Anatomy and chemical composition of Pinus pinea L. bark

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Anatomy and chemical composition of Pinus pinea L.

bark

Elsa Nunes, Teresa Quilhó, Helena Pereira

To cite this version:

Original

article

Anatomy

and

chemical

_composition

of Pinus

_pinea

L. bark

Elsa Nunes

a

Teresa

_Quilhó

_b

Helena

Pereira

a

Centro de Estudos _{Florestais, Departamento} de

_Engenharia

Florestal, Instituto

_Superior

de

_Agronomia,

Universidade Técnica de Lisboa, 1399 Lisboa Codex,

Portugal

b

Centro de Estudos de

_Tecnologia

_Florestal, Instituto de

_{Investigação}

Científica

_Tropical,

1399 Lisboa _Codex,

_Portugal

(Received 21

_{September 1998; accepted}

23 March 1999)

Abstract - The

_{secondary phloem}

of Pinus _pinea L. bark has sieve cells and axial and radial

_parenchyma,

but no fibres. Resin ducts are _present in fusiform rays.

Styloid crystals,

starch

_granules

and tannins occur inside sieve and

_parenchyma

cells. The

_rhytidome

of

P. pinea bark has a variable number of

_{periderms forming scale-type}

discontinuous

_layers

over

_{expanded parenchyma}

cells. The

phellem comprises

two to four

_layers

of thick-walled cells and the

_phelloderm

a

_layer

of two or three thin-walled cells with inclu-sions and sometimes a

_layer

of

_expanded

cells. Ash content of P. _pinea bark is low and the

_pH

is

_slightly

acidic. Total extractives

amount to 19.1 % and tannins to 7.2 % of o.d.

_weight.

Content of

_lignin

and

_{unhydrolysable phenolic}

acids is 37.5 % and of

polysac-charides 36.8 %, with the

_following

monosaccharide

_{composition: glucose}

44.6 %, mannose 18.2 %,

xylose

20.7 %,

galactose

7.6 % and arabinose 8.9 %. (© Inra/Elsevier, Paris.)

Pinus

_pinea

L. / bark / anatomy / chemical

_composition

Résumé - Anatomie et

_{composition chimique}

de l’écorce de Pinus

_pinea

L. Le

_phloème

secondaire de Pinus pinea L. contient des cellules criblées, du

_parenchyme

axial et radial mais pas de fibres. Les canaux résinifères

apparaissent

dans les rayons fusiformes. Des cristaux

_styloïdes,

des

_granules

d’amidon et des tanins ont été observés dans les cellules criblées et le

_parenchyme.

Le

_rhytidome

renferme un nombre variable de

_{péridermes qui}

forment des couches discontinues en écailles sur des cellules de

_{parenchyme élargies.}

Le

_{phellème comprend}

de deux à _quatre couches de cellules à

_{paroi épaisse}

et le

_phelloderme

une couche de deux à trois cellules à

paroi

mince avec inclusions et,

_parfois,

une couche de cellules

_élargies.

La teneur minérale de l’écorce de Pinus

_pinea

est faible et le

_pH

_légèrement

acide. Les extractibles

_{correspondent}

à 19,1 % et les tanins à 7,2 % (masse

anhydre).

La teneur en

_lignine

et en acides

_phénoliques

non

_{hydrolysables}

est de 37,5 %, tandis que celle des

polysaccharides

est de 36,8 % avec la

_composition

suivante:

_glucose

44,6 %, mannose 18,2 %,

xylose

20,7 %,

galactose

7,6 % et

ara-binose _8,9 %. _{(© Inra/Elsevier, Paris.)}

Pinus

_pinea

L. / écorce / anatomie /

_{composition chimique}

1.

Introduction

Pinus

_pinea

L.

(stone

pine)

is a

_pine

_species

of

eco-nomic

_{importance, growing}

in southern

_Europe

espe-cially

in the western Mediterranean

countries,

namely

*

Correspondence

and

_reprints

deftec@

_{mail.telepac.pt}

in the Iberian

_peninsula

and in France. P.

