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Procedia Engineering 42 (2012) 1682 – 1691

20th International Congress of Chemical and Process Engineering CHISA 2012

25 – 29 August 2012, Prague, Czech Republic

Solute-matrix and solute-solute interactions during

supercritical fluid extraction of sea buckthorn leaves
M. Sajfrtova a*, H. Sovova
Institute of Chemical Process Fundamentals of the ASCR, v.v.i., Rozvojová 135, 16502 Prague, Czech Republic

Abstract

In this work, the SFE was applied to extract selected medicinal substances from sea buckthorn (Hippophae
rhamnoides L.) leaves at different conditions (pressure 20-28 MPa, temperature 40-60 °C and ethanol concentration
in CO2 0-6.9 wt. %) influencing solvent power of CO2. Interest was focused on the leaf oleoresin (total extract) and
its minor components (fat soluble vitamins and carotenoids). The yield of polar component was still steadily
increasing at the moment when the extraction is almost finished as non-polar components are exhausted and the
increase in total extract is very low. Out of the operation variables, the ethanol concentration in CO2 showed the
strongest effect on the extraction. The final yields of polar components were increasing with rising ethanol
concentration in CO2. The extraction of non-polar components (vitamins and β-carotene) and polar lutein was
synchronised with oleoresin, whereas the extraction of non-polar lycopene, a component strongly affected by
entrainer, was not influenced by the extraction of oleoresin. The solute-solute interactions were expressed either as a
delay of the initial extraction period for some poorly soluble components against the more soluble ones or, on the
contrary, as a synchronous extraction of the components similar in structure.

© 2012 Published by Elsevier Ltd. Selection under responsibility of the Congress Scientific Committee
(Petr Kluson) Open access under CC BY-NC-ND license.

Keywords: Supercritical fluid extraction; sea buckthom leaves; solute-solute interaction; carotenoids

* Corresponding author. Tel.: +420220390202; fax: +420220920661.

E-mail address: [email protected].

1877-7058 © 2012 Published by Elsevier Ltd. Open access under CC BY-NC-ND license.
doi:10.1016/j.proeng.2012.07.561
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1. Introduction

Natural medicinal substances for nutraceuticals, food, cosmetics and pharmaceuticals become much
sought after nowadays. Supercritical fluid extraction with carbon dioxide (SFE) is one of the “green”
technologies for their separation. It uses neither toxic solvents nor high temperatures and its energy
consumption is lower than that of conventional techniques.
Sea buckthorn (Hippophae rhamnoides L.) is a deciduous shrub which fruit has a unique composition
with high levels of nutritionally and medicinally important components and a high potential as a dietary
and medicinal supplement. The chemical composition and the processing of sea buckthorn berries have
been reviewed [1]. As a by-product of sea buckthorn fruit processing, the leaves are a very cheap and
easy accessible source of carotenoids, flavonoids, tocopherols, free and esterified sterols, triterpenols, and
isoprenols [2-4]. Only few studies are available on their pharmacological effects, in contrary to fruits and
seed. Sea buckthorn leaves ethyl alcohol extract was reported to possess antioxidant and
immunomodulatory [5], anti-inflammatory [6], anti-stress and adaptogenic activity with no toxicity [7].
While the SFE of lipids from sea buckthorn berries and seeds has been studied by several researchers
[8-10] and is carried out on large scale, the technique has not been applied to sea buckthorn leaves,
according to our knowledge.
Although oleoresin extraction from various plants is described by many models in the literature, there
is no universal procedure to evaluate the extraction course of minor components. The same component
can indeed show a different relationship to the matrix and its relationship with other components may
vary. This is due to different structural properties and chemical composition of any plant or part thereof.
The seeds contain a significant proportion of lipids, the main component of the oleoresin from leaves are
cuticular waxes, while the roots are mostly formed by the polysaccharide matrix.
In this study, the evaluation was done by comparing the extraction course of the individual
components (α- and γ-tocopherol, vitamin K1, β-carotene, lycopene and lutein) of sea buckthorn leaves
depending on the extraction conditions. Furthermore, attention was focused on the possible interaction of
minor components during the extraction and on the correlation of their yields with the total extract
(oleoresin). The extraction yields of sea buckthorn leaf oleoresin were measured gravimetrically to
describe the course of the extraction. The composition of extract samples was analysed by HPLC.
carotene).

