2',4,4'-Trlhydroxydihydrochalcone from Viburnum davidil

This compound first appeared as an unknown on chroma• tograms examined for phenolic acids. Fresh leaf material of

V. davidii was extracted repeatedly with boiling 80$ ethanol.

The alcoholic extracts were pooled and evaporated to dryness under an air jet. The residue was taken up in hot water and filtered through Celite filter aid. The filtrate was hydrolyzed with either 2N HC1 at 100° for 1-2 hours or ernulsin

(ca. 25 mg per 100 ml of extract) at room temperature for one day. After hydrolysis, the aglycone was extracted with ether and the ether fraction was concentrated and spotted on Whatman No, 3. MM paper. These chromatograms were developed two dimenslonally with the organic layer of benzene:acetic acidrwater (10:7:3) followed by 2$ formic acid in water for the second direction. The compound was located under ultra• violet light and eluted with 95$ ethanol. The ethanol was evaporated leaving a residue which crystallized slowly from water. This crystalline material was dissolved in 95$ ethar.ol and banded on cellulose MN 300 G (Mackerey, Nagel and Company,

Germany) thin-layer plates and run in 20$ formic acid to remove trace impurities. The correct band was located under ultraviolet light and eluted with 95$ ethanol. The ethanol soluble residue was recrystallized several times from water to give pale yellow crystals (Bohm and Glennie, 1969). 35

The glycoside was obtained by taking the water soluble extract from Celite filtration and extracting it continuously with ethyl acetate in a continuous liquid-liquid extractor.

The ethyl acetate soluble fraction was banded.on Whatman No,

3 MM paper and chromatographed in the following solvents:

2% formic acid in water; t-butanol:acetic acid:water (3:1:1); and ethyl acetate:formic -acid:water (20:15:10). After each chromatographic separation the correct band was located under ultraviolet light, eluted, and banded on fresh paper before running in the next solvent.

(1) Identification of the Sugar Residue

Small portions of the glycoside were hydrolyzed by heating with 2N HC1 for one hour after which the reaction mixture was extracted exhaustively with ether. The remain• ing aqueous solution was concentrated and spotted on Avicel

(microcrystalline cellulose) plates using a number of common sugars as standards. The solvents used were n-butanol:acetic acid:water (4:1:2.2) and n-butanol:benzene:formic acid:water

(100:19:10:25). The positions of the sugars on the plates were determined by spraying with a periodate-benzidine reagent

(Hollman, 1964, p. 183).

The periodate-benzidine test for sugars was carried out in the following manner: 0.228 gm H^lO^ (periodic acid)

were dissolved in 10 ml Ho0 and this was diluted to 200 ml 36

with acetone. 0.184- gm of benzidine were dissolved in 0.6

ml glacial acetic acid plus 4.4 ml H20 and then diluted to

100 ml with acetone. The chromatogram was sprayed with the periodic acid solution and then air dried briefly before spraying with the benzidine solution. On drying, the back• ground is colored deep blue, the chromatogram remaining white or yellow at the positions where the periodate oxidiz- able sugars occur.

(J) Determination of the Glucose - Aglycone' Ratio

A portion of the glucoside was hydrolyzed with acid as in the sugar residue determination. This mixture was then extracted exhaustively with ether. The ether extract was evaporated to dryness, the residue taken, up in 95% ethanol, made to a standard volume, and aliquots therefrom analyzed spectrophotometrically. This analysis was carried out at

278 nm on a. Beckman DU spectrophotometer. Results of this analysis were compared to a standard curve prepared by measur• ing the absorbance of different concentrations of crystalline

2',4,4'-trihydroxydihydrochalcone in 95$ ethanol.

The aqueous solution, free of the phenolic compound, was concentrated using a rotary evaporator and the residual material meide to a standard volume using distilled water.

The concentration of glucose present was determined using the procedure of Nelson (1944). 37

. (K) Base Cleavage of the Aglycone

Twenty-five mg of the aglycone and 2-3 ml of 50$

NaOH were heated at 100°C for two hours. The mixture was

acidified with 6N HC1 and extracted several times with ether

using a. separatory funnel. After the ether was evaporated

the residue was taken up in a small amount of 95$ ethanol

and spotted on Avicel plates for chromatography. These plates

were developed two dimensionally in the organic layer of

benzene:acetic acid:water (10:7:3) and 2$ formic acid. After

air drying they were examined under ultraviolet light and

sprayed with diazotized p-nitroaniline. These chromatograms

were compared to chromatograms, treated in the same manner,

of the uncleaved aglycone, resorcinol and phloretic acid.

The standards and the cleavage products were spotted

on Whatman No. 1 paper and run in the benzene:acetic acid:

water and formic acid solvents. After air drying the spots

were located under ultraviolet light and eluted with 95$

ethanol. Ultraviolet spectra were run of all compounds using

a Unicam SP 800 Recording Spectrophotometer.

(L) ^C Precursor Studies

Two micrpcuries of labelled precursors phenylalanine- 14 14 14 U-" C, cinnamic acid -2- C, and p-coumaric acid -2- c were

dissolved separately in about 3 ml of water. The acids were 38

converted to their sodium salts by adding sufficient IN NaOH to keep the pH of the solutions about ?. These solutions were administered through cut stem ends of V. davidii shoots which had 3 pairs of leaves. The plants were allowed to metabolize for 24 hours under cool-white fluorescent lamps.

When the labelled precursors had been absorbed (about 1 hour) distilled water was added to the feeding vials for the remainder of the metabolic period. The plant material was extracted and the aglycone isolated by the procedures previously des• cribed.

The radioactivity of the aglycone from each experiment was determined in the following manner. Three aliquots of the aglycone vrere placed in counting vials and 15 ml of counting solution was added. The counting solution contained 0.05 gm

POPOP, 1,4 di[2-(5-phenyl oxazolyze)J benzene from Kent Chemi• cals Ltd., Vancouver, 4 gm PPO, 2,5 diphenyloxazole, from

Fraser Medical Supplies Ltd., Vancouver, 625 ml toluene and

375 ml ethanol. The samples were then counted in a Nuclear

Chicago Liquid Scintillation Counter (?20 Series).

(M) Identification of Flavonoids

The ethyl acetate soluble fractions from the plant extracts were spotted separately on. Avicel plates and chroma• tographed two dimensionally in t-butanol:acetic acid:water

(3:1:1) and. 15$ acetic acid. These chromatograms were air 39

dried and examined under ultraviolet light. The spots were

circled and described in all respects. The plates were then,

exposed to NH-^ and any changes noted. As a permanent record

these chromatograms were then copied on tracing paper with

a full description of the spots, These Avicel chromatograms

gave identical patterns as found on. Whatman No. 3 MM chromato•

grams .

Aliquots of the ethyl acetate soluble material were

spotted on Whatman No. 3 MM paper and developed two dimension-

ally in the t-butanol:acetic acidiwater and acetic acid sol•

vents. The spots were located under ultraviolet light, cut

out and eluted with 95$ ethanol. Ultraviolet spectra were

run on these ethanol solutions in the following manner.

First, a spectrum of the ethanol solution of the spot was

obtained; then a small amount of solid anhydrous sodium acetate was added to the cell and shaken to produce a saturated solu•

tion (the excess solid settled to the bottom) and a spectrum

taken, To this same solution a small amount of solid boric

acid was added, the cell again shaken and the spectrum run,

A fresh sample of the ethanol solution was taken and 1 drop

of 0.1N NaOH added and after shaking a spectrum run. The final reagent to be used was AlCl^; 2 drops of a saturated

AlClj solution in 95$ ethanol were added to a fresh sample before the spectrum was run.

The remaining ethanolic solutions were evaporated 40

to dryness under an air jet and the residue hydrolyzed by heating with 2N HC1 for 30 minutes, After cooling, this aqueous, solution was extracted with ethyl acetate using a separatory funnel. Both fractions were taken to dryness under an air jet. The residue from the ethyl acetate frac• tion was taken up in a minimum of ethanol and spotted on

Whatman Nc. 3 MM paper for chromatography in t-butanol:acetic acid:water and acetic acid. The aglycone spot was located under ultraviolet light, cut out, eluted, and spectra run using the same reagents as used above for the glycosides.

The water soluble fractions were spotted on Avicel plates with standard sugars and run in one dimension in each of these solvents:' butanol: acetic acid:water (4:1:2.2) and buta.nol: pyridine: water (75:15:10). After the plates were air dried they were sprayed with a naphthoresorcinol spray made by dissolving 20 mg of naphthoresorcinol in 10 ml of ethanol containing 0.2 ml cone HgSO^. The plates were then heated in a drying oven to develop the color of the sugar spots. RESULTS

(A) Distribution of Phenolic Acids

The properties of the phenolic acids identified in

this, study appear in Table II. Identification was made by

comparison to the following standard phenolic acids: p-

coumaric (4—hydroxycinnamic), p-hydroxybenzoic, caffeic

(3,4-dihydroxycinnamic), vanillic (3-methoxy-4—hydroxy-

benzoic) , ferulic (3~methoxy-4-hydroxycinnamic), proto-

catechuic (3,4 dihydroxybenzoic ) , sinapic (3, 5-d-imethoxy-

4-hydroxyc.innamic) and phloretic (4--hydroxydihydrocinnamic ) .

