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anthocyan pigment are present as well as plastid pigments. The two former go into
solution in the water and the glucoside in time crystallizes out. In either case the
glucoside can be filtered off and tested as in the previous experiment. A positive
result will be given in each case.

Kaempferol occurs in the flowers of a species of Larkspur (Delphi-
nium consolida) (Perkin and Wilkinson, 25) and Prunus (Perkin and
Phipps, 24) and in the leaves or flowers of several other plants. It has
the formula :




HO



Myricetin and fisetin are two other flavones which have been found
in species of Sumac (Rhus) and other plants. They have respectively
the formulae :




OH



HO



Myricetin




OH



o.



AROMATIC COMPOUNDS AND [CH.



THE ANTHOCYAN PIGMENTS.

These pigments are the substances to which practically all the blue,
purple and red colours of flowers, fruits, leaves and stems ,are due
(Wheldale, 3). They occur in solution in the cell-sap and are very
widely distributed, it being the exception to find a plant in which they
are not produced. As members of a group, they have similar properties,
but differ somewhat among themselves, the relationships between them
being much the same as those between the various flavone and flavonol
pigments. They occur in solution in the cell-sap but occasionally they
crystallize out in the cell. They are present in the plant in the form of
glucosides, and in this condition they are known as anthocyanins ; as
glucosides they are readily soluble in water and as a rule in alcohol
[except blue Columbine (Aquilegia), Cornflower (Centaurea Gyanus) and
some others] but are insoluble in ether and chloroform. The glucosides
are hydrolyzed by boiling with dilute acids, and the resulting products,
which are non-glucosidal, are termed anihocyanidins (Willstatter and
Everest, ^5). The latter, in the form of chlorides, are insoluble in ether,
but are generally soluble in water and alcohol. The anthocyanins can
be distinguished from the anthocyanidins in solution by the addition of
amyl alcohol after acidification with sulphuric acid. The anthocyanidins
pass over into the amyl alcohol, the anthocyanins do not. The antho-
cyanins and anthocyanidins themselves (with one exception) have not
yet been crystallized, but of both classes crystalline derivatives with
acids have been obtained (Willstatter and Everest, 35).

In considering the reactions of anthocyan pigments the difference
between those given by crude extracts and those of the isolated and
purified substances must be borne in mind. With acids the anthocyan
pigments give a red colour: with alkalies they give, as a rule, a blue or
violet colour when pure, but if flavone or flavonol pigments are present
(as may be the case in a crude extract) they give a green colour, due to
mixture of blue and yellow. In solution in neutral alcohol and water
many anthocyan pigments lose colour, and this is said to be due to the
conversion of the pigment into a colourless isomer which also gives a
yellow colour with alkalies (Willstatter and Everest, 35) ; hence even a
solution of a pure anthocyan pigment may give a green coloration with
alkali due to mixture of blue and yellow. The isomerization can be
prevented or lessened by addition of acids or of neutral salts which form
protective addition compounds with the pigment. With lead acetate



vn] OXIDIZING ENZYMES 99

anthocyan pigments give insoluble lead salts, blue if the pigment is
pure, or green, as in the case of alkalies, if it is mixed with flavone or
flavonol pigments, or the colourless isomer.

When anthocyan pigments are treated with nascent hydrogen, the
colour disappears but returns again on exposure to air. It is not known
what reaction takes place.

Expt. 101. The reactions of anthocyanins and anthocyanidins. Extract petals of
the plants mentioned below with boiling alcohol in a flask. Note that the anthocyan
colour may disappear in the alcoholic extract. Filter off some of the alcoholic extract
and make the following tests (a) and (6) with it :

(a) Add a little acid and note the bright red colour.

(b) Add a little alkali and note the green colour.

The decolorized petals, after filtering off the extract, should be warmed with a
little water in an evaporating dish. The colour is brought back if pigment is still
retained by them.