2. Experimental part

2.1. Materials

The leaves of sea buckthorn. were harvested from plants grown in the Czech Republic. Leaves were
dried at room temperature and grinded in a coffee mill prior to the extraction. The average particle size
leaves measured by sieve analysis of the particles after extraction was evaluated as the Sauter mean
diameters of 0.17 mm.
Carbon dioxide of purity better than 99.9 % was purchased from Linde Gas. Ethanol for spectroscopy
(Lachema Neratovice, CR) was used as entrainer. Antioxidant 2,6-di-tert-butyl-p-cresol (BHT) was
purchased from Fluka Chemie AG. Methanol and terc-butylmethylether (Chromservis s.r.o., CR) were
used as a mobile phase for chemical analysis. Chromatographic standards of β-carotene, lycopene, lutein,
α- and γ-tocopherol and vitamin K1 were purchased from Sigma (Sigma-Aldrich, Steinheim, Germany).
1684 M. Sajfrtov and Helena Sovova / Procedia Engineering 42 (2012) 1682 – 1691

2.2. Supercritical fluid extraction

The SFE apparatus used in this work was described in detail in a previous work [11]. Extraction
columns of 150 mL (I.D. 30 mm) volume was filled with 10 g of plant particles placed between layers of
glass beads serving as solvent flow distributors. The extractor was immersed in a temperature-controlled
water bath. CO2 was pressurised by compressor NovaSwiss 560.0007 and controlled by the pressure
regulator unit NovaSwiss 560.0009 to operating pressure. In experiments with entrainer, the stream of
CO2 was before entering the extractor mixed with ethanol pumped at a constant flow rate by high-
pressure pump LCP 4020.3 ECOM, CR. The solution was expanded to ambient pressure in a heated
micrometer valve behind the extractor and the extract fractions were collected in empty glass traps at
ambient temperature, weighed and stored for chemical analysis. At least five samples were taken in each
experimental run to describe the course of extraction. The amount of gaseous solvent leaving the trap was
measured using a gas meter.
The carbon dioxide flow rate was adjusted to 0.5 g.min-1. The extraction pressure was 20 MPa and 28
MPa, the extraction temperatures were 40 °C and 60 °C and the ethanol concentration in CO2 varied from
0 to 6.9 wt. %. To protect the components against degradation, the small amount (0.05 g into 100 mL) of
commercial antioxidant (BHT) was added into ethanol. Most of experiments were finished after 80 g
CO2.g leaves-1 passed through the extraction column.

2.3. HPLC analysis

The HPLC apparatus consisted of chromatograph HP 1090M and by UV-vis DAD detector (Hewlett-
Packard, Waldbron, Germany). Analytic column with stationary phase Develosil C30-UG 250x4 mm I.D
(Nomura Chemical Co. Ltd., Anada-Cho Seto, Japan) was protected by pre-column of size 10×4 mm I.D
with the same stationary phase.
The CO2 extracts were dissolved in 5 ml of methanol – tert-Butyl methyl ether mixture and filtered.
Non-linear multistep gradient with mobile phase methanol – tert-Butyl methyl ether – ammonium acetate
solution (0.2 %) was applied: 12 min 95:0:5 (v/v/v) isocratic; gradient 12 min 80:15:5 (v/v/v) and
gradient 6 min 30:65:5 (v/v/v). Chromatographic conditions were as follows: the flow rate of the mobile
phase was 1 mL.min-1, the ambient temperature was 20-23 ºC, and UV detection was performed at λ =
245 nm (vitamin K1), 275 nm (lycopene), 295 nm (tocopherols) and 480 nm (lutein, β-carotene).

