Extraction, Purification Procedures and HPLC-RI Analysis
of Carbohydrates in Olive (Olea europaea L.) Plants
A. R o m a n i 1 / A. Baldi 1 / M. Tattini .2 / F. F. Vincieri 1
2l I ) . ~partimento di Scienze Farmaceut~che,
9 . .
Umvers~ta ,
degh Stud~, Vm G. Cappom 9, 50100, Firenze, Italy
. .
Istituto sulla Propagazione delle Specie Legnose, Consiglio Nazionale delle Ricerche, Via Ponte di Formicola 74,
50018, Scandicci, Firenze, Italy
Key Words
Liquid chromatography
Olive tissues
Oligosaccharide quantitation
Refractive index detection
Summary
Qualitative and quantitative analyses of carbohydrates
In olive (Olea europaea L.) tissues have been carried
out by HPLC-RI. Sample purification was by two
SUccessivesolid-liquid extractions to remove completely
plant phenolics and pigments. Five carbohydrate peaks;
SUcrose (staehyose + raffinose + sucrose), glucose,
galactose, fructose (fructose + myo-inositol) and
raannitol were detected when plant extracts were run
on a Sugar SC 1011 column operating at 75 ~ using
Water as eluent 0.5 ml min-1. The use of two serial
Sugar SC 1011 columns operating at 90 ~ and eluting
!he plant extracts with H20-CH3CN (95/5 v/v) enables
!dentification and quantification of nine carbohydrates,
tncluding tetra and tri-saccharides.
Introduction
Carbohydrates play a key role in the plant world
ranging from the supply to the skeleton for aminoacid
and protein synthesis to the involvement in fruit quality
and composition. High-performance liquid chromatog-
raphy (HPLC) in carbohydrate analysis of fruit species,
especially of olive (Olea europaea L.) plants, has not
been widely used [4, 9, 14, 15].
Recently, qualitative and quantitative analysis of solu-
ble carbohydrates of olive leaves has been performed
by Combined TLC and HPLC [6]. However, the use of
two SUccessive chromatographic techniques adversely
affects quantitation [9, 11].
ItPLC analysis of carbohydrates in Olea europaea
Should be theoretically complicated by the presence of
Qhrornatographia Vol. 39, No. II2, July 1994 Original
0009-5893/94/07 0035-05 $ 3.00/0 9 1994 Friedr. Vieweg & Sohn Verlagsgesellschafl mbH
. .
large amounts of plant phenolics and plant pigments [1,
2, 8, 13]. These compounds interfere in sugar analysis
when using a RI detector and accurate sample clean-up
become essential.
In addition, as previously reported [6] olive tissues
contain a large number of soluble carbohydrates that
also complicate qualitative analysis.
Among the fruit species, the olive represents a case of
current interest since mannitol, instead of sorbitol and
or sucrose has been found to represent the main
component of the soluble carbohydrate pool [5, 12].
Recently it has been observed that mannitol is found
near to sites of QO 2 assimilation, in mesophyll cells, but
it is not the trang~orted sugar. Phloem sap principally
comprises higher~\oligosaccharides, namely stachyose
and raffinose [6]. Mannitol should be regarded as a
storage soluble carbohydrate, having high compatibili-
ty with the cytoplasm function [6, 7]. In this regard
detection and quantitation of.higher oligosaccharides
become essential in the sugar metabolism of olive
plants.
The purposes of this work, carried out on different
olive tissues (leaves and stem), was to develop both
extraction and purification (fractioning) procedures
enabling qualitative and quantitative analysis of a
complex sugar matrix by HPLC and RI detection.
Experimental
Sampling Procedure
One-year old olive (Olea europaea L. cv. Leccino)
plants were grown in a sand culture system in a green-
house (rain-max. temps. 16-26 ~ from Sept. 15th to
Oct. 15th. The plants were daily supplied with a full
strength Hoagland solution [10]. At the end of the
culture period leaves and stem were collected. There
were apical and basal leaves and the corresponding
stem tissues. The tissues were immediately stored at
- 8 0 ~ then lyophilised and finally crushed under
liquid nitrogen (Mixer Mill MM2, Retsch, Ge(many).
Four replicates for each plant tissue were collected.
35
Extraction and Purification Procedures
100-300 mg plant tissue were extracted overnight in
10-30ml 75 % E t O H (adjusted to p H 7 ) at room
temperature and the homogenate filtered through
Whatman 42 paper. Extraction steps were repeated 3
times. The 30-90 ml solution was then evaporated to
dryness at room temperature under reduced pressure
(Rotavapor Buchi R-144, Switzerland) and diluted with
1 ml, pH 7, water (Milli Q grade, Millipore Italia).
The samples were fractionated using liquid-solid ex-
tractions carried out by eluting samples through:
a) pre-packed 3 ml Bond-elut CH cartridge (Varian,
CA, USA), a reverse-phase cyclohexyl resin, insert-
ed on a Bond-elut SAX cartridge (Varian), a qua-
ternary-amine, strong anion-exchange resin;
