Journal of Chromatography A, 1447 (2016) 26–38

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Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Selective enrichment in bioactive compound from Kniphofia uvaria by

super/subcritical fluid extraction and centrifugal partition
chromatography夽
Johanna Duval a , Emilie Destandau a , Virginie Pecher b , Marion Poujol b ,
Jean-François Tranchant b , Eric Lesellier a,∗
a
Univ. Orléans, CNRS, ICOA, UMR 7311, F-45067 Orléans, France
b
LVMH Recherche, 185 Avenue de Verdun, F-45800 Saint-Jean-de-Braye, France

a r t i c l e i n f o a b s t r a c t

Article history: Nowadays, a large portion of synthetic products (active cosmetic and therapeutic ingredients) have
Received 30 September 2015 their origin in natural products. Kniphofia uvaria is a plant from Africa which has proved in the past
Received in revised form 9 March 2016 by in-vivo tests an antioxidant activity due to compounds present in roots. Recently, we have observed
Accepted 11 April 2016
anthraquinones in K. uvaria seeds extracts. These derivatives are natural colorants which could have
Available online 12 April 2016
interesting bioactive potential. The aim of this study was to obtain an extract enriched in anthraquinones
from K. uvaria seeds which mainly contains glycerides.
Keywords:
First, the separation of the seed compounds was studied by using supercritical fluid chromatography
Supercritical fluid chromatography (SFC)
Centrifugal partition chromatography (CPC)
(SFC) in the goal to provide a rapid quantification method of these bioactive compounds. A screening of
Super/subcritical fluid extraction (SFE) numerous polar stationary phases was achieved for selecting the most suited phase to the separation
Anthraquinone of the four anthraquinones founded in the seeds. A gradient elution was optimized for improving the
Selective enrichment separation of the bioactive compounds from the numerous other families of major compounds of the
extracts (fatty acids, di- and triglycerides).
Besides, a non-selective and green Supercritical Fluid Extraction (SFE) with pure CO2 was applied to
seeds followed by a Centrifugal Partition Chromatography (CPC). The CPC system was optimized by using
the Arizona phase system, to enrich the extract in anthraquinones. Two systems were selected to isolate
the bioactive compounds from the oily extract with varied purity target. The effect of the injection mode
for these very viscous samples was also studied.
Finally, in order to directly apply a selective process of extraction to the seeds, the super/subcritical
fluid extraction was optimized to increase the anthraquinone yield in the final extract, by studying var-
ied modifier compositions and nature, as well as different temperatures and backpressures. Conditions
suited to favour an enrichment factor bases on the ratio of anthraquinone and trilycerides extracted are
described.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction increasingly recovered, as they represent an attractive economical

Vegetable natural resources represent an incredible tank of
bioactive molecules for numerous different applications such as
cosmetic, food-processing or pharmaceutical fields. These veg-
etable natural resources are today little valued and by-products are
often non-recovered. However, for several years, wastes have been
夽 Selected paper from 42nd International Symposium on High Performance Liquid
Phase Separations and Related Techniques, 21–25 June 2015, Geneva, Switzerland.
∗ Corresponding author.
E-mail address: eric.lesellier@univ-orleans.fr (E. Lesellier).
option in order to decrease or eliminate these by-products, while
generating a new benefit for corporations. Consequently, the devel-
opment of vegetable resources has become an important topic and
challenge for corporations. Methods need to be developed and opti-
mized for an important production of new value-added products,
while limiting the environmental impact in terms of energy, toxic-
ity and consumption of solvent. One possible solution is to exploit
the entire plant. For instance, only one part of the Kniphofia uvaria
plant is currently used in luxury cosmetic products.
The genius Kniphofia is a plant from Asphodelaceae family
according to phylogenetic classification. It comes from Africa and
is known for its tubular red or orange flowers and presents a little

http://dx.doi.org/10.1016/j.chroma.2016.04.029
0021-9673/© 2016 Elsevier B.V. All rights reserved.
 J. Duval et al. / J. Chromatogr. A 1447 (2016) 26–38 27

Table 1
Characteristics of different stationary phases tested for separation of target compounds (anthraquinones).