Evaporate the remainder of the alcoholic extract to dryness and note that the
anthocyan colour returns. Dissolve the residue in water and continue the following
tests, taking a little of the solution in each case :

(c) Add ether and shake. The anthocyan pigment is not soluble in ether.

(d) Add acid. A bright red colour is produced.

(e} Add alkali. A bluish-green or green colour is produced which may pass to
yellow.

(/) Add basic or normal lead acetate solution. A bluish-green or green precipitate
is produced.

(g) Add a little sulphuric acid and then amyl alcohol and shake : the latter does
not take up any of the red colour, indicating that the pigment is in the anthocyanin
(glucosidal) state.

(h] Heat a little of the solution on a water-bath with dilute sulphuric acid and
then cool and add amyl alcohol. The colour will pass into the amyl alcohol, indicating
that the pigment is now in the anthocyanidin (non-glucosidal) state.

(i) Acidify a little of the solution with hydrochloric acid and add small quantities
of zinc dust. The colour disappears. Filter off the solution and note that the colour
rapidly returns again.

For the above reactions it is suggested that the following flowers be used as
material : magenta Snapdragon (Antirrhinum majus\ brown Wallflower (Cheiranthus
Cheiri\ crimson Paeony (Paeonia officinalis\ magenta "Cabbage" Rose, Violet
( Viola odorata), but the majority of coloured flowers will serve equally well.

Though the above represent the reactions and solubilities given by
the greater number of anthocyan pigments, it will be found that all are
not alike in these respects.

Expt. 102. Demonstration that anthocyanins may be insoluble in alcohol but soluble
in water. Extract petals of any of the species mentioned belosv with boiling alcohol
and note that they do not lose their colour. It will be found that the pigments are
either completely or largely insoluble in alcohol, but are soluble in water. Test the

72



100 AROMATIC COMPOUNDS AND - [GEL

water extract as in Expt. 101 (c)-(i). Also take equal quantities of the water extract
in two evaporating dishes. To one add sodium chloride. Note (as mentioned above,
see p. 98) that the colour fades less rapidly from the extract containing the salt. The
..flowers of the following species can be used ": blue Larkspur (Delphinium), Cornflower
(Centaurea Cyanus], blue Columbine (Aquilegia).

There is a small group of plants belonging to some allied natural
orders, of which the anthocyan pigments give chemical reactions still
more different from the general type already described, though they
nevertheless resemble each other. Such, for instance, are the pigments
of various genera of the Chenopodiaceae [Beet (Beta), Orache (Atriplex)^
Amarantaceae (Amaranthus and other genera), Phytolaccaceae (Phyto-
lacca) and Portulacaceae (Portulaca). These anthocyan pigments are
insoluble in alcohol but soluble in water : they give a violet colour with
acids, red to yellow with alkalies, and a red precipitate with basic lead
acetate.

Expt. 103. Reactions of the Beet-root (Beta vulgaris) pigment. Take some Beet-
root leaves, tear them into small pieces and put them into alcohol. Allow the leaves
to stand for some time and note that the chlorophyll is extracted but the red pigment
is insoluble. Then pour off" the alcohol and add water : the red pigment goes into
solution. Filter off the solution and make the following tests :

(a) Add acid. The pigment turns violet.

(b) Add alkali. The pigment becomes redder and finally turns yellow.

(c) Add basic lead acetate. A red precipitate is formed.

(d) Acidify with hydrochloric acid and add zinc dust. The colour disappears, but
on filtering off from the zinc it does not return again.

Anthocyan pigments may also occur in leaves, and this is veiy obvious
in red-leaved varieties of various species such as the Copper Beech, the
Red-leaved Hazel, etc.