_pinea

is a

species

of

_{ecological importance}

in these

_regions

and

and it is

_expanding

due to the choice of this

_species

for many afforestation

projects.

The stone

_pine

is used

main-ly

for the

_production

of the edible

_pine

nut but also for timber and the trees _may be

_tapped

for resin. The

impor-tance of nut

_production

has increased with the recent use

of

_{grafting, allowing}

fruit

_production

after 6 _years. The bark of P.

_pinea

has not

_previously

been

charac-terised,

although

the _anatomy and chemical

_composition

of its wood has

_already

been studied

[2, 6, 12].

Detailed

descriptions

of the bark of other Pinus

_species,

_{e.g. P.}

echinata,

P.

taeda,

P.

_palustris

and P.

_rigida,

have been

made

_{[7, 8]}

and data on

_pine

bark chemical

_composition

have been summarised

[5].

A

_description

of P.

_pinaster

bark _anatomy and chemical

_composition

has

_recently

been

_published

_[9].

In this paper we describe the

_anatomy

and chemical

composition

of P.

_pinea

bark.

2.

Materials

and methods

The material used for

_analysis

was obtained from commercial P.

_{pinea plantations}

from one site in the south of

_Portugal

_(Albufeira).

Ten trees

_{approximately}

25 years old were

_randomly

selected and bark

_samples

were taken at breast

_height.

For determination of the chemical

_composition,

a

composite sample

was

_{prepared by mixing aliquots}

of

the bark of each tree

_sampled.

The

_{composite sample}

was milled and the

_{granulometric}

fraction 40-60 mesh

was used for

_analysis.

The chemical

_composition

was determined

_using

standard methods for wood

_analysis

[15]

with

_{adaptations reported}

for cork chemical

_analysis

[11]

and used

_previously

for the

_analysis

of maritime

pine

bark

_[9].

Extractives were determined

_using

successive

extrac-tions with

_{dichloromethane,}

ethanol and water. Suberin

content was determined on _{extractive-free bark: 1.5 g} were reacted under reflux with a sodium methoxide solu-tion in methanol

(250

mL methanol and _{2.7 g}

_Na)

for 3 h and further with 100 mL methanol for 15 min after

filtra-tion; the combined filtrates were acidified

to

_pH

6 with a

2 M

_sulphuric

acid solution in

_methanol,

_evaporated

to

dryness

and the residue

_suspended

in water; the

alcohol-ysis

products

were extracted three times with 200 mL

chloroform,

dried over sodium

_sulphate

and

_evaporated

to

_dryness.

The desuberinised residue was

_hydrolysed

and the

_hydrolysate

used for

_separation

and

quantifica-tion of monosaccharides as alditol acetates

_{by gas}

chro-matography.

The residue of

_hydrolysis

was

_weighted

as

lignin

and

_phenolic

acids.

Total

_phenols

and tannins were determined in the

extracts obtained

_by

successive extractions with ethanol

and water and

_{spectrophotometric}

measurement at 765

nm after reaction with Folin-Ciocalteu

_reagent

_using

gal-lic acid for calibration

[11]. The determination of

tannin

content used

_{methylcellulose}

as absorbent: 10 mL

extract were added to 10 mL of a 0.04 % solution of

methylcellulose

of

_high

substitution

_degree,

8 mL of a

saturated ammonium

_sulphate

solution and 25 mL water; after 20 min this was filtered and the

_phenol

content in

the filtrate determined as

_previously.

Tannin content was calculated as the difference between total

phenols

and

phenols

remaining

after tannin

_absorption.

Total

_nitrogen

was determined

_by

the

_Kjeldahl

method. The

_pH

was measured in a

_suspension

of _{2 g} bark in 100 mL distilled

water.

The _anatomy studies were carried out

_using

the bark of the individual trees

_{sampled. Microscopic}

sections of

10 _{μm in thickness} were

_prepared

for

_optical

_microscopy

using

a Reichter

_sliding

microtome after

_penetration

with

DP 1500

_polyethylene

_glycol

_[14],

and stained with a

triple staining

with

chrysodine

pyronine

and astra blue. Sudan 4 was used for suberin detection. Observations were also made

_{by light}

_microscopy

on dissociated

ele-ments. The

_samples

were macerated in acetic acid and

hydrogen peroxide

1:1 at 60 °C for 48 h and the

macerat-ed material was stained with astra blue. The

_terminology

used for the anatomical

_description

_mainly

followed

Trockenbrodt [17].