Column name Stationary phase Supplier Column Dimensions (mm) Particle size (␮m)

(␮m)
YMC polyamine II Polyamine bonded silica YMC 250 × 4.6 5
Discovery HS PEG Poly(ethylene glycol)bonded silica Supelco 250 × 4.6 5
Luna Hilic Crosslinked propanediol bonded silica Phenomenex 250 × 4.6 5
BEH—Xbridge amide Trifunctional amide bonded on hybrid silica Waters 250 × 4.6 3.5
Viridis silica Silica Waters 250 × 4.6 5
TSK-gel amide 80 Polyamide gel bonded on silica Tosoh 150 × 4.6 3
Kinetex Hilic Core-shell silica Phenomenex 150 × 4.6 2.6
Atlantis Hilic Silica Waters 250 × 4.6 5
ACE CN-ES Cyanoalkyl ACE 250 × 4.6 5
Nucleosil 100-5 NO2 4-nitrophenyl Macherey-Nagel 250 × 4.6 5
Cosmosil Hilic 1H-1,2,4-Triazole bonded silica Nacalai Tesque 250 × 4.6 5
ACE 5C4 Butyl bonded silica ACE 250 × 4.6 5
Ascentis express OH5 5Hydroxyl group bonded silica Supelco 150 × 4.6 2.7
Accucore HILIC Core–shell silica Thermo 150 × 4.6 2.6
YMC pack PVA-SIL NP Poly(vinyl alcohol) bonded silica YMC 250 × 4.6 5
BEH-Xbridge Hilic Organic/inorganic hybrid silica Waters 250 × 4.6 5
Viridis SFC hybrid 2EP 2-Ethylpyridine bonded silica Waters 250 × 4.6 5

assisted microwave extraction (MAE), ultrasonic extraction (UE),

Fig. 1. Analytical strategy to enrich the Kniphofia uvaria extract from seeds.
more than 70 species. In Europe, Kniphofia is used for ornamental
gardening whereas in Africa, it is used for medicinal applications.
Indeed, rhizomes of Kniphofia foliosa are used for treatment of
abdominal cramps or to eradicate endoparazites [1]. The phyto-
chemistry of Kniphofia has been thoroughly studied and it appeared
to contain anthraquinones, flavonoids and alkaloids, which have
bioactivities including anticancer or antimalarial activities [2–4].
However, only few studies about this plant were published. A valu-
ation of different parts of this plant (e.g. seeds) had been performed
by the Supercritical Fluid Extraction (SFE) and demonstrated by
in-vivo tests an antioxidant activity.
Many studies report the extraction of lipids from vegetable
matrices such as seeds, most of them reporting the same two
methods, namely Folch and Soxhlet. However, these methods are
performed at high temperatures; require a long extraction time
with hazardous organic solvents (methanol and chloroform). As
a consequence, quicker and greener extraction techniques are
needed [5,6]. Among them, the most frequently used techniques are
pressurized fluid extraction (PFE) and supercritical fluid extraction
(SFE) [5].
Microwaves have been used to extract lipids from vegetable
matrices. However, to achieve the extraction of lipids, the solvent
must have a sufficient dielectric constant able to absorb microwave
energy, together with some lipophilicity [7,8].
Pressurized fluid extraction is a method largely employed for the
extraction of bioactive compounds from plants. This method has
already been applied for extraction of several kinds of interesting
compounds such as phenolic compounds, essential oils or lipids.
This extraction consists to use a pressurized liquid solvent above
its atmospheric boiling point to enhance compound solubility and
mass transfer properties from the plant to the extracting solvent
[5,9]. Obviously, the choice of solvent depends of the polarity of
target molecules [10,11].
Centrifugal partition chromatography (CPC) is a technique that
can be used to purify and concentrate natural bioactive com-
pounds from plant extracts. This method is based on continuous
liquid-liquid partition chromatography in which compounds are
separated according to their partition coefficient between two
immiscible liquid phases. These two phases are represented as a
stationary phase and a mobile phase. The stationary phase is main-
tained in column thanks to the rotational speed, and the mobile
phase crosses the stationary phase to extract compounds present in
the stationary phase. The main advantages of CPC are no irreversible
adsorption, total sample recovery at the end of the experiment, a
wide loading capacity with a possibility of scale-up [5]. Generally
applied to organic solvent extracts, there are only few papers on the
fractionation of vegetable oils on semi-preparative or preparative
scales [12–14].
In the recent years, the number of studies related to supercrit-
ical fluid extraction of active ingredients from plant materials has
increased. SFE mainly uses supercritical carbon dioxide, as non-
toxic solvent. One of the main advantages of super/subcritical fluid
extraction (SFE) is the ability to control the solvent power and so the
selectivity of the extraction by parameters such as pressure, tem-
perature or the addition of a modifier in the extracting supercritical
fluid [15].
The term subcritical refers to a liquid at temperatures between
the atmospheric boiling point and the critical temperature, whereas
in the supercritical state, experimental conditions are higher than
the critical temperature and pressure.
Different molecular families of compounds can be extracted by
SFE such as fatty acids, alcohols, flavonoids, terpenes, phenolic com-
pounds, sterols, or triglycerides. Several studies have showed the
28 J. Duval et al. / J. Chromatogr. A 1447 (2016) 26–38