b) pre-p~ :1 3 ml Bond-elut SAX cartridge (Varian,
CA, USA);
c) 10 ml poly-prep column (Bio-Rad, CA, USA) filled
with 500 mg of Bio-Rex 5 (Bio-Rad), an anion-
exchange resin (100-200 mesh, chloride form).
The cartridges were actived by 5 ml MeOH and
conditioned by adding 10 ml pH 7 water. The elution
was performed using an additional 10 ml pH 7 water.
Finally the eluate was evaporated to dryness under
vacuum and then diluted with pH 7 water.
Analytical Equipment
Analyses were carried out using a binary LC pump 250
(Perkin Elmer, USA) equipped with an automatic
injection system (ISS 101, Perkin Elmer).
A Waters column heater module (Waters Division,
Millipore, Milford, MA, USA) controlled by a Temper-
ature Control Module (Waters) maintained the column
at 75 ~ The column was a 8 x 300 mm Shodex Sugar
SC 1011 (Showa Denko Europe GmbH, Germany)
equipped with a Guard-Pak Insert Sugar Pak II
(Waters). The mobile phase was water, Milli Q grade,
at 0.5 ml rain- 1
We also tested the performance of two serial SC 1011
columns operating at 90 ~ using H 2 0 - C H 3 C N 95 : 5,
as eluent at 0.20 ml rain- 1.
A refractive index detector was employed (LC-30 RI,
Perkin Elmer), while a HP 1040M Diode Array
Detector (Hewlett Packard, USA) was used for moni-
toring the presence of substances other than sugars in
the purified samples.
Qualitative and quantitative analysis of soluble carbo-
hydrates was performed with the help of a work-
station; HP 9000 (Hewlett Packard, USA).
Identification and Quantitation of Soluble
Carbohydrates
The identity of soluble carbohydrates was confirmed
using authentic carbohydrate standards (Sigma, USA).
Identification of galactinol was after Madore et al. [9]
and Mitchel et al. [11]. Sorbitol, a sugar not detected in
36
olive tissues, was used as internal standard and added
to the crushed material. The recovery was estimated for
each carbohydrate. Thus 0.25, 0.50, 0.75 and 1.0 ml
1 mg ml- 1 carbohydrate solutions were fractioned and
analysed as described for samples. Recovery ranged
from 92 to 99 %. Calibration curves were performed
for stachyose, raffinose, sucrose, galactinol, glucose,
galactose, fructose, myo-inositol, mannitol and sorbi-
tol. The recovery and calibration curves for galactinol
were constructed after preparative HPLC using cucum-
ber extracts.
Results and Discussion
Sample clean-up,.using both Bond-Elut SAX cartridge
and Bio-Rex 5 resin was satisfactory when analysing
stem tissue. Following the fractionation of olive leaves
by both SAX cartridge and Bio-Rex 5 resins, the final
colour of the solution was light yellow-green. The
presence of chlorophyll a and b was confirmed by
absorption measurements, at 645 and 663 nm, on the
leaf extract. Furthermore, detectable amounts of flavo-
noids in the leaf extract were observed by thin-layer
chromatography, as previously described [8]. This
finding agrees with the presence of large amounts of
flavonoids, flavoinoid-glycosides and bi-flavonoids in
olive leaves [8].
A satisfactory cleaning-up of the leaf tissue was by
serial utilisation of CH and SAX cartridges. In this case
the major part of both plant phenolics and plant
pigments were fixed by the cycloexyl resin in the c H
cartridge, as observed by the absence of any colour in
the SAX cartridge. Furthermore, following the two
solid-phase extractions, diode array analysis of the leaf
extract did not detect compounds other than carbohy-
drates and the final solution only constituted the sugars
reported in Figure 1 and Table I.
Both extraction and purification steps, carried out
using pH 7 water, did not cause sucrose hydrolysis as
confirmed by the behaviour of a sucrose standard
solution treated in the same way as olive tissues.
Qualitative and Quantitative Analysis of
Carbohydrates in Olive Tissues
Use of a single SC 1011 column. In Table I, amounts
and retention times of carbohydrates in both olive
leaves and stem, using a single SC 1011 column, are
reported. We clearly detected glucose, galactose, fruc-
tose (fructose + myo-inositol) and mannitol. We also
detected an un-resolved peak having a retention time
of 11.2 min; it comprised (see Figure 2B) three co-
eluting carbohydrates, namely, stachyose, raffinose and
sucrose (Figure 1). Quantitative data is reported i~
Table I as mg sucrose.
The peak at 17.2 min was assigned to fructose, but
samples enriched with both fructose and myo-inositol
always increased the peak at Rf 17.2 min. This result
agrees completely with findings of previous experi-
Chromatographia Vol. 39, No. 1/2, July 1994 Original
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Figure 1
C h r o m a t o g r a m o f 20 p.l leaf extract, a f t e r B o n d - e l u t - C H + B o n d e l u t - S A X solid-liquid e x t r a c t i o n .
C o n d i t i o n s as T a b l e I. Sorbitol internal s t a n d a r d .