Expt. 104. Extraction of anthocyan pigment from the Red-leaved Hazel. Extract
some leaves of the Blood Hazel (Corylus Avellana var. rubra) with alcohol. Filter off
and evaporate the solution to dryness. Add water. Pour a little of the crude mixture
in the dish into a test-tube and add ether. There will be a separation into a green
ethereal layer containing chlorophyll, and a lower water layer containing anthocyan
pigment. Filter the extract remaining in the dish and with the filtrate make the
tests already given in Expt. 101 (c)-(i).

The leaves of the Copper Beech (Fagus sylvatica var. purpurea) can also be used.

In many flowers, the cells of the corolla may contain yellow plastid
(see p. 39) in addition to anthocyan pigments. The colour of the petals
is in these cases the result of the combination of the two, and is usually
some shade of brown, crimson or orange-red, as in the brown-flowered
variety of Wallflower (CheirantJius Clieiri).



VIl]



OXIDIZING ENZYMES



Expt. 105. Demonstration of the presence of anthocyan and plastid pigments
together in petals (see also Expt. 100). Extract petals of the brown-flowered variety of
Wallflower with alcohol. Filter, and evaporate the extract to dryness. Take up
with water and add ether. Pour the mixture into a separating funnel. The plastid
pigment will pass into solution in the ether, and the anthocyan pigment will remain
in the water. Test the aqueous solution as in Expt. 101 (c)-(t).

The following may be used as material : ray florets of bronze or crimson Chrys-
anthemum, ray florets of Gaillardia,&ud orange-red flowers of Nasturtium (Tropaeolum
majus}.

Anthocyanins and anthocyanidins have been isolated from various
species. The pigments themselves with one exception have not been
obtained in the crystalline state, but crystalline compounds with acids
have been prepared both of the glucosidal and non-glucosidal forms.

All the pigments so far described appear to be derived from three
fundamental compounds, pelargonidin, cyanidin and delphinidin, of
which the chlorides are represented thus :




OH H

Pelargonidin chloride




Cyanidin chloride




OH



Delphinidiu chloride

It has been suggested, at least in the case of cyanidin, the pigment
of the Cornflower (Gentaurea Cyanus), that the pigment itself is a neutral
substance, purple in colour and of the following structure (Willstatter,
33, 36):




102'



COMPOUNDS AND



[CH.



Further, that the blue pigment of the flower is the potassium salt
of the purple, and the red acid salt, cyanidin chloride, depicted above, is
a so-called oxonium compound of the purple.

Pelargonidin, moreover, has been prepared synthetically (Willstatter
and Zechmeister, 45).

The above three pigments, either as glucosides or in the form of
methylated derivatives, are found in a number of plants which are listed
below (Willstatter, etc., 33-46). The sugar residues or methyl groups
may, of course, occupy different positions, thus giving rise to isomers :



Callistephin
Pelargonin



Pelargonidin.



Monoglucoside of pelargonidin
Diglucoside of pelargonidin



Flowers of Aster (Callistephus chinensis]

Flowers of Scarlet Geranium (Pelargoniu

zonale), pink var. of Cornflower (Centaure

Cyanus} and certain vars. of Dahlia

variabilis]



Cyanidin.



Asterin

Chrysanthemin
Idaein
Cyanin



Mekocyanin
Keracyanin
Peonin



Monoglucoside of cyanidin
Monoglucoside of cyanidin
Monogalactoside of cyanidin
Diglucoside of cyanidin



Diglucoside of cyanidin
Rhamnoglucoside of cyanidin
Diglucoside of peonidin (cyanidin
monoethyl ether)



Flowers of Aster (Callistephus chinensis)
Flowers of Chrysanthemum (C. indicum]
Fruit of Cranberry ( Vaccinium Vitis-Idaea
Flowers of Cornflower (Centaurea Cyanus

Rosa gallica and certain vars. of Dahl

(D. variabilis}

Flowers of Poppy (Papaver Rhoeas]
Fruit of Cherry (Prunus Cerasus)
Flowers of Paeony (Paeonia officinalis)



Delphinidin.