The distribution of these compounds in the Capri-

foliaceae appears in Table III. The plant number refers to

the plant collection number, a, b,-and c refer to the treat•

ment: a-acid hydrolysis, b-base hydrolysis and c-unhydrolyzed

The unknowns listed here appear on the chromatograms but were not positively identified. A diagram of a chromatogram with all compounds found in this study is shown in Figure 1.

This corresponds to the chromatogram of Ibrahim and Towers

(I960) and shows the unknowns found in this study.

Unknown 1 was found only in the basic hydrolysis of

all plants examined; it occurred in small quantities on the

chromatograms and any attempt to isolate crystalline quantiti 42

of it failed. An ultraviolet spectrum was run of chromato- graphically pure material and it absorbed at 303 nm. Unknown

2 occurred only on basic hydrolysis but in just a few plants examined (Table III). Unknown 4 occurred in hydrolyzed and unhydrolyzed extracts of Linnaea borealis var. americana and unknown 5 was found in hydrolyzed and unhydrolyzed ex• tracts of Lonicera syringantha var. grandiflora. PIGURE 1 PIGURE 1 - Continued

1. p-Coumaric acid

2. Protocatechuic acid

3. Caffe ic acid

4. Hydroxybenzoic acid

5. Ferullc acid

6. Vanillic acid

7. Syringic acid

8. Phloretic acid

9. Unknown 1

10. Unknown 2

11. Unknown 3

12. Unknown 4

13. Unknown 5 TABLE II

FLUORESCENCE. COLOR REACTIONS AND Rf VALUES OF PHENOLIC ACIDS

UV Fluor• Color reaction Rf UV Fluor• escence with diazotized

Acid escence with NH-^ p-nitroaniline Bz:HOAc:H?0 2% formic

1. p-Coumaric Dark blue Bright blue Blue 18 ?i,33L

2. p^Hydroxybenzoic None None Pink 15 62

3. Caffeic Blue. Light brown fades 3 Yellow-green to white 58,29

4. Vanillic None Purple 50 None 52 5. Ferulic Blue Blue-green 53 62,25 Bright blue 6. Protocatechuic None Light brown 3 50 None 7. Light blue fades Sinapic Green 65 50,15. Green to white

8. Phloretic None None Purple-violet 40 79

9. Unknown 1 None None Blue fades 4 12 to red TABLE II - Continued

UV Fluor• Color reaction Rf UV Fluor• escence with diazotized Acid escence with NHo p-nitroanilme Bz iHOAc;H?0 2% formic

10. Unknown 2 Dark blue Dark blue Gold 14 42

11. Unknown 3 None None Blue fades 15 77 to gold

12. Unknown 4 None None Pink 23 81

13. Unknown 5 Blue Blue Yellow 20 70/35

a-The values given represent Rf x 100 in the organic phase of benzene:acetic acid:water (10:7:3) and 2% formic acid.

b-These values represent the cis and trans isomers of the cinnamic acids. TADLE III

DISTRIBUTION Or PHENOLIC ACIDS

SIN P H B PRO TO VAN PHLOR UNK 1 UNK 2 UNK 3 UNK 4 UNK PCAb abc abc abc . a h c a b

+ + + + + 4 4 + + 4 4 4 4

+ 4 + + + + + 4 4 4 4 4 •il Ab«li.i A gramHm*. + + + 4 4 4 52 AbrlU X utran^itlora + + • 4 4 4 4 4

28 K,,l»h.-..» ••.mal.ili. 4 + + 4 4 4

4 4

35 Mnnac.i hfir«:.ilia sap. 4 4 -i + + + 4 4 + 4 4 4- 4 4 4 4 4 4

13 T + + + 4 + 4 4 + 4 4 4

4 4 4 4 4 4 sp. 4 + + + + 4

19 l^onit.'era invclocr.tta + + • + + + 4 + 4 4 4 4

+ 4 + + + 4 + 4 4 4 4

4 4 57 L^jz-cra U:*i>Wrii + 4 4 + + * 4 4 50 Lor.it: cr* rr>.VKI:it 4- + 4 4 4 + + 53 4 + 4 4 + + 4 4 .4 4

23 Lor.-fera pttida + + . + + 4 + 4 4 4 4

40 Lonicera oblon gifolia + 4 + + + + 4 4 4 4 4

10 Lonic c r ;i [Ji 1 c.11a + + 4 +

3- Lonic era .^mpcvirens + +

58 Lonice r.i sv rin i',.iniha va gr.ir.di flora

tarta r ica var . alba 4 + + + + + 4 4

18 4 + 4 4 4 + 4 4 4 4

31 + + + +' 4 4 TABLE nr

DISTRIBUTION OF PHENOLIC ACIDS

P H B PROTO VAN PHLOR UNK 1 UNK 2 UNK 3 UNK 4 UNK 5 PCA b CAF FER a. b c a b c a b e a b c a b c a b c a b c a b c a b c a b c a b c a b c

30 Sambucua coerulea + + + + +

22 Sambucua racemosa + + + + '- + +

Sambucua canadennig + + +4 + + + + + +

41 Sambucua canadensis + + + + + + + + + + +++' + +

42 Sambucua nigra + + + + + + + + + + + +

2 1 Sambucun racemosa var. pubona

17 Samhurus r.ictmo5a var . + + • + +

6 Symphoricarpos albus + + +• + + T + + + + +

36 Sy m ph o r i ct r p o a + + + + + + occidentals ' + + + + + + + + + + + 54 Triostrum far tn_-aii + + + + + + +

55 Triosteum pimiatifidura + + + + + • + + + + + + 45 Triosteum ninnaturn + + + + + + + + + + + + + 2b Viburnum X burkwoodii + + + + ' + + + + + 5 Viburnum caricsii + + + + + + 3 Viburnum davidii + + + + + + + +

8 Viburnum dcnt;iUtm + 4 + + + + + + +

29 Vi bur num dentatum • + + + + + + + +

^ Viburnum lantana + + + + + + + + +

27 Viburnum opulim var . ii.iiiiiin i i + + 4 Viburnum opulua var. ruseuiti + + + +

co TABLE I II

DISTRIBUTION OF PHENOLIC ACIDS

PCAfc CAF F E R SIN. PHB PROTO VAN PHLOR UNK I UNK Z UNK 3 UNK 4 UNK 5 abc abc abc abc abc abc abc abc' abc abc abc a b c a b c

IS Viburnum odule + + . 4 4 4 4 T + + + +

61 Viburnum prunifolium + 4 4 4 4 4 + + t + + +

33 Vihurmim rccocmtiim + 4 4 4 4 4 . 4 + + + + • * 1 + +

I V-hu rrnitn rnvtwUnhvllum 4 4 4 + + + T + +

7 Viburnum Bargcnti + + 4 4 4 4 + T + * • % Viburnum tinus + + 4 4 4 + + T •+ + + U Viburnum trilohum 4 + 4 4 4 4 + + + + +

3? Viburnum t ri lohurn + + 4 4 4 4 + + + + + + +

59 Viburnum urcftotalum + 4 '+* 4 4 + + + + + . + +

16 Weii?<:la "Brirttol Ruby" X "Abel Car rie TC" hvbrida 4 + 4 4 4. 4 4 + + + + + + +

2 4 Wei^l,! floribunda 4 + 4 4 4 4 + + + +

25 Wei nr. ta flo rida + 4 4 4 + + + + +

47 V/tii y Ii ho rtcnsi a + 4 4 4 4

51 Wi-i^i-U h'/brida var. 4 nn'ga * 4 4 * 4

60 Wci^cla praecojc + 4 4 4

56 Wrigfila florid a forma varia^ata + 4 + 4 4 4

(a) PCA - coumaric acid; CAF - caffeic acid; PER - {erulic acid; (b) a .- acid hydrolysis

S!N - sinapic acid; PRO TO - protocatechuic acid; VAN - vanillic acid; b - base hydrolysis

SYR - syringic acid; PHLOR - phloretic acid; UNK - unknown. , c - neutral - no hydrolysis 50

(B) Plants Containing a Hydrogen Cyanide Producing Compound

All plants collected were tested for HCN producing compounds but the only positive tests were obtained with No.

17, 21, 22 and 42. These were all from the genus Sambucus.

Of the species of Sambucus tested only two gave negative results, S, coerulea and S. canadensis, The remaining taxa gave positive results: S, racemosa ssp.' racemosa, S. racemosa ssp. pubens, and S, nigra. As a point of interest the fruit; of S. racemosa ssp. racemosa was tested for HCN containing compounds but no trace of HCN could be detected.