T a b l e !. R e t e n t i o n t i m e s a n d a m o u n t s ( p m o l (g dry w e i g h t ) - 1) o f s o l u b l e c a r b o h y d r a t e s in d i f f e r e n t
tissues o f olive plants. C h r o m a t o g r a p h i c c o n d i t i o n s : 8 x 300 m m S C 1011 c o l u m n at 75 ~ flow rate
0.5 ml rain- l H 2 0 solvent.

Retention Sugar Apical Basal Apical Basal

time leaves z leaves stem stem

11.2 Sucrose* 7.97 + 0.96 8.43 + 0.81 11.12 _+ 0.95 11.54 + 1.32
14.0 Glucose 75.27 + 4.15 79.32 + 5.98 70.77 +_ 6.17 68.32 _+ 4.16
15.6 Galactose 3.89 + 0.61 4.45 + 0.78 5.17 _+ 0.98 3.32 + 0.64
17.2 Fructose** 9.16 +_ 0.32 7.55 + 0.36 10.23 _+ 0.56 9.39 + 0.44
19,8 G l u c u r o n i c Ac.
21.8 Mannitol 65.65 +- 4.12 51.14 + 3.98 63.11 _+ 5.00 40.95 + 2.74

* S t a c h y o s e + r a f f i n o s e + s u c r o s e r e p o r t e d as m g s u c r o s e
** F r u c t o s e + m y o - i n o s i t o l r e p o r t e d as m g f r u c t o s e
z D a t a m e a n o f four m e a s u r e m e n t s (+__SE)

rnents [4, 6, 9, 11] where the separation of fructose and The calibration curve for sorbitol was also constructed
raYo-inositol in the carbohydrate pool did not occur. since HP integration software correct the quantitation
l~inally we observed a peak at 19.8 min corresponding of each soluble carbohydrate by mean of this curve. In
to the retention time of glucuronic acid. We did not fact, small variations accompyining the extraction and
Carry out any molecular identification of this com- cleaning-up procedures can be usefully corrected, using
pOUnd; however, enriched samples in glucuronic acid the different response factor for the tested sugars, by
trlcreased the peak at 19.8 min. means of the calibration curve and response factor of
9A. relationship between relative additions ofglucuronic sorbitol.
acid and the area of the peak at 19.8min was not The use of a single SC 1011 column was unable to
Observed. There were (data not shown) difficult-to- detect or separate all the carbohydrates recently found
control equilibria between dissociated (co-eluting with in olive tissues using other analytical procedures [6]
SNvent peak) and undissociated forms (retention and our results agree with the findings of Drossopoulos
19.8 rain) of glucuronic acid. This largely complicated and Niavis [5] and of Priestley [12].
the quantitation of glucuronic acid and, for this reason, The use o f two serials S C 1011 columns. In Figure 2A a
~Vehave reported in Table I only the retention time of typical chromatogram of a 201al injection of 0.1%
this compound. standard solution of stachyose, raffinose, sucrose,
~Uantitation of soluble carbohydrates was carried out glucose, galactose, fructose, myo-inositol, glucuronic
Or each carbohydrate, using calibration curves for the acid, mannitol and sorbitol is reported. The chromato-
flange 0-35 lag. Regression coefficients (n = 5) ranged gram was obtained using two serial 8 x 300 mm Sugar
torn 0.9989 through 1.00 using a linear model9 SC 1011 columns, operating at 90 ~ and eluted with

Q h r o m a t o g r a p h i a V o l . 39, N o . 1/2, July 1994 Original 37

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4~ 50 GO 70 8~ EB~ 1OO
Tiros (rain.)
Figure 2
A) C h r o m a t o g r a m of 20 I.tl 0 . 1 % carbohydrate mixture, using two serial 8 300 mm SC 1011 columns at
90 ~ flow rate 0.20 ml rain- 1 H 2 0 _ C H 3 C N (95 : 5 v/v) solvent.
B) Typical chromatogram of 20 pl olive leaf extract, after Bond elut CH + Bond elut SAX. Conditions as
Figure 2A. Sorbitol internal standard.