3. Results and discussion

3.1. Extraction yield

The extraction of fat-soluble vitamins (α- and γ-tocopherol, vitamin K1) and plant pigments (β-
carotene, lutein and lycopene) from sea buckthorn leaves was studied under various conditions. The most
represented vitamin in the extracts was α-tocopherol, its average yields were 22 and 35 times higher than
the yields of γ-tocopherol and vitamin K1, respectively. Tocopherols β- and δ- were not found in the
leaves. Most experiments were performed at temperature 40 °C and pressure 28 MPa, in which the effect
of increasing ethanol concentrations in CO2 from 0 to 6.9 wt. % was studied. The influence of
temperature increase to 60 °C and pressure reduction to 20 MPa was measured at ethanol concentration in
CO2 4.3 wt. %. The experimental conditions and their labeling in graphs are summarized in Table 1.
 M. Sajfrtov and Helena Sovova / Procedia Engineering 42 (2012) 1682 – 1691 1685

Table 1. The overview of extraction conditions and the final yields of sea buckthorn leaf oleoresin and its minor components

Experiment Nr./ Mark 1/Ƈ 2/z 3/„ 4/Ÿ 5/¸ 6/¨

Extraction conditions
p [MPa] 28 28 28 28 28 20
T [°C] 40 40 40 40 60 40

Ethanol in CO2 [wt. %] 0 1.5 4.3 6.9 4.3 4.3

-1
Yields [mg.g leaves ]
Extract 74.6 77.4 80.1 91.1 86.7 88.9

β-Carotene 0.16 0.20 0.18 0.16 0.18 0.14

Lycopene 0.019 0.026 0.040 0.041 0.034 0.036

Lutein 0.025 0.040 0.054 0.063 0.043 0.042

α-Tocopherol 3.12 3.49 2.90 3.09 3.81 3.42

γ-Tocopherol 0.15 0.19 0.18 0.06* 0.21 0.10*

Vitamin K1 0.085 0.083 0.073 0.094 0.100 0.086

* Too low values probably due to analytical error

As apparent in Table 1, the lowest extraction yield of oleoresin was obtained using pure CO2.
However, the increased concentration of ethanol in CO2 led to co-extraction of polar components and
increase of oleoresin yield from 74.6 to 91.1 mg.g leaves-1. As expected, the amount of ethanol added to
CO2 did not affect the yield of non-polar vitamins. Both increase of temperature to 60 °C and reduction of
pressure to 20 MPa led to a slight increase of yields of oleoresin and all vitamins (in case of neglecting
the result of γ-tocopherol at experiment Nr. 6). The β-carotene yield was influenced neither by the
addition of ethanol in CO2 nor by the temperature but the reduced pressure of 20 MPa caused a decrease
in its yield from 0.18 to 0.14 mg.g leaves-1. The extraction of lycopene was strongly influenced by the
ethanol concentration in CO2 despite its non-polar character. The addition of 6.9 wt. % of ethanol to CO2
led to doubling the lycopene yield compared to the extraction with pure CO2. As expected the yield of
polar lutein was strongly dependent on the concentration of ethanol in CO2; its yield with 6.9 wt. %
ethanol in in CO2 was tripled compared to the yield with pure ethanol Reducing the pressure and the
temperature increase caused a slight decrease in the yield of lycopene and lutein.

3.2. Component interactions

The correlations of monitored components during the extraction were determined by plotting the yields
of compared components obtained in course of the extraction against each other.
The dependence of the γ-tocopherol and vitamin K1 yields on the α-tocopherol yield obtained in course of
the extraction (Figure 1) shows that both components are extracted always in a certain ratio to the
prevailing α-tocopherol. After removing the failed experiments, it could be possible to interpolate the
plotted points by linear line. This correlation is probably given by the similar structure of all three
vitamins.
1686 M. Sajfrtov and Helena Sovova / Procedia Engineering 42 (2012) 1682 – 1691

Fig. 1. The correlation of γ-tocopherol and vitamin K1 with α-tocopherol in course of extraction from sea buckthorn leaves; for
meaning of symbols see Table 1

The extraction of carotenoids was slower than the extraction of more soluble tocopherols. It illustrates
the Figure 2 where the yields of β-carotene against α-tocopherol are plotted. The yields of carotenoids
initially grew very slowly (β-carotene) or not at all (lutein), but after completion of α-tocopherol
extraction it started to rise rapidly.