Violanin
Delphinin

Ampelopsin

Myrtillin

Althaein

Petunin

Malvin

Oenin



Rhamnoglucoside of delphinidin
Diglucoside of delphinidin -f

p-hydroxybenzoic acid
Monoglucoside of ampelopsidin

(delphinidin monomethyl ether)
Monogalactoside of myrtillidin

(delphinidin monomethyl ether)
Monoglucoside of myrtillidin

Diglucoside of petunidin (delphi-
nidin monomethyl ether)

Diglucoside of malvidin (delphi-
nidin dimethyl ether)

Monoglucoside of oenidin (delphi-
nidin dimethyl ether)



Flowers of Pansy ( Viola tricolor)

Flowers of Larkspur (Delphinium consolida

Fruit of Virginian Creeper (Ampelopsis quir
quefolia]

Fruit of Bilberry ( Vaccinium Myrtillus)

Flowers of deep purple var. of Hollyhoc
(Althaea rosea)

Flowers of Petunia (P. violacea)
Flowers of Mallow (Malva
Fruit of Grape ( Vitis mnifera)



vii] OXIDIZING ENZYMES 103

Of the methylated compounds, myrtillidin and oenidin may be re-
presented thus:

Cl





HO H Hd H

Myrtillidin Oenidin

Expt. 106. Preparation and reactions of pelargonin chloride. Extract the flowers
from two or three large bosses of the Scarlet Geranium (Pelargonium zonale) in a
flask with hot alcohol. Filter off and concentrate on a water-bath. Then pour the
hot concentrated solution into about half its volume of strong hydrochloric acid. On
cooling, a crystalline precipitate of pelargouin chloride separates out. Examine under
the microscope and note that it consists of sheaves and rosettes of needles. Filter
off the crystals, take up in water and make the following experiments with the
solution :

(a) Add alkali. A deep blue-violet eolour is produced.

(b) Take two equal quantities of solution in two evaporating dishes. To one add
as quickly as possible some solid sodium chloride. The colour in the solution without
salt will rapidly fade owing to the formation of the colourless isomer in neutral
solution : this change is prevented to a considerable extent in the solution containing
salt owing to the formation of an addition compound of the pelargonin with the
sodium chloride which prevents isomerization (see p. 98). To portions of the water
solution (without sodium chloride) which has lost its colour add respectively acid
and alkali. The red colour returns with acid owing to the formation of the red acid
oxonium salt : with alkali a greenish-yellow colour will be produced due to the
formation of the salt of the colourless isomer. If alkali is added to the portion of the
pigment solution containing the sodium chloride, it will be found that it still gives
a violet colour.

(c) Add sulphuric acid and arnyl alcohol. The alcohol does not take up the
colour. Add amyl alcohol after acidifying another portion of the solution with
sulphuric acid and heating on a water-bath. The alcohol now abstracts some of the
colour. This shows that the glucoside pelargonin exists in the first case, but is
decomposed into the non-glucosidal pelargonidin after heating with acid.

(d) Acidify with hydrochloric acid and add zinc dust : the colour disappears and
returns again after filtering.

Expt. 107. Preparation of the acetic acid salt of pelargonin. Make an alcoholic
extract of petals as in Expt. 106. Evaporate down and pour into glacial acetic acid
instead of hydrochloric acid. The crystals of the salt formed are smaller and more
purple in colour than those of the chloride.

Expt. 108. Preparation and reactions of crude peonin chloride. Extract the petals
of one or two flowers of the Crimson Paeony (Paeonia officinalis] with 95-98%
alcohol. Filter and evaporate nearly to dryness. Then add some methyl alcohol and



104 AROMATIC COMPOUNDS AND [CH.

pour into a little strong hydrochloric acid. Then add ether to the mixture in a
separating funnel. A crude precipitate of peonin chloride will separate out after a
time, which may be more or less crystalline. Filter off this precipitate, take up in
water and make the following experiments :

(a) Take two equal quantities of the solution in two evaporating dishes. To one
add solid sodium chloride as in Expt. 106 (6). Then neutralize both portions carefully
with very dilute sodium carbonate solution until the colour changes slightly to
purple. The colour will fade more rapidly in the solution without sodium chloride
on account of the formation of a colourless isomer, as in the case of pelargonin
chloride. The water solution after standing will give a green colour with alkali owing
to admixture with the yellow salt of the isomer.