95 : 5 (v/v) water-acetonitrile, at 0.2 ml min- 1. These inositol. It was the first time that myo-inositol was
sugars with the addition of galactinol were also detect- detected, and in appreciable amounts, in olive leaves.
ed in leaf tissue of olive plants (Figure 2B), confirming This fact assumes a particular relevance, since inositol
the results of Linda and Madore [6]. metabolism in plants, especially the biosynthesis of
The identity of galactinol, that co-eluted with glucose, inositol lipids, is assuming increasing interest for plant
when using a single SC 1011 column at 75 ~ was physiologists.
confirmed by injecting an extract of cucumber [9, 11] The presence of the component, having the retentioJa
and following the increment of the galactinol peak in time of glucuronic acid, was again observed by the
the analysed samples. Verbascose, a penta-saccharide serial use of two SC 1011 columns, as a peak at 75.8 mi~
detected in mesophyll tissue of Olea europaea by Linda (Figure 2B).
and Madore [6] was not detected in the test samples.
Retention of verbascose was within that of the eluent Carbohydrate Composition o f Olive Tissues
peak. Relative amounts of the detected sugars changed
It should be mentioned that an excellent separation was according to the type of olive tissue. The sucrose pool
obtained between the peaks of fructose and,myo- (stachyose + raffinose + sucrose) was lower in leaves

38 C h r o m a t o g r a p h i a Vol. 39, No. 1/2, July 1994 Original

 / The use of a single column enable quantification of 5
BO
~
70 : o
6O
50
| ,=,
E 30
o
c
o
~
m studies involving synthesis of sugars in plants under
20
i i 1 i F i
1 2 3 4 /5
Oligosaccharides
Figure 3
Relative amounts (mg/g dry weight) of soluble carbohydrates
from olive leaves collected 15th October. Data mean of four
replicates.
than in stems; mannitol was relatively higher in apical
parts of both shoots and leaves (Table I).
Glucose was detected in higher amounts than mannitol,
in agreement with previous results of Linda and
Madore [6]. These findings do not fit with the results of
Drossopoulos and Niavis [5] and Priestley [12]; the
authors cited, in carbohydrate metabolism of Olea
europaea, assigned a minor role to glucose with respect
to that of sucrose and mannitol. In this regard, it should
be mentioned that the qualitative and quantitative
analysis of carbohydrates of the experiments cited
largely differed from our conditions. Their analyses [5,
12] were mostly carried out using colorimetric determi-
nations.
Furthermore, relative amounts of glucose and mannitol
largely depend on the harvest time. We detected in
leaves of olive plants harvested at the end of Novem-
ber, comparable mannitol and glucose concentrations
(data not shown).
Finally, the use of two serial columns operating at 90 ~
With 5 % of acetonitrile in the eluent ( C H 3 C N - H 2 0
5/95) enable us to estimate the relative influence of
each carbohydrate on the soluble carbohydrate pool of
the leaf (Figure 3). The minor role, in Olea europaea
Species, of stachyose and raffinose, as well as the
marginal role of sucrose to leaf mesophyll sugar pool is
definitively demonstrated here (Figure 3).
Conclusion
]'he suggested procedures of extraction and purifica-
tion were able to give a fraction containing exclusively
Soluble carbohydrates from difficult-to-work olive tis-
SUes, usually very rich in phenolics and pigments
[1, 2, 8].
Chromatographia Vol. 39, No. 1/2, July 1994
soluble sugars, namely, the sucrose pool (stachyose,
raffinose and sucrose), glucose, galactose, fructose +
c
myo-inositol and mannitol. These sugars represent
more than 90 % of total soluble carbohydrates in the
tested olive tissues (Figure 3).
This rapid H P L C method is particularly suitable for
o .... stress conditions in which glucose and mannitol seems
to play the unique role of osmotica [3, 7].
i J i The use of two serial columns and RI detection is
6 7 8 9 suggested for experiments concerning carbohydrate
metabolism and transport, in studies of compartmenta-
tion of photosynthates. The analysis time is increased,
but we have a one-step analysis giving good compound
quantitation compared to the use of 2 or 3 different
analytical techniques during the same analysis [6], that
interfere on sugar quantitation.
Further investigations are in progress to characterize
completely and quantify the compound having the
same retention time as glucuronic acid.
References
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[12] C.A. Priestley, J. Horticultural Science 52, 105 (1977).
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Received: Nov 4, 1993
Revised manuscript
received: Marll, 1994
Accepted: Apr 11, 1994
Original 39