Fig. 2. The correlation of β-carotene with α-tocopherol in course of extraction from sea buckthorn leaves; for meaning of symbols
see Table 1
 M. Sajfrtov and Helena Sovova / Procedia Engineering 42 (2012) 1682 – 1691 1687

Fig. 3. The correlation of carotenoids in course of extraction from sea buckthorn leaves; for meaning of symbols see Table 1

A similar behavior exhibited β-carotene and lycopene in relation to the less soluble lutein, as shown in
Figure 3, where one can also observe the effect of addition of ethanol to CO2 on the extraction of
carotenoids. Lutein was extracted in a certain ratio to β-carotene until β-carotene was present in the plant.
Extraction of lycopene was independent of other carotenoids and was affected in particular by the
concentrations of ethanol in CO2.
1688 M. Sajfrtov and Helena Sovova / Procedia Engineering 42 (2012) 1682 – 1691

3.3. Correlation of component with oleoresin

The correlations of monitored components with total extract (oleoresin) during the extraction were
determined by plotting the yields of compared components obtained in the course of the extraction against
the yield of oleoresin.

Fig. 4. The correlation of vitamins with total extract in course of extraction from sea buckthorn leaves; for meaning of symbols see
Table 1

When the incorrect data of γ-tocopherol are neglected, it can be seen in Figure 4 that the extraction
process all vitamins correlated with the oleoresin extraction, especially at the beginning of experiment,
when the easily soluble non-polar compounds are extracted. This relationship ceases to be obvious with
 M. Sajfrtov and Helena Sovova / Procedia Engineering 42 (2012) 1682 – 1691 1689

the growing yield of oleoresin, since the extraction of these three vitamins is complete when the total
extract is rising.

Fig. 5. The correlation of carotenoids with total extract in course of extraction from sea buckthorn leaves; for meaning of symbols
see Table 1

The extraction course of individual carotenoids was different, as shown in Figure 5. In contrast to β-
carotene and lutein, which yields correlated with the total extract irrespectively of the conditions, the
extraction of lycopene was affected by concentration of ethanol in CO2 and independent on the total
extract or other components.
1690 M. Sajfrtov and Helena Sovova / Procedia Engineering 42 (2012) 1682 – 1691

4. Conclusion

The extraction yields of individual components of sea buckthorn leaves were measured at equilibrium
conditions to examine the effects of pressure, temperature and ethanol concentration in CO2 on their final
yields as well as the solute-matrix and solute-solute interactions.
The published papers on the SFE from plants are focused on the yield of oleoresin and its composition
under different conditions, but data on the extraction kinetics is very limited and mostly concern only
oleoresin or one of its components. In this work, however, the extraction rate of more components was
examined simultaneously and the main contribution is the description of their interactions and correlation
with oleoresin.
An intense increase in the yield of polar component lutein was observed when the extraction of
oleoresin seemed to be almost finished. In contrast, the extraction of non-polar vitamins and β-carotene
was already complete at this time. Addition of ethanol to CO2 significantly facilitates the dissolution of all
components, but the final yields of non-polar components are not affected. Increasing temperature has a
positive effect on the extraction of vitamins and oleoresin.
Extraction of non-polar components (vitamins, β-carotene) and polar lutein proceeded in parallel with
oleoresin while the nonpolar lycopene, which shows a strong dependence on the ethanol concentration in
CO2, is affected neither by the extraction of oleoresin nor other components. The extraction course of
carotenoids is influenced by the extraction of vitamins, which are extracted simultaneously. A delay in
extraction of β-carotene is caused by initial extraction of more soluble α-tocopherol. Similar behavior
exhibit also β-carotene and lycopene in relation to the less soluble lutein.
The results extend the knowledge of SFE of medicinal substances from plants and provide information
about the most suitable conditions and extraction time for the extraction of given substance, which can be
used to control the concentration of certain components in the extract as desired.

Acknowledgements

The financial support by the Technology Agency of the Czech Republic via grant TA01010578 is
gratefully acknowledged.

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