(6) Add alkali. A deep blue colour is produced. A crude extract of the fresh
petals made as in Expt. 101 will give a green or bluish-green colour with alkali owing
to the presence of the accompanying flavone.

(c) Add amyl alcohol and sulphuric acid. No colour is taken up by alcohol. The
pigment is present as the glucoside peonin. Boil another portion with sulphuric
acid and add amyl alcohol. The pigment is partly hydrolyzed and the peonidin goes
into solution in the alcohol.

(d] Eeduce another portion with zinc dust and hydrochloric acid. The colour
returns after filtering.

In considering the anthocyan pigments, the question now arises
What is the chemical significance of the various shades in the living
plant ? Apparently the same pigment may be present in two flowers
of totally different colours, as in the blue Cornflower and the magenta
Rosa gallica. It has been suggested that in such cases the pigment is
modified by other substances present in the cell-sap: thus it may be
present in one flower as a potassium salt, in another as an oxonium salt
of an organic acid, and in a third in the unaltered condition. But exactly
how these conditions are brought about is not clear. In one or two
cases, moreover, where there is a red or pink variety of a blue or purple
flower, the variety, when examined, has been found to contain a different
pigment and one less highly oxidized than that in the species itself.
The above phenomena are exemplified in the Cornflower (Centaur ea
Cyanus). The flowers of the blue type contain the potassium salt of
cyanin, the purple variety, cyanin itself, while those of the pink variety
contain pelargonin.

The mode of origin of anthocyan pigments in the plant is as yet
obscure. It has been suggested (Wheldale, 29) that they have an
intimate connexion with the flavone and flavonol pigments, which can
be seen at once by comparing the structural formula of quercetin with
that suggested for cyanidin :



VII]



OXIDIZING ENZYMES



105





Quercetin



All the anthocyan pigments so far isolated, however, have been
found to contain the flavonol, and not the flavone, nucleus.

Just as in the case of the flavone and flavonol pigments, some of
the anthocyan pigments are specific, while others, on the contrary, are
common to various genera and species. Also more than one anthocyan
pigment may be present in the same plant.

It will be pointed out later that small amounts of a substance iden-
tical with cyanidin are said to be formed by reduction of quercetin with
nascent hydrogen, but this does not necessarily prove that the formation
of anthocyan pigments in the plant takes place on the same lines. If
we compare the formulae for a number of anthocyan with flavone and
flavonol pigments, it is seen that they may be respectively arranged in
a series, each member of which differs from the next by the addition of
an atom of oxygen :



Luteolin, kaempferol and fisetin C 15 H 10 O 6
Quercetin C 15 H 10 O 7
Myricetin C 15 H 10 O 8



Pelargonidin C 15 H 10 O 5
Cyanidin C 15 H 10 6
Delphinidin C 15 H 10 O 7



The relationship between these two classes of substances in the
plant can only be ascertained by discovering which flavone, flavonol and
anthocyan pigments are present together, and then to determine whether
the relationship is one of oxidation or reduction, a problem which has
not yet received adequate attention (Everest, 6).