use of SFE for the valuation of varied compounds from seeds [15].
Although SFE is more used for lipophilic solutes, it is also possible
to extract compounds with a higher polarity such as polyphenols,
by adding a polar co-solvent such as ethanol [15–17].
Finally, Supercritical Fluid Chromatography (SFC) is an
environmentally-friendly alternative to liquid chromatography,
and can be used to quantify the compounds extracted. The
mobile phase has a low viscosity and a high diffusivity com-
pared to the liquid mobile phase in high-performance liquid
chromatography (HPLC). Thanks to these particular properties, SFC
can be done at high flow rates to reduce the analysis time, or
with long column lengths to increase the efficiency. So, SFC can
advantageously replace non-aqueous reverse-phase liquid chro-
matography (NARP-LC) methods, which are usually employed for
hydrophobic compounds such as triglycerides [18]. Indeed, iso-
meric separation is easier, and non-polar compounds as lipids can
be analysed without the need for derivatization (unlike gas chro-
matography (GC)). Thermolabile compounds that are unsuited to
GC can also be easily analysed with SFC. Consequently, for the anal-
ysis of vegetable oils or extracts of oleaginous plants, SFC coupled to
ultraviolet (UV) and mass spectrometry (MS) detections is a pow-
erful tool to characterize and identify compounds.
The aim of this work is to develop methods to extract or con-
centrate the minor bioactive compounds from seeds, which is an
oleaginous media (Fig. 1). A first oily extract from K. uvaria seeds
obtained by a non-selective supercritical fluid extraction using pure
CO2 was first provided by LVMH-Recherche. The extract, which
contains a wide amount of free fatty acids (FFA), diacylglycerides
(DAG) and triacylglycerides (TAG), presents a biological activity.
Its color is due to the presence of anthraquinones. To improve
the amount of colored and bioactive compounds from this non-
selective SFE oily extract, an enrichment of anthraquinones was
performed. First, the separation of triglycerides was carried out
using centrifugal partition chromatography (CPC). In a parallel step,
a second CPC system was achieved to fractionate more specifically
anthraquinones from all glyceride families. A second approach was
studied by using super/subcritical carbon dioxide with ethanol or
ethanol/water mixture as a co-solvent, to selectively extract the
targeted anthraquinones directly from vegetable seeds. In all cases,
the amounts of anthraquinones extracted and of varied glycerides
were analyzed using SFC-UV with varied analytical conditions with
regards to the studied compound family.

2. Experimental

2.1. Chemicals

All solvents used were purchased from VWR (Darmstadt,

Germany) and were reagent-grade for HPLC. Aqueous phases
(CPC studies) was prepared with pure water produced by a
Milli-Q water (18 M) system (Millipore, Bedford, MA, USA).
Anthraquinone standards were purchased from Extrasynthese
(Genay, France): Aloe emodin, emodin, rhein, juglone, alizarin,
chrysophanol, physcion and plumbagin. For extraction, the CO2 was
supplied by Linde (Munich, Germany) and presents a reagent grade
for analytical conditions. For analytical chromatography, the CO2
bottle was supplied by Messer (Bad Soden, Germany) with a purity
of 99.995%.

2.2. Supercritical fluid chromatography Fig. 2. Representation of the filling of extraction cell.

Stationary phases studied in this work are listed in Table 1,

along with their characteristics. A first screening in isocratic con-
ditions (CO2 /Methanol 90/10) was carried out with 17 stationary
phases based on pure silica gel or having varied polar bonded
 J. Duval et al. / J. Chromatogr. A 1447 (2016) 26–38 29

Fig. 3. Chromatographic profiles of 4 clusters of columns with a mobile phase CO2 /MeOH (90/10), T = 25 ◦ C, Pout = 15 MPa, Flow rate = 3 mL min−1 , Vinj = 1 ␮L at 210 nm, (A)
XBridge BEH Amide (B) Luna HILIC (C) Viridis BEH 2-Ethylpyridine (D) ACE-5-C4 columns.