3. Results and

discussion

The stone

_pine

_{(Pinus pinea L.)}

has a thick scale bark of a _strong brown reddish colour.

The bark _anatomy of Pinus

_pinea

L. shows three structural

_layers

from cambium to the outside

_(figure

_1a,

b):

the

_secondary

_phloem,

the innermost

_periderm

and the

_rhytidome

_(which

includes a variable number of

peri-derms).

The anatomical characteristics observed were similar for all trees. The _anatomy is similar to that of the

bark of P.

_pinaster

_[9]

and other _{Pinus spp.}

_[7,

_8].The

secondary phloem

includes sieve cells

_(Sc),

axial

parenchyma

(p)

and rays (r)

(figure

1a,

b).

No fibres were found and this _agrees with

_general

observations for

the genus Pinus

[4].

Sieve cells are

_elongated

with

_unlignified

thin walls

and with many lateral sieve areas

_(figure

_2).

The sieve

cells nearest to the cambium are

_radially

_aligned

but this

alignment

is

_subsequently

lost

_by

distortion towards the outside

_through

cell

_collapse

due to loss of

_turgidity

_[7].

This distortion is

_particularly

obvious near the innermost

The

_parenchyma

cells

_(p)

are

_thin-walled,

approxi-mately

circular in cross section and scattered.

_They

have

styloid crystals

(c)

(figure

3)

and starch

_granules.

It has

been

_reported

for southern

_pine

[7]

that

_styloid

_crystals

are

_composed

of calcium oxalate and that

_they

are abun-dant

_throughout

the innerbark and

_{rhytidome, deposited}

as a metabolic

_by-product.

The

_{phloem rays}

(r)

are similar to the

_{xylem rays,}

uniseriate and homocellular. Albuminous cells form the

margins

of some _rays. Fusiform _rays with resin ducts are

also observed

_(figure

_4).

_Rays

extend

_{linearly through}

the

_{non-collapsed phloem,}

but near the

_periderm

the

alignment

is lost. The transition from

_{non-collapsed}

to

collapsed phloem

is

_gradual

and

_{signalled by}

the

col-lapse

of sieve cells and loss of their radial and

_tangential

alignment,

the distortion of rays and the

expansion

of

parenchyma

cells. These

_changes

_represent the

adjust-ment of the

_{secondary phloem}

to the radial tree

_growth,

as

_reported

for P.

_{pinaster [9]}

and for other

_species

of the _{genus Pinus}

[7, 8]

as well as _{for other genera}

_{[1, 13,}

16].

The

_periderm

(Pr)

is made up of

phellem

(Pm),

phel-logen

and

_phelloderm

_(Pd)

_(figures

_{1a, 5).}

In each

perid-erm, the

_samples

showed a variable number of cells in

the

_phellem

and

_{phelloderm layers.}

The

_phellem

includes

layers

of two to four

_{radially aligned}

thick-walled and

sclerified cells

_(Scl),

_{sometimes with many} inclusions

(figure

5).

A band of suberised cells

_alternating

with

thick-walled cells was not

_observed,

as described for

other Pinus spp.

[7].

The

_phelloderm,

located to the

inte-rior of the

_phellogen,

has two

_types

of cells: near the

phellogen,

two to three

_layers

of thin-walled cells with

numerous

_inclusions;

then one

_layer

of

thin-walled,

radi-ally expanded

cells

_(figure

_{5, arrow)}

_{and may} be con-fused with cells from the

_{expanded parenchyma}

_(ex)

(figure

5).

The variable number of cells in the

_phellem

and

_{phelloderm layers}

in each

_periderm

_{agrees with} Howard

[7]

who found that in the bark of southern

_pine

the amount of tissue in the

_{periderm may vary within}

a

single

sample.

In P.

_pinea,

similar to P.