Two dried herbarium specimens were tested but only one gave a positive result, S. glauca Nutt. (herbarium speci• men #74611). S. melanocarpa Gray (specimen #65656) yielded no HCN on emulsin hydrolysis. No significance can be placed on the negative results obtained from the dried herbarium specimen as the HCN producing compound, if present in the plant, could have been destroyed by'pressing and drying,

Since no fresh material was available to confirm the negative result it will be disregarded.

The isolation techniques yielded a very small amount of material which was not sufficiently pure to determine spectra and melting points. On hydrolysis the compound 51 released hydrogen cyanide and the ether soluble portion agreed with authentic benzaldehyde in Rf values and color after being sprayed with 2, 4-dinitrophenylhydrazine. Since the cyanogenetic glucoside, sambunigrin (Figure 2), has been previously isolated and described by Bourquelot and Danjou

(1905) no further attempts were made to isolate it in this study.

(D) Distribution of Chlorogenic Acid Isomers

Of the phenolic compounds present in the Caprifolia- ceae the caffeic acid bearing compounds appear to be present

in the highest concentration. As seen in Table II caffeic acid was present in every plant in both acid and base hydrolyz- able forms and appeared in the unhydrolyzed extracts of many plants.

The chlorogenic acid isomers were identified by Rf values and their similar behaviour patterns under ultraviolet and with ammonia as compared to standard compounds. Also, oh hydrolysis they yielded caffeic acid and quinic acid,

identified by chromatography with standards. The Rf values for the isomers are given in Table IV". TABLE IV

Rf VALUES OP CHLOROGENIC ACID ISOMERS

Acid Rf'

3- caffeoylquinic acid 55

4- caffeoylquinic acid 65

5- caffeoylquinic acid 4-5

3.4- dicaffeoylquinic acid 76

3.5- dicaffeoylquinic acid 83

4,5-dicaffeoylquinic acid 72

a-The Rf values x 100 in acetophenone; methyl ethyl ketone (3:1). 53

Until recently, workers with chlorogenic acid Isomers did not determine the position of attachmment of the caffeic acid on the quinic acid molecule. It is well known that chlorogenic acid is 3-caffeoylquinic acid but other caffeoyl• quinic acids of indeterminate structures were given names such as "Band 510" and "isochlorogenic acid." Scarpati and

Guiso (1964) reported that "isochlorogenic acid" consists of a mixture of the three dicaffeoylquinic acid isomers: 3,4- dicaff eoylquinic3, 5-d-icaff eoylquinic and 4, 5-d-icaff eoyl• quinic acids. Neochlorogenic acid is 5-caffeoylquinic acid

(Scarpati and Esposito, 1963) and cryptochlorogenic acid

(also known as "Band 510") is 4-caffeoylquinic acid (Grod- zinska-Zaokwieja et al, 196?).

The distribution of these chlorogenic acid isomers as revealed in this study is presented in Table V. It can readily be seen that there is a large number of these isomers distributed throughout the Caprifoliaceae. How do these

isomers come about? The caffeoyl residues can migrate in both the monocaffeoyl and dicaffeoylquinic acids. Scarpati and Guiso (1964) boiled a sample of any one of the mono- caff eoylquinic acids in phosphate buffer at pH7-7.2 and found a mixture of three monocaffeoylquinic isomers. The same procedure carried out on any of the dicaffeoylquinic esters produced a mixture of the three dicaffeoylquinic esters. TABLE V

DISTRIBUTION OF THE ISOMERS OF CHLOROGENIC ACID

FOUND IN THE CAPRIFOLIACEAE

Plant Name Chlorogenic Acid isomers

3CQAC 4CQA 5CQA 3 . 4DQA 3 , 5DQA " 4, 5DQ&

14. Abelia X grandiflora + + + +

4-9. Abelia X grand if lora +

52. Abelia X grandiflora -f +

48. Diervilla rlyularls + + + +

28. Kollrwltzia amabilis + + +

11. Linnaea borealis ssp, longiflora + + + 35. Linnaea borealis ssp. americana + +

13. Lonicera ciliosa + + + + 46. Lonicera sp. +

19.. Lonicera involucrata + + + TABLE V - Continued

Plant Name Chlorogenic Acid Isomers

3CQA H-CQA 5CQA 3, H-DQA 3. 5DQA 4-, 5DQA

37. Lonicera involucrata

57. Lonicera ledebourii + . +

50. Lonicera maackii + +. +

53. Lonicera myrtillus var. depressa + +

23. Lonicera nitida + . + +

4-0. Lonicera obiongifolia + + + '+••'.

10. Lonicera plleata .

32. Lonicera sempervirens + +

58. Lonicera syringantha var. grandiflora + +

20. Lonicera tartarica var. alba + + +

18. Lonicera utahensis + + +

31. Lonicera xylosteum + + . + . + TABLE V - Continued

Plant Name Chlorogenic Acid Isomers

3CQA 4CQA . 50QA . 3, 4DQA 3. 5-DQA 4, 5DQA

30, Sambucus coerulea + +

22. Sambucus racemosa ssp. racemosa +

34. Sambucus canadensis + +

41. Sambucus canadensis . +• + • .

42. •Sambucus nigra +

21, Sambucus racemosa ssp. pubens + +

17. Sambucus racemosa ssp. racemosa + ' + -t- +

.6, Symphoricarpos albus .+ + . +

36. Symphoricarpos occidentalis + + +..'.+

54. Triosteum fargesii + + ' •

55. Triosteum pinnatifidum

45. Triosteum sinuatum + • + +

26. Viburnum burkwoodii + + + TABLE V - Continued

Plant Name Chlorogenic Acid Isomers

4-CQA 5CQA 3. 4DQA 3, 5DQA 4, 5DQA

5. Viburnum carlesii + + + +

3. Viburnum davidii + +

8. Viburnum dentatum + +

29. Viburnum dentatum var. venosum + . + + +

2. Viburnum lantana + +

27. Viburnum opulus var. nanum + +

4. Viburnum opulus var. roseum + +

33. Viburnum opulus var. roseum + + 15. Viburnum pauciflorum + + +

61. Viburnum prunifolium + + 33. Viburnum recognitum + +

1. Viburnum rhytidophyllum

7. Viburnum sargenti + TABLE V - Continued

Plant Name Chlorogenic Acid Isomers

3CQA 4CQA 5CQA 3. 4DQA 3, 5DQA 4, 5DQA

9, Viburnum tinus • + 12. Viburnum trilobum + + + 39. Viburnum trilobum + + + 59. Viburnum urceolatum 4- + 16. Weigela "Bristol Ruby" X. "Abel Carriere" Mix + + + +

24. Weigela floribunda -I- + + 25. Weigela florida + + 47. Weigela hortensis + + + 51. Weigela hybrida var, nivea + + 60. Weigela praecox + + + 56. Weigela variagata + + +

a-3CQA - 3-caffeoylquinic acid; 4CQA - 4-caffeoylquinic acid; 5CQA - 5-caffeoylquinic acid; 3,4DQA - 3,4-dicaffeoylquinic ^ acid; 3.5DQA - 3. 5-dicaffeoylquinic acid; 4.5DQA - 4,5- oo dicaffeoylquinic acid. 59

Since Scarpati and Guiso (1964) observed migration of the caffeoyl moiety to different positions on the quinic acid colecule by boiling in phosphate buffer the standards were subjected to the extraction procedure used in this study.

Mo migration of caffeoyl groups could be found. After the chromatograms were fumed with ammonia only the starting material could be detected. This would suggest that the isomers found are the ones actually present in the plant.with no random migration occurring.

All plants had chlorogenic acid present and it is also of interest to note that with the exception of Sambucus racemosa ssp. racemosa the genus Sambucus did hot have any dicaffeoylo,uinic acids present. There were other caffeic acid containing compounds present in members of the Capri- foliaceae but they were in trace amounts and no attempt was made to isolate them.

In a study on the browning of plant tissues and the presence of chlorogenic acid and its isomers in Viburnum

(Imaseki, 1959), it was shown that the 12 species studied had chlorogenic acid present. Cryptochlorogenic acid was present in eight species, neochlorogenic acid was present in eleven and "isochlorogenic acid" (mixture of the dicaffeoyl• quinic esters) was present in ten species tested. In this paper Imaseki also commented on the apparent random distribu• tion of the chlorogenic acid isomers and their biosynthetic 6o

relationships but was unable to offer any definite suggestions as to the significance of this random distribution.

(E) Identification of Scopoletin in Hydrolysates of Weigela Species

Scopoletin (6-methoxy-7-hydroxy-coumarin) has been identified as a major phenolic constituent in hydrolysates of seven sx>ecies or hybrids of Weigela. There are W, "Bristol

Ruby" X "Abel Carriere" Mix, W. floribunda, W. florIda, W. hortensis, W. hybrida var. nivea, W, variagata and W. praecox.