A reaction which is of interest in connexion with the relationship
between the above two classes of pigments is that which takes place
when solutions of some flavone or flavonol pigments are treated with
nascent hydrogen. If an acid alcoholic solution of quercetin is treated
with zinc dust, magnesium ribbon or sodium amalgam, a brilliant magenta
or crimson solution is produced, and this solution gives a green colour
with alkalies (Combes, 5). The red substance thus produced has been
termed " artificial anthocyanin " or allocyanidin. The product is not a



106 AROMATIC COMPOUNDS AND [CH.

true anthocyan pigment but has, it is suggested, an open formation
(Willstatter, 36):




OH



It is said, however, to contain small quantities of a substance iden-
tical with natural cyanidin from the Cornflower (Willstatter, 36). The
fact that small quantities of a natural anthocyan pigment can be obtained
artificially from a hydroxyflavonol by reduction does not necessarily imply
that one class is derived from the other in the living plant.

From the above reaction of quercetin the result follows that when
many plant extracts [most plants (see p. 94) contain flavone or ttavonol
pigments] are treated with nascent hydrogen, artificial anthocyan pig-
ment is produced. Moreover, it seems probable that if the yellow
pigments acted upon are in the glucosidal state, and if the reduction
takes place in the cold, allocyanin (the glucoside of allocyanidin) is
formed and the product is not extracted from solution by amyl alcohol.
But if the flavone is non-glucosidal, or if the solution is boiled before or
after reduction, then allocyanidin (non-glucosidal) is formed and is
extracted by amyl alcohol.

Expt. 109. Formation of allocyanidin from, quercetin. Make an alcoholic solution
of a little of the glucoside of quercetin prepared from either Narcissus or Cheiranthus
(see Expts. 98 and 100). Acidify with a little strong hydrochloric acid and heat on a
water-bath. Add a little zinc dust from time to time. A brilliant magenta colour
due to allocyanidin is produced. To a little of this solution add some alkali : a green
colour is produced. If the alcohol and hydrochloric acid are evaporated off, and a
little water and sulphuric acid added, on shaking up with amyl alcohol, all the
allocyanidin passes into the arnyl alcohol. (The distribution of the allocyanidin in
the amyl alcohol is greater with aqueous sulphuric acid than with aqueous hydro-
chloric acid.)

Expt. 110. Formation of allocyanin from quercetin. Make a suspension of the
glucoside of quercetin from Cheiranthus or Narcissus (see Expts. 98 and 100) in about 2N
sulphuric acid, and then add zinc dust (or a drop of mercury about the size of a pea
and a little magnesium powder) in the cold. The red or magenta colour is gradually
developed, Divide the coloured solution into two parts in two test-tubes. Boil one
for 5-10 minutes. Then add amyl alcohol to each. In the unboiled test-tube the
amyl alcohol extracts no colour, since allocyanin is present. In the boiled test-tube
allocyanidin is taken up by the amyl alcohol as in Expt. 109.



vii] OXIDIZING ENZYMES 107

Expt. 111. Formation of allocyanin and allocyanidin from plant extracts. For this
purpose the yellow varieties "Primrose" or "Cloth of Gold" of the Wallflower
(Cheiranthus Cheiri) can be used. The flowers are pounded in a mortar, extracted
with cold water, the water extract acidified with sulphuric acid, and zinc dust (or
mercury and magnesium powder as above) added. A red coloration is slowly
developed. To some of the red solution add aruyl alcohol. The colour is not
abstracted (allocyanin). Boil another portion. The allocyanin is thus converted into
allocyanidin which is then taken up on addition of amyl alcohol.



THE OXIDIZING ENZYMES.

There is a certain group of enzymes of which we have most informa-
tion in their connexion with aromatic substances. These are the oxi-
dizing enzymes.

The presence of such enzymes in plants was long 'ago associated with
the following phenomena. If the expressed juices, or water extracts of
the tissues, of some plants are added to a solution of guaiacum gum, in
the presence of air, a deep blue colour is obtained in a short time. On
the other hand, expressed juices, or water extracts, of other plants added
to guaiacum solution produce no blue colour. On addition, however, of
a few drops of hydrogen peroxide, in the latter case, the blue colour
rapidly develops. Plants are said to contain an oxidase when the extracts


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