_pinaster

and other Pinus spp., the

periderm

is

discontinuous,

following

an

irregu-lar

_path

around the stem. The

_periderms

form

_layers

with

edges curving

outwards to _{merge with older periderms;}

the

_phloem

is thus isolated in a

_scale-type

_pattern and the

outer bark forms a scalebark. The

_rhytidome

(Rt)

has a variable number of

_periderms

_(on

_{average three}

perid-erms)

that

_{overlap enclosing}

a

_phloem

tissue that is

mainly

made up

by

expanded parenchyma

cells _many times

_larger

than their

_original

diameters

_(figure

_5).

This

type of cell

_change

at the outside of the innermost

perid-erm has been observed in P.

_{pinaster [9],}

in southern

pines

[7]

and also in other _genera

_[3].

These

_expanded

parenchyma

cells

_(ex)

_occupy a

_large

volume fraction of the

_rhytidome

and contribute to _{its porous} structure;

they

are sensitive to fracture and determine the external

mor-phology

of the

_rhytidome,

which is

_{kept together by}

the sclerified cells

_(Scl)

of the

_periderm.

The

_rhytidome

of

P.

_pinea

has a

_high

amount of material

_deposited

in the

lumens,

mainly

tannins,

which

_gives

the bark its _strong reddish brown colour.

The chemical

_composition

of

_pine

bark is summarised in table I. Extractives amount to

_19,1

% and include

mainly polar compounds

extracted

_by

ethanol and water

(14.0

% of o.d.

_bark);

_{approximately}

half of the

extrac-tives

_(7.2

% of o.d.

_bark)

are of

_phenolic

character and

correspond

to tannins which amount to 96 % of total

phenolics

(table II).

The

_microscopic

observations show that tannins are

_deposited

in the cell lumens of the

rhytidome

(figure

5).

The suberin content is

low,

in

accordance with the small number

_{of phellem}

cells found

in the

_rhytidome

and the fact that

_they

were thick-walled

and sclerified

_(figure

_5).

_{Consequently, lignin}

and

38 % of the o.d. bark. The monomeric

_composition

of

polysaccharides

(table III),

which

_correspond

on _average

to

_{approximately}

37 % of

_pine

bark,

shows a

predomi-nance of

_glucose.

In the

_{hemicelluloses,}

_xylans

and man-nans have almost similar

_importance,

20.7 and 18.2

%,

respectively,

of total monosaccharides. Arabinose is

pre-sent in considerable amounts

₍₉

% of

_{monosaccharides).}

In

_comparison

to the chemical

_composition

of other

Pinus

_species

_{[5, 9],}

P.

_pinea

bark shows a content of

extractives similar to P.

_sylvestris

and P. taeda

_(20.7

and 18.3

%,

_{respectively), higher}

than P.

_pinaster

_{(11.4 %)}

and lower than P. elliotti

(35.8 %).

The content of total

polysaccharides

is lower than in P.

_pinaster

and P.

sylvestris.

The

_proportion

of

_xylans

is

_higher

than in P.

sylvestris

(12.6

% of total

_{monosaccharides)}

and lower

than in P.

_pinaster

_{(26.1 %).}

The bark of stone

_pine

is

_slightly

acidic

_(pH

_4.5)

and total

_nitrogen

content is 0.76 %.

4. Conclusions

The bark

_anatomy

of P.

_pinea

L. is similar to that of

P.

_pinaster

and other Pinus _spp. The

_{secondary phloem}

of P.

_pinea

has sieve cells and axial and radial

parenchy-ma, but no fibres. Resin ducts are _present in fusiform

rays and

styloid

crystals,

starch

granules

and tannins

occur inside sieve and

_parenchyma

cells. The

_rhytidome

of P.

_pinea

has a variable number of

_periderms

as

scale-type discontinuous

_layers

over

_{expanded parenchyma}

cells. The

_phellem

has a small number of thick-walled and small suberised cells.

The chemical

_composition

of P.

_pinea

bark shows a considerable amount of tannins and a

_relatively

low

con-tent of

_{polysaccharides.}

Acknowledgements:

The authors thank Cristiana Alves and Cristina Leonor for their technical assistance.

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