The isolated scopoletin agreed with authentic scopoletin in all the tests performed. Rf values in the organic phase of benzene:acetic acids water (10 s7;3) and 2% formic acid were 0.38 and 0.34 respectively. The melting points were: isolated 201-202°C, authentic 201.5-20'3°C, mixed 200-201°C and literature 204°C (Handbook of Chemistry and Physics, p. C-26l). Under ultraviolet light scopoletin from both sources had a bright blue fluorescence which turned bluish white when exposed to ammonia. The infrared spectra of both agreed in all respects and both compounds gave peaks at 346nm in the ultraviolet spectra.

(F) The Isolation and Identification of 2',4,4'- Trihyaroxydihydrochalcone from Viburnum davidii

The isolation techniques yielded a pale yellow 61

crystalline product which, after purification and thorough drying, had a melting point of 158--l6o°C. Melting points were taken on a Thomas Hoover capillary melting point apparatus'.

The ultraviolet spectrum was virtually identical to that of resacetophenone (2 *,41-dihydroxyacetophenone). Also, the spectral "behavior of the compound in the presence of AlCl-^ was characteristic of an o-hydroxyl:etone. Spectral data are presented in Table VI.

The infrared spectra of the natural aglycone and the synthetic compound were identical. Mass spectral analysis gave a molecular weight of 258 which corresponds with c2.^14.•

Integration of the NMR spectrum showed a total of 14 protons in agreement with the mass analysis,

A complex series of bands centered at 7.0 T on the

NMR spectrum integrated as four protons and was interpreted as theand p -protons of a dihydrochale one. Three phenol1c protons could be accounted for at 1,90, 0.45 &nd -2.85T, the latter representing the strongly hydrogen bonded phenolic hydrogen. One of the two remaining phenolic functions can be assigned to the 4 position of ring-B. This assignment is based upon the nature of the aromatic protons of that ring. The doublet centered at 2.99~vfis assigned to the pro• tons on carbons 2 and 6 while the doublet at 3.34T is assigned to the protons on carbons 3 and 5. TABLE VI

ULTRAVIOLET SPECTRAL CHARACTERISTICS OF 2',4,4'-

TRIHYDROXYDIHYDROCHALCONE AND RELATED COMPOUNDS

ETOH ETOH/NaOH ETOH/A1C1-, a Natural aglycone • 2 78 314 336 306 356 Synthetic aglycone 278 314 337 306 356 Resacetophenone 277 313 336 305 352 Natural glycoside . 278 314 nd 278 314

a-all figures in nm

b-nd - not determined 63

Both of these groupings exhibit characteristic ortho-splitting.

A single proton exhibiting . ortho-splitting is centered at

2.39 "y and has been assigned to position 6'. Centered at

3.66'Y is a single proton exhibiting m-splitting while at

3.76T a final single proton is observed also exhibiting

m-splitting. These protons are assigned to positions 5"

and 3', respectively. These results suggest 2 *,4,4'-trihydroxy-

dihydrochale one as the most likely structure of the compound.

Confirmation of this suggestion came from an alkali degrada•

tion of- the compound. The treatment of the compound with

hot concentrated base resulted in the formation of resorcinol

and 4-hydroxyphenylpropionic acid (phloretic acid).

Insufficient amounts of the glycoside were obtained

to carry out a melting point or obtain an infrared spectrum

but ultraviolet spectral examination was carried out (Table.

VI). After acid hydrolysis and removal of the aglycone by

ether extraction the hydrolysis mixture was analyzed for sugars

by thin layer chromatography in the two solvent systems.

The only sugar detectable was glucose. An experiment with

j9 -glucosidase also showed glucose as the sole sugar.

Estimation of the ra.tio of aglycone to glucose by the method

of Nelson gave a value of 1.36 aglycone to 1.00 glucose.

This analysis was repeated several times and each time there

was less glucose than aglycone. This would suggest that the

naturally occurring compound is a monoglucoside. The point 64

of attachment of the glucose was determined by examination of the ultraviolet spectral characteristics of the compound,

In the presence of AlCl^ the aglycone exhibited the typical hypsochromic shift of an o-hydroxyacetophenone (Table VI) whereas the glucoside showed no change. This absence of a shift indicates that the glucose is attached at the 2'- position and leads to the conclusion that the naturally occurring compound can be fully described as 2'-^ -D-glucosy- loxy 4,4-' -clihydroxydihydroc hale one . The structure of the aglycone is shown in Figure 2.

A preliminary study of the biosynthesis of this 14 dihydrochalcone was undertaken. The three G labelled precursors administered to V. daviu11 were incorporated in the following amounts phenylalanine 2.11$, cinnamic acid

2.35$. and p-coumaric acid 2.89$. The incorporation of these compounds into the aglycone is about equal. If hydroi:y- lation of a C^-C^-C^ intermediate cccurred in the formation of the aglycone then p-coumaric acid would presumably be a much poorer precursor than the nonhydroxylated precursors.

This would suggest involvement of an intermediate at the

C^-C-^-C^ level which already has a hydroxyl function at position -4. 65

(G) Identification of Flavonoids

The flavonoids of the Caprifoliaceae identified in

this study show.a simple pattern. The aglycones that have

been identified are the.two flavonols, kaempferol and quer-

cetin, and the two flavones apigenin and luteolin (Figure 2).

It is their distribution pattern and glycosylation pattern

which allows them to be used as comparative phytochemical

markers. The biflavonyl, amentoflavone., was found in two

species of Viburnum and the dihydrochale one, 2',4,4'-tri-

hydroxydihydrochalcohe was found in one species of Viburnum .

.only.

The flavonoids were identified by their ultraviolet

spectral properties and by chromatography of the glycosides

and the individual aglycones and sugars. The chromatography

solvents t-butanol:acetic acids water (3:1:1) and 15$ acetic

acid were found to be exceptionally good for the separation

of the flavonoids found in this study. Some spots overlapped

but by reducing the amounts of plant extract spotted on the

chromatograms better resolution was obtained. Forestal solvent

(^acetic acid: cone HC1: water (30: 3 :10) jwas tried and gave poor

separation but was used occasionally to test the chromato•

graphic purity of the compounds separated in the t-butanol

solvent. n-Butanol:pyridine:water (75:10:15) was also used

to check purity. 66

The ultraviolet spectral shifts induced hy chelating or ionizing reagents aided in identification of the flavonoids.

The procedures used are discussed fully by Jurd (1962, 1969) and by Schroeder (1967).

The following is a description of the flavonoids found in this study. The glycosides and aglycones were chroma.tographed on Whatman No, 3 paper and the chromato• graphic Rf values are listed in Table VII. The sugars were chromatographed on Avicel thin layer plates. The chromato• graphy of the sugar moieties involved comparison with the commonly found sugars: glucose, galactose, rhamnose, arabinose and xylose,, This aided sugar identification as most sugars could be excluded on chromatographic differences. FIGURE 2

2 \ 4,4'-Trihydroxydihydrochalcone . Sambunigrin

Amentoflavone TABLE VII

Rf VALUES OP FLAVONOIDS

TBAC HOAcd TBA • HOAc TBA HOAc

.1. Quercetin 3-rhamnoglucoside 44e 57 44 53

II Quercetin 3-glucoside 49 43 37 (P.69)

III Quercetin 3,7-diglucoside 22 58 13 66

IV Kaempferol 3-rhamnoglucoside 58 53 5^ (P.69)

V Kaempferol 3-glucoside 70 46 43 (p.69)

VI Kaempferol 3-d.iglucoside 38 61

VII Luteolin 7-rhamnoglucoside 28 26 26 30

VIII Luteolin 7-glucoside 4l 16 43 16

IX Luteolin 5-glucoside 70 11 07 (p.48)

X Apigenin 7-rhamnoglucoside 33 50

XI Apigenin 7-glucoside 60 25 61 23

XII Apigenin 5-glucoside 31 21 TABLE VII - Continued

Mabry Harborne

TBA HOAc TBA HOAc TBA HOAc

Amentoflavone • 86 15 91 - -

Quercet.in 60 00 87 03 -

Kaempferol 80. 08 79 04 - - Luteolin 85 07 77 08 - -

Apigenin 85 11 87 11 _ ' —

a - Mabry, T.J,

b - Harborne, 1967

c - TBA - t-butanol:acetic acid:water (3:1:1)

d - HOAc - 15$ acetic acid

e - All Rf values x 100 70

I. Quercetin 3-rhamnoglucoside

Rf of sugars: glucose rhamnose

n-'butanol:acetic acid:water (4:1:2.2) 18 45

n-butanol:pyridine!water (75:10:15) 9 38

With naphthoresorcinol spray glucose gave a blue color while

rhamnose gave a turquoise color. Under ultraviolet light

the glycoside absorbed turning yellow with ammonia while the aglycone was yellow under ultraviolet light becoming more

intense with ammonia.

Spectral Properties: (Maxima and shifts in nm)

ETOH NaOAc Boric AlClo Na OH II I II I 7.1 I II JI II

Glycoside 258 350 +6 +8 -.-6 +25 +15 +65 +2;5 +

Aglycone 255 374 +6 +8 +10 + 8 +56 +32 71

II. Quercetin 3-glucoside

Rf of sugars: glucose

n-butanol:acetic acid:water (4:1:2.2) 18

n-butanol:pyridine:water (75:10:15) 9

With naphthoresorcinol spray the glucose gave a blue color.

Under ultraviolet light the glucosjde absorbed turning yellow with ammonia while the aglycone was yellow under ultraviolet light becoaing more intense with ammonia.

Spectral properties: (Maxima and shifts in nm)

ETOH NaOAc Boric AlCl^ NaOH II I II I II I II ^1 II I

Glucoside 258 360 +6 +8 +5 +22 +15 +60 +18 +50

Aglycone 255 3?4 +6 +8 +7 +10 + 8 +56 +32 72

III. Quercetin 3,?-diglucoside

Rf of sugars: glucose

n-butanol:acetic acid:water (4:1:2,2) 18

n-butanol:pyridine:water (75:10:15) 9

With the naphthoresorcinol spray the glucose gave a blue color. Under ultraviolet light the diglucoside absorbed turning yellow with ammonia while the aglycone was yellow under ultraviolet light becoming more intense with ammonia.

Spectral properties: (Maxima and shifts in nm)

ETOH NaOAc Boric A1C1- NaOH II I II I II I II JI II I

Glycoside 258 360 0 +8 +5 +18 +15 +40 +22 +49

Aglycone 255 374 +6 +9 +7 +10 +15 +55 +33

The amount of glucose present was estimated directly from chromatograms after spraying vith naphthoresorcinol. 73

IV. Kaempferol 3-rhamnoglucoside

Rf of sugars: glucose rhamnose

n-butanol:acetic acids water (4:1:2.2) 19 45

n-butanol:pyridine:water (75:10:15) H 38

With the naphthoresorcinol spray glucose gave a blue color

while rhamnose gave a turquoise color. Under ultraviolet

light, the glycoside absorbed turning gold with ammonia while:

the aglycone was yellow-green under ultraviolet light becoming

more intense with ammonia.

Spectral properties: (Maxima and shifts in nm)

ETOH NaOAc Boric AlClo NaOH II I II I II I II 1 II I

Glycoside 268 356 +2 0 0 0 +7 0 + 9 +5^ Aglycone 268 368 +2 0 0 0 +5 - +15 +54 7^

V. Kaempferol 3-glucoside

Rf of sugars: glucose

n-butanol:acetic acid:water (4:1:2.2) 19

n-butanol:pyridine:water (75*10:15) H

With the naphthoresorcinol spray the glucose gave a blue

color. Under ultraviolet light the glucoside absorbed turn•

ing gold with ammonia while the aglycone was yellow-green

under ultraviolet light becoming more intense with ammonia.

Spectral properties.: (Maxima.and shifts in nm)

ETOH NaOAc Boric AlClo NaOH II I II I II I II I II I

Glycoside 268 356 +2 0 0 0 +7 0 + 9 +5^

Aglycone 268 368 +2 - +4 0 +4 0 +15 +60 75

VI. Kaempferol 3-diglucoside Rf of sugars: glucose

n-butanol .-acetic acid: water (4:1:2.2) .19 n-butanol:pyridine:water (75:10:15) 11

With the naphthoresorcinol spray the glucose gave a blue color. Under ultraviolet light the glycoside absorbed turn• ing gold with ammonia while the aglycone was yellow-green under ultraviolet light becoming more intense with ammonia.

Spectral properties: (Maxima and shifts in nm)

ET-OH NaOAc Boric AlClo NaOH II I II I II I II I II I

Glycoside 268 356 +20.00 +7 0 + 9 +54

Aglycone 268 368 +2 0 +4 -0 +5 0 +15 +54

The amount of glucose present was estimated directly from the chromatogram after spraying with naphthoresorcinol.

To determine the amount present the spots were compared by their relative size and intensity but because of different molar color intensities this process does not allow absolute results but relative comparisons can be made (Maier and

Metzler, 1967). 76

VII. Luteolin 7-rhamnoglucoside

Rf of sugars: glucose rhamnose

n-butanol:acetic acid-.water (4:1:2.2) 19 45

n-butanol:pyridine:water (75:10:15) 11 38

With the naphthoresorcinol spray glucose gave a blue color while rhamnose gave a turquoise color. Under ultraviolet light the glycoside absorbed turning yellow with ammonia while the aglycone absorbed under ultraviolet light remaining un• changed with ammonia.

Spectral properties: (Maxima and shifts in nm)

ETOH NaOAc Boric AICI3 NaOH IT I II I II . I II I II I

Glycoside 255 353 0 0 +7 +20 +22 +48 0 +55

Aglycone 255. 350 +4 0 +8 +20 +20 +20 +20 +60 268a

a-Shoulder or inflection 77

VIII. Luteolin 7-glucoside

Rf of sugars: glucose

n-butanol:acetic acidswater (4:1:2.2) 19

n-butanol:pyridine:water (75:10:15) .11

With the naphthoresorcinol spray glucose gave a blue color.

Under ultraviolet light the glucoside absorbed turning yellow with ammonia while the aglycone absorbed under ultraviolet light remaining unchanged with ammonia.

Spectral properties: (Maxima and shifts in nm)

ETOH NaOAc Boric AICI3 NaOH II I II I II I II I II I

Glucoside 255 353 0 - +5 +25 + 5+30 - +35

Aglycone 255. 350 +4 0 +8 +20 +20 +20 +20 +60 268a

a-Shoulder or inflection 78

IX. Lute ol in 5-glucosid-e

Rf of sugars: . glucose

n-butanol:acetic acid:water (4:1:2.2) 19

n-butanol:pyridine:water (75:10:15) 11

With the naphthoresorcinol spray glucose gave a blue color.

Under ultraviolet light the glucoside absorbed turning yellow with ammonia while the aglycone absorbed under ultraviolet

light remaining unchanged with ammonia.

Spectral properties: (Maxima and shifts in nm)

ETOH NaOAc Boric AICI3 NaOH II I II I II I II I II I

Glucoside 255 352 +2 0 0 +20 0 +51 - +51

Aglycone 255^ 350 +4 0 +8 +20 +20 +20 - +50 368

a-Shoulder or inflection 79

X. Apigenin 7-rhamnoglucoside

Rf of sugars: glucose rhamnose

n-butanol:acetic acid:water (4:1:2,2) 19 45

n-butanol:pyridine:water (75:10:15) 11 38

With the naphthoresorcinol spray glucose gave a blue color

while the rhamnose gave a turquoise color. Under ultraviolet

light the glycoside absorbed turning yellow with ammonia

while the aglycone absorbed remaining unchanged with ammonia..

Spectral properties: (Maxima and shifts in nm)

ETOH NaOAc Boric AICI3 Na0H II I II I II :: II I II I

Glycoside 268 336 00 3 0 +6+ 4 0 +54

Aglycone 269 336 +9 0 0 0 +12 +45 +28 +64 80

XI. Apigenin 7-glucoside

Rf of sugars: glucose

n-butanol-.acetic acid:water (4:1:2.2) 19

n-outanol:pyridine:water (7.5:10:15) H

With the naphthoresorc inol spray glucose gave a blue color.

Under ultraviolet light the glucoside absorbed turning yellc:

with ammonia while the aglycone absorbed under ultraviolet

light remaining unchanged with ammonia.

Spectral properties: (Maxima and shifts in nm)

ETOH NaOAc Boric AICI3 NaOH II I II I II I II I II I

Glucoside 268 336 0 0 3 0 +12 +6 0 +52

Aglycone 269 336 +9 0 0 0 +12 +45 +12 +64 81

XII, Apigenin 5-glucoside • •

Rf of sugars: .glucose

n-butanol:acetic acid:water (4:1:2.2) 19 n-butanol:pyridine:water (75:10:15) 11

With the naphthoresorcinol spray glucose.gave a blue color.

Under ultraviolet light the glucoside absorbed turning yellc with ammonia while the aglycone absorbed under ultraviolet light remaining unchanged with ammcnia.

Spectral properties: (Maxima and shifts in nm)

ETOH. NaOAc Boric AlClo NaOH II I II I II I II JI II I Glucoside 268 336 +2 0 0 0 0 0 +23 +54

Aglycone 269 336 + 9 0. 0 0 +10 +45 +28 +61 82

XIII, Amentoflavone Spectral properties: (Maxima and shifts in nm)

ETOH NaOAc Boric A1C13 NaOH II I II I II I II JI II I

269 336 +9 - +9 0 +12 +45 +10 +65

The compound was unaffected by acid hydrolysis and no sugar could be found. The Rf values of the amentoflavone were the same as above and no change in the spectral properties occurred. It appears that amentoflavone occurs in the free state. Using the above solvent systems and spectral proper• ties amentoflavone could not be distinguished from apigenin. Using n-butanol: 2N NH^OH (1:1) on Avicel plates amentoflavone (Rf:0.90) could be distinguished from apigenin (Rf:0.80). The isolated amentoflavone agreed in all respects with authentic amentoflavone provided by Dr. Horhammer of Munich. 83

Table VIII shows the distribution of these flavonoids in the plants studied. The plants are arranged according to genus and the flavonoids are an-anged according to their aglycone, Q3H, Q3G and Q3,?D are quercetin derivatives,

K3R, K3G and K3D are kaempferol derivatives, L7R, L7G and

L5G are luteolin derivatives, A7R, A7G and A5G are apigenin derivatives and Ament. is amentoflavone, It is interesting to note that amentoflavone, which is the biflavonyl made up of two apigenin units, is found in Viburnum carlesii and

V. X burkwoodii but apigenin itself is not found in either the free or bound form in this genus. Horhammer et al (1965) found amentoflavone in the bark of Viburnum prunifolium but careful checking in this study did not reveal It in the leaves of V. prunifolium. TABLE VIII

DISTRIBUTION OF THE FLAVONOIDS IN THE CAPRIFOLLACEAE

No. Plant Name Q3Ra Q3G Q3.7D K3R K3G K3D L7R

14. Abelia X grandiflora + + +

4-9. Abelia X grandiflora + + +

52. Abelia X grandiflora

16. Weigela "Bristol Ruby X Abel Carriere" + +

24. Weigela floribunda + +

? c 4. •- — --o - •-

47. Weigela hortensis + +

51. Weigela hybrida var, nivea + +

56. Weigela variagata + +

6o. Weigela praecox + +

48. Diervilla rivularis + +

28. Kolkwitzia amabilis + TABLE VIII - Continued

No. Plant Name Q3R Q3G Q3, 7D K3R K3G K3D L7_R L_7G Lj>G A_7R .A7G AJG Ament.

54. Triosteum fargesii + +

55. Triosteum pinnatifidum + +

45. Triosteum sinuatum . +

11. Linnaea borealis ssp. longiflora + + +

35. Linnaea borealis . ssp, americana +

23. Lonicera nitida + + +

58. Lonicera. syringantha var. grandiflora + + +

10. Lonicera pileata + + + +

13. Lonicera clliosa + + + +

31. Lonicera xylosteum + + + +

50. Lonicera maackii + • + + +

53. Lonicera myrtillus var. depressa + + + CO TABLE VIII - Continued

No. Plant Name Q3R Q3G Q3,7D K3R K3G K3D L7R L7G L^G A7R A7G

57. Lonicera ledebourii + + + +

19. Lonicera. involucrata + + + + . +

37. Lonicera involucrata + + + + +

40. Lonicera oblongifolia + + + + +

32. Lonicera. sempervirens + + . + +

46. Lonicera sp. + + + +

20. Lonicera tartarica var. alba + + + +•

18, Lonicera utahensis + + + + 4- + +

36. Symphoricarpos occidentalis + +

o. Symphoric arpos albus + + + +

3. Viburnum davidii + +

9. Viburnum tinus +

12. Viburnum trilobum + + CO ov TABLE VIII - Continued

K3R K3G L7R No. Plant Name Q3R Q3G Q3f' K3B L7G LJ>G A?_R A?G AJjG Ament.

15. Viburnum pauciflorum +

33. Viburnum recognitum + +

61. Viburnum prunifolium + +

4. Viburnum opulus var. roseum + + +

7. Viburnum sargenti + + +

27. Viburnum opulus var, nanum + + +

38. Viburnum opulus var, roseum + + +

29. Viburnum dentatum var. venosum + + +

39. Viburnum trilobum +

59. Viburnum urceolatum + + +

1. Viburnum rhytidophyllum + +

8. Viburnum dentatum + + + co TABLE VIII - Continued

No. Plant Name Q3R Q3G Q3,7D K3R K3G KJD L_7R L7G LJ$G A7R A7_G AJ>G Ament,

2. Viburnum lantana + + + +

5. Viburnum•carlesli + + + + +

26. Viburnum X burkwoodil •+ + + + +

30. Sambucus coerulea .+ . +

41. Sambucus canadensis + +

42. Sambucus nigra + +

21. Sambucus racemosa ssp. oubens + +

17. Sambucus racemosa ssp. racemosa + + +

22. Sambucus racemosa ssp. racemosa + + + + • +

a - Q3R - quercetin 3-rhamnoglucoside: Q3G -quercetin 3-glucoside: Q3.7D - quercetin 3, 7-d.iglucoside; K3R - kaempferol 3-rhamnoglucos ide: K3C - kaempferol 3-glucoside: K3D - kaempferol 3-diglucoside: L7R - luteolin 7-rhamnoglucoside; L7G - luteolin 7-glucoside; L5G - luteolin 5-glucoside; A7R - apigenin 7-rhamnoglucoside: oo

Ament. - amentoflavone. . . .. A — l* ~ i J _ DISCUSSION

Phenolic compounds, especially the flavonoids, have come to be widely used as taxonomic markers in the last fif• teen years, Bate-Smith (1963, p. 127) includes with the flavonoid compounds not only substances having the true flavonoid structure but also closely related compounds such as the stilbenes, cinnamic acids, and coumarins by virtue of their being formed by a common pathway. Four excellent books on chemical taxonomy in plants and the importance of flavonoid molecules were produced by Swain (1963, 1966),

Alston and Turner (1963) and Harborne (1967).

For a class of chemical compounds to be useful in plant taxonomy it is desirable that they have the following characteristics: chemical complexity and structural variability, physiological stability, wide spread distribution, and easy and rapid identification (Harborne, 1967). Flavonoids dis• play immense structural variation. There are at least a dozen classes of flavonoids each with variation in its degree of hydroxylation, methylation and glycosylation, The flav• onoids are more widely distributed throughout vascular plants than any other secondary metabolite. They have been found in every family examined for their presence. 90

Flavonoids are amongst the most physiologically stable compounds found in plants. They are not actively concerned in cellular metabolic processes and would not be expected to vary a great deal in concentration so long as physIologioal conditions were constant. There is some quantitative vari• ation in leaf materials as environmental factors vary but this is net considered to be a serious drawback. Large numbers of plants can be screened for their flavonoid content because of the ease and speed of identification. Chromatographic properties of flavonoids and their fluorescent behavior under ultraviolet light are very characteristic and these data can be found in the literature (Harborne, 1967). Also the ultraviolet absorption spectra of flavonoids are distinct and characteristic and much information is available in the literature.

The cyanogenetic compound sambunigrin is used as a chemotaxonomic marker in this study despite the fact that it is not phenolic. Sambunigrin is a secondary metabolite and hence has most of the chemical properties required as a taxonomic marker. It does not have structural variation throughout the Caprifoliaceae as the flavonoids do but rather has a constant structure and is found only in one genus,

Sambucus. Sambunigrin is related to the phenolics by the fact that it arises from the shikimic acid pathway. 91

(A) Distribution of Phenolic Acids

The common phenolic acids are found distributed throughout the angiosperms (Harborne and Corner, 196la;

Runeckles and Woolrich, 1963; Birkofer, 1961). 'In this study of the Caprifoliaceae a similar distribution pattern of the phenolic acids was found (Table III). This distribu• tion pattern was of no diagnostic value in determining the relationship of one taxon to another. The few acids which were present in the unhydrolyzed fraction occurred in much higher concentrations in the hydrolyzed extracts. The bound forms of these acids could be sugar conjugates. The most common form of these acid-sugar conjugates reported in the plants examined -was the glucose ester (Harborne and Corner,

196lb). Stroh e_t al (1962) reported three glucose esters in Sambucus nigra; they identified two of these compounds as l-(caffeoyl)- j% -D-glucose and l-(feruloyl)-.^ -D-glucose.

Since a larger number of acids were released on basic hydro• lysis than on acid hydrolysis it seems reasonable to suggest, that these acids are bound as sugar esters. Caffeic acid occurs mainly in the chlorogenic acid group of conjugates.

However, its presence as a sugar ester cannot be ruled out.

(B) Plants Containing Hydrogen Cyanide Producing Compounds

In the Caprifoliaceae the only plants giving a 92

definitely positive test for cyanogenetic compounds are in

the genus Sambucus. Gibbs (1958) reports that in testing

plants from the genus Viburnum a positive test was obtained.

These results were distinctly different from those obtained

from Sambucus. In. this study no positive results were obtained

from any Viburnum. The compound from Viburnum producing the

positive results for Gibbs is probably different from the

usual cyanogenetic compounds, as indicated by the different

type of result obtained. The HCN producing compound in Sam•

bucus is sambunigrin.

Render divided the genus Sambucus into two sections:

Sect. Eusambucus Spach. - cyme flat, umbel-like; pith white; and Sect. Botryosambucus Spach, - cyme paniculiform; pith usually brown. In Sect. Eusambucus are S. coerulea and S.

canadensis which contained no HCN producing compounds and

S. nigra and S. glauca which gave a positive reaction. In

Sect. Botryosambucus, S. racemosa ssp. racemosa a.nd S. race• mosa ssp, pubens all contain the cyanogenetic compound.

In this study only the leaf material was tested except for

the fruit of S. racemosa ssp, racemosa which contained no cyanogenetic compound.

The chlorogenic acid isomers appear to be spread at 93

random throughout the Caprifoliaces.e with one exception as shown in Table V. This exception is the genus Sambucus which contains only the monocaffeoyl quinic acids except for S. racemosa ssp. racemosa which contained two dicaffeoyl quinic acids. In this study Sambucus is the only genus in the family which contains virtually only the monocaffeoyl quinic isomers and also contains the cyanogenetic glucoside sambunigrin.

In Sambucus nigra, Stroh et al (1962) found that caffeic acid was bound to glucose in the form of 1-(caffeoyl)-

P -D-glucose. There is, however, no reason to believe that glucose esters are restricted to the genus Sambucus in the

Caprifoliaceae as they are reported widely in higher plants,

(D) Presence of Scopoletin in Hydrolysates of Weigela Species and Hybrids

Coumarins have wide distribution throughout the

Angiospermeae and several are found in the Caprifoliaceae.

Fraxin was found in Diervilla (Charaux, 1911; McCullagh et al, 1929) and esculetin was found in Symphoricarpos spp.

(McCullagh, 1929). Plouvier (1951) suggested the presence of esculetin or some similar glucoside in Weigela on the basis of fluorescence of the plant extracts. He did not identify the fluorescent compound which was probably scop• oletin as no esculetin was found in the present study.

It is of interest to note that scopoletin is found 94

in no other genus of the Caprif oliaceae, not even the close].y

related Diervilla. Fraxin (8-glucesyloxy-6,?-dihydroxycoumarin) has been reported from D. canadensis (Charaux,

1911) and D. diervilla (McCullagh jet al.,1929) but fraxin

or its aglycone fraxetin were not found in D. rivularls in

this study. Esculetin (6,7-dihydroxycoumarin) was reported

from Symphoricarpos occidentalis and.S. rivularis. No real relationships can be drawn among these three genera because

of the fact that they all contain coumarins. The coumarins all have different hydroxylation patterns and the.enzymes

responsible for their formation could be an expression of

parallel evolution.

(E) Distribution of Flavonoids in the Caprifoliaceae

The flavonoids found in the Caprifoliaceae exhibit an interesting pattern from quercetin 3-rhamnoglucoside, which is widely distributed, to amentoflavone which is very restricted In its distribution in the angiosperms.

The biflavonyl amentoflavone has been found to occur

in three species of Viburnum. It was found in the leaves of V. carlesii and V. X burkwoodli in this study and in the bark of V. prunif olium (Horhammer et_ al, 1965) , In this study amentoflavone was not found in the leaves of V. prunifolium; the bark was not examined. Amentoflavone could be restricted to the bark and not be present in the leaves in 95

a situation similar to the cyanogenetic glucoside being present in the leaves of elderberry but not iri the fruit.

Another possibility is the geographical factor. Horhammer et al collected their plant, material in Germany while the

V. prunifolium of this study came from the Montreal Botanical

Gardens.

Amentoflavone is formed from two molecules of apigenin by carbon-carbon coupling at the 8 - and 3' - positions.

Several biflavonyls based on this structure have been found distributed in the gymnosperms with the exception of the

Pinaceae 3.nd Ephedraceae (Harborne, 1967) . In the angiosperms only two isolated occurrences of biflavonyls have been re• ported. One is the presence of hinokiflavone in Casuarina stricta (Casuarinaceae) and the other is amentoflavone in

Viburnum. It has been suggested that the presence of biflav• onyls represent a primitive character in plants but their occurrence in the relatively highly advanced genus Viburnum would seem to weaken this hypothesis.

It has been suggested, however, that Viburnum is the most primitive genus in the Caprifoliaceae. The presence of the supposedly primitive amentoflavone in three species would add weight to this suggestion. In Egolf's study (1962) he suggested that the species with n = 8 were the most primi• tive as they showed primitive characters, V. sieboldil being the most primitive. Viburnum carlesii is one of the most 96

primitive of the n .= 9 group, Thomas (1921) suggested that

species with n = 9 were the more primitive in phylogeny;

the species with n = 8 having arisen by the loss of a chromo•

some. If amentoflavone Is truly a primitive compound then

this would suggest that the n = 9 group, including V. carles i and V. prunifolium (all n = 9), is the more primitive. Until more is known about the distribution of biflavonyls in the angiosperms this remains a matter of speculation.

In all species of Sambucus examined in this study both quercetin 3-rhamnoglucoside and quercetin 3-glucoside are present. Kaempferol 3-diglucoside, luteolin 7-glucoside and apigenin 5-glucoside are present in S. racemosa ssp. racemosa. The breakdown, of the genus Into sections accord•

ing to Rehder is shown in Table IX. TABLE IX

FLAVONOIDS OF SAMBUCUS

Plant Flavonoids

Q3R Q3G K3D L7G A5G Sam.

Sect. Eusambucus

S. canadensis + t

S. coerulea + -f-

S. nigra + t +

Sect. Botryosambucus

S. racemosa ssp. pubens

S. racemosa ssp. racemose. + + +

Q3R - quercetin 3-rhamnoglucoside; Q3G - quercetin 3-glucoside; KjD -kaempferol 3-diglucoside; L?G - luteolin 7-glucoside,- A^G - apigenin 5-glucoside; Sam.- sambunigrin. 98

It can be readily seen from Table IX that there appears to be a transition stage between the sections, with the presence of sambunigrin in one species of Eusambucus and the presence of only quercetin glycosides in one species of Botryosambucus. It would be of value to have more species of Sambucus examined for their flavonoid and sambunigrin content tc find the extent of this transition. Another worth while study would be a chemotaxonomic study of the natural hybrids which occur in Sambucus. A study of this nature could make good use of carotenoid content as well • as the flavonoid content as the carotenoids have been studied by Huneck e_t al (1965) and Laurie e_t al (1964),

The other arboraceous' genus, Viburnum, also contains quercetin 3-rhamnoglucoside and quercetin 3-glucoside in all species examined. Egolf (1962) suggested that V. sieboldii is one of the most primitive viburnums and that V. carles ii,

V. lantana and V, dentatum are all relatively primitive.

Rehder put all these species except V. dentatum in Sect.

Thyrososma and placed V, dentatum in Sect. Odontotinus.

The flavonoids found in this study are: quercetin 3-rhamnoglucoside, quercetin 3-glucoside, quercetin 3,7-diglucoside, luteolin 7-rhamnoglucoside, luteolin 7-glucoside and amentoflavone. Table X is Rehder's breakdown of the genus and the flavonoids found in each species. TABLE X

FLAVONOIDS OF VIBURNUM

Plant Flavonoids

Q3R Q3G Q3,7D L7R L7G Ament,

Sect. Lantana

V. carles 11 + + + +

V. X burkwoodii + +

V. lantana + + +

V. rhytidophy1lum + +

V. urceolatum + +

Sect. Lentago

V. prunIfolium +

Sect. Tinus

V. davldii + +

V. tinus + 4'

Sect. Odontotinus

V. dentatum + +

V, dentatum var, venosum + + +

Sect, Opulus

V. pauc iflorum +

V, trilobum + +

V. sargenti + + + 100

TABLE X - Continued

Plant Flavonoids

Q3R Q3G Q3,7D L7R L?G Ament

V. opulus var. roseum +

V. opulus var. nanum + +

Q3R - quercetin 3-rhamnoglucoside, Q3G - quercetin 3-glucoside, Q3.7D - quercetin 3,7-diglucoside, L?R - luteolin 7-rhamnoglucoside, L7G - luteolin 7-glucoside, Ament. - amentoflavone. 101

Egolf considered V. opulus the most advanced of the species of Viburnum with nine chromosomes, along with V. trilobum and V, rhytidophyIlum. Render, placed V. opulus and V. trilobum in Sect. 9, Opulus, but placed V, rhytido• phy Hum in Sect. 2, Iantana, which contains the more primitive species. The presence of luteolin 7-glucoside in V, rhytidophyllum along with other species in Sect. Lantana would support its position there rather than with the species found in Sect. Opulus which contain only flavonols. -

. The flavonoids found in all species of Weigela examined are restricted to quercetin 3-rhamnoglucoside and quercetin

3-glucoside. These two compounds were the only flavonoids found in the closely related species of Diervilla. In the : monospecific genus Kolkwitzia a single flavonoid, qtiercetin

3-rhamnoglucoside, was found. The flavonoids in the Abelia hybrids examined present an interesting picture. The hybrids examined were all Abelia X grandiflora but different flavonoid patterns were evident. Two hybrids contained quercetin 3- rhamnoglucoside, quercetin 3-glucoside and luteolin 7-rhamno• glucoside while the third contained only apigenin 7-glucoside.

Weberling (1966) found that Kolkwitzia had a closer affinity to Abelia than to Lonicera on morphological proper• ties. The presence of quercetin glycosides in Kolkwitzia and Abelia and their absence from most species of Lonicera would appear to confirm this suggestion. Linnaea borealis 102

ssp. longlflora contains quercetin 3-rhamnoglucoside, quer• cetin 3-glucoside, luteolin ?-glucoside, apigenin 7-glucosio.e, and. apigenin 5-glucoside while L. borealis spp. americana contains both the 3-rhamnoglucoside and glucoside of quer• cetin as well as kaempferol 3-diglucoside. The consistent presence of quercetin glycosides in Linnaea and their absence from most species of Lonicera would support Weberling's idea that Kolkwltzia and Abelia should be placed with Linnaea rather than with Lonicera.

The treatment of the genus Lonicera by Rehder with the flavonoid content of each species examined is as shown in Table XI. The flavonoids found In Lonicera are: quercetin 3-rhamnoglucoside, quercetin 3-glucoside, quercetin

3,7-diglucoside, kaempferol 3-rhamnoglucoside, kaempferol

3-glucoside, luteolin 7-rhamnoglucoside, luteolin 7-glucosid.e, luteolin 5-glucoside and apigenin 7-glucoside. TABLE XI

FLAVONOIDS OF LONICERA

Plant Flavonoids

Q3R Q3G Q3,7D K3R K3G L7R L7G L5G' AJ7_G

Subgenus I Chaecerosus

Sect. Isoxylosteum

L. myrtillus var.

depressa + + . + +

Sect. Islka

L. utahensis + + + • + + .+ +

L. obiongifolia + ++•+'+

L. involucrata + + + •+•• +

L. ledebourii +++,.+

Sect. Coboxylosteum

L. tartarica var. alba + + + + + L. xylosteum .+••+++ TABLE XI - Continued

Plant Flavonoids

Q3R Q3G Q3,7D K3R K3G L?R L7G L5G A7G

L. maackii + + +

Subgenus II Perichymenum

L. sempervlrens . + +

L. ciliosa + + +

Q3R - quercetin 3-rhamnoglucoside, Q3G - quercetin 3-glucoside, Q3,?D - quercetin 3,7-diglucoside, K3R - kaempferol 3-rhamnoglucoside, K3G - kaempferol 3-glucoside, L7R - luteolin 7-rhamnoglucoside, L-7G - luteolin 7-glucoside, L5G - luteolin 5-glucoside, A7G - apigenin 7-giucoside.

r—1 O -Cr 105

Lonicera utahensis, with two glycosides, of quercetin

and one of kaempferol and L. tartar lea, var. alba with two

glycosides of both quercetin and kaempferol, show the most

primitive characteristics of the Lonicera spp. examined.

According to Harborne (1967) flavonols (quercetin and kaemp•

ferol) are more primitive characteristics than flavones

(luteolin and apigenin). It is interesting to note almost

complete replacement of flavonols by flavones in this genus.

In the species of Triosteum examined only flavones were found.

In T. fargesii and T. plnnatlfidum luteolin 7-glucoside and

apigenin 7-glucoside were found and only luteolin 7-glucoside

was found in T. sinuatum.

The position of Triosteum with Viburnum in the Tribe

Viburneae (Fritsch, 1888) is probably not correct. Hutchinson

(1967) suggested that Triosteum is probably a reduced Lonicera

and placed it in its own tribe, the Triosteae. The finding

of the 7-glucosides of flavones and the absence of flavonol;;;

would place the genus Triosteum closer to Lonicera than

Viburnum. Harborne has suggested that the replacement of

flavonols by flavones is a loss mutation and since only

flavones are found in Triosteum it would support Hutchinson's

suggestion.

The two species of Symphoricarpos examined•contained

only flavones. S. albus contains the 7-rhamnoglucoside of

kaempferol and apigenin while S. occidental is contains only 106.

the 7-glucosides of kaempferol and apigenin. The presence of flavones only in Symphoricarpos places it among the more advanced genera in Caprifoliaceae. It would be of value to have more species of Symphoricarpos available for study to confirm this hypothesis.

Figure 3 is a polygonal representation of the paired affinity indices of each of the ten genera of this study to all others. This relationship can be arrived at by the follow• ing equation:

characters in common for genera 1 and 2 paired affinity = ; x 100

total characters in 1 and 2

The values obtained can then be plotted on a polygonal graph to give a visual representation of the data. FIGURE 3

Abelia 2. Diervilla

I Kolkwitzia 4.. Linnaea 108

Figure 3 - Continued

e 6

i i

7, Symphoricarpos 8. Triosteum 109

Figure 3 - Continued

I I

9. Viburnum 10. Weigela Figure 3 - Continued

Numbers on polygonal graphs represent

1. Abelia

2. Diervilla

3. Kolkwitzia

4. Linnaea

5. Lonicera

6. Sambucus

7. Symphoricarpos

8. Triosteum

9. Viburnum

10. Weigela Ill

Sixteen characters were used to obtain the graphs in Figure 3. They were: the 13 flavonoids found in this study, the presence of sambunigrin in Sambucus, the virtual absence of the dicaffeoylquinic acid in Sambucus and the presence of scopoletin in Weigela. The characters used were the presence or absence of compounds of known structure.

No attempt was made to draw comparisons using unidentified chromatographic spots. The distribution of the individual mono- and di-caffeoylquinic acids was not used as not enough is known about their distribution throughout the angiosperms.

No great taxonomic significance can be attached to these individual acids until more is known about their distribution patterns. The flavonoids, on the other hand have been exten• sively studied and their value as taxonomic markers proven

(Harborne, 19^7).

The polygonal graphs show that Abelia, Diervilla,

Kolkwitzla and Weigela have more than 50 per cent of their compounds in common. Symphoricarpos and Triosteum also have a large number of compounds in common. The presence of flav• ones and absence of flavonols in these genera makes them the most advanced in the family. It can also be seen that Sam• bucus has very few biochemical characters in common with any other genus in the family.

The genus Sambucus is related to other genera in the Caprifoliaceae but its relationship is probably a distant 11?.

one. Hoch (1892) placed it in its own family, the Sambuc- aceae, Fritsch (1892), Hutchinson (1948) and Takhtajan (1969) have questioned the wisdom of placing it with the Caprifolia• ceae. The hear lack of dicaffeoylquinic acids in the genus and the.presence of the cyanogenetic glycoside, sambunigrin., would tend to set it aside from the; otherwise homogeneous family. The evidence found in this study would support the separation of the genus Sambucus from the Caprifoliaceae.

Net a great deal can be said about the relationship • of members of the Caprifoliaceae to the members of other families cn a phytochemical basis. Insufficient data are available on the occurrence or absence of compounds in neighboring families to be able to draw reliable conclusions at this time. More studies of a comprehensive nature with extensive collection and positive identification of plant material must be carried out.

Ferguson (1966) suggested that Viburnum and Sambucus show affinities to the Cornaceae on the basis of pollen morphology. This would be supported by the presence of kaempferol, quercetin and caffeic acid in seven species and quercetin in four other species of the Cornaceae (Heg• nauer, 1964). Since these compounds are very widely distrib• uted throughout the angiosperms their presence in both Corn• aceae and Caprifoliaceae cannot be taken as a measure of direct relationship; A study to determine the form in which 113

these compounds occurred in the Cornaceae would be of value and some lines of relationship may then possibly be assessed.

In the Rubiaceae, kaempferol has been reported in three species and quercetin reported in nine species (Har• borne, 1967), Again, before any conclusions can be drawn on a comparative basis much more ttfork has to be carried out..

Plants have to be screened carefully and the presence or absence of compounds recorded before phytochemical results can be of any value.

Hutchinson (19^8) suggested that Viburnum may poss• ibly be related to Hydrangea on the basis that species in both genera have outer floxfers of the cyme that are sterile and enlarged. Kaempferol 3-galactoside has been reported to occur in Hydrangea macrophylla (Harborne, 1967) but since this is the only report from Hydrangea no conclusions can be drawn.

The present study has shown the capabilities of com• parative phytochemistry. The virtual absence of the dicaffeoylquinic acid isomers, the presence of sambunigrin and the flavonoid distribution in Sambucus would support Its separation from the Caprifoliaceae. The flavonoid distribution in Sambucus,

Viburnum and Lonicera add support to Rehder's breakdown of these genera into sections. The discovery of amentoflavone in Viburnum carlesii supports Egolf's work suggesting that viburnums in which the base chromosome number is nine are the more primitive. 114

The removal of Triosteum from the tribe Viburneae, where Fritsch placed it, is supported by the lack of flavonols in this genus. The presence of flavones only in Triosteum would place it among the most advanced genera of this family.

It is evident that comparative phytochemistry can be a great aid to the taxonomist. It deals with another field of gene controlled factors, namely the chemical con• stituents of the plants. Now that the ground work has been done and compound identification techniques improved•to the point of rapid and easy manipulation, comparative phyto• chemis try will have a more significant role to play in plan-: systematics. REFERENCES

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