Table 2 Table 3
Super/subcritical fluid extraction tested conditions. Resolution of two major chromatographic peaks with Viridis BEH 2EP, Luna HILIC
and XBridge BEH Amide at 430 nm using the gradient at 20%.
Solvent Percentage (v/v) (%) Temperature (◦ C) Pressure (MPa)
BEH-2-EP Luna HILIC BEH-Xbridge amide
CO2 100 30 10
20 Peak 1 Peak 2 Peak 1 Peak 2 Peak 1 Peak 2
60 10
20 tr(min) 10.65 17.83 10.08 13.03 11.82 13.55
Résolution 14.9 6.3 4.9
CO2 /EtOH 95/5 30 10
20
60 10
20 column oven compatible with 150 mm length columns and another
70/30 30 10 column oven compatible with 300 mm length columns. The detec-
20
tion system is a photodiode-array (PDA) detector. The injector
60 10
20 valve comprised a 10 ␮L loop. Chromatograms were recorded with
Empower® 3 software.
CO2 /EtOH/H2 O 95/4/1 30 10
20
60 10
2.2.2. Analysis of CPC collection with jasco equipment
20
70/24/6 30 10 Analytical chromatographic separations were carried out using
20 equipment purchased from Jasco (Tokyo, Japan). The system was
60 10 equipped with one CO2 pump model 2080-CO2 Plus and a second
20 HPLC pump 2080. Then, the supercritical fluid and modifier were
mixed in a dynamic chamber PU 4046 (Pye Unicam, Cambridge,
UK). The injector valve was purchased from Rheodyne and com-
ligands to select the best suited chromatographic column for the prised a 20 ␮L loop (model 7125 Rheodyne, Cotati, CA). Columns
anthraquinone quantification. A non-polar stationary phase, a C18- were thermostated with an oven (Jetstream 2 Plus, Hewlett-
bonded silica phase (Kinetex C18) was also used for glyceride Packard, Palo Alto, CA). The detector was a Gilson UV 151 detector
analysis (see details below). (provided by Waters, Milford, MA) equipped with a pressure-
Chromatographic separations were carried out using two differ- resistant stainless steel cell. After the detector, the outlet column
ent instruments. pressure was controlled by a Jasco BP-2080 Plus back pressure regu-
lator (BPR) which was heated at 60 ◦ C to avoid ice formation during
2.2.1. Analysis of SFE extract with UPC2 equipment the CO2 depressurization.
The first equipment was an ACQUITY UltraPerformance Conver- For the chromatographic separation of anthraquinones, the
gence ChromatographyTM (UPC2) system from Waters (Millford, separation was performed with the Viridis BEH 2-Ethylpyridine
MA, USA). The system was equipped with a binary solvent delivery column at 430 nm, the eluent was methanol/CO2 . A gradient from
pump compatible with mobile phase flow rates up to 4 mL min−1 2% to 30% methanol was performed in 14 min with a flow rate of
and pressures up to 414 bar, an autosampler that included par- 3 mL min−1 . Then, the modifier percentage was decreased down to
tial loop volume injection system, a backpressure regulator, one 2% from 19 to 21 min. The injected volume was 20 ␮L.
30 J. Duval et al. / J. Chromatogr. A 1447 (2016) 26–38

Fig. 4. Comparison of UV chromatograms of the extract of Kniphofia uvaria (30 000 ppm) at different wavelengths: 210 (blue, positive base line shift)) and 430 nm (red,
negative baseline shift)) with Viridis BEH 2EP (250 × 4.6 mm, 5 ␮m) (A), Luna HILIC (250 × 4.6 mm, 5 ␮m) (B), XBridge BEH Amide (250 × 4.6 mm, 3.5 ␮m) (C), Mobile phase
(A) CO2 , (B) MeOH, gradient elution: 0–5 min: 2% B, 5–14 min: 2–30% B, 14–19 min: 30% B, 19–21 min: 30–2% B, 3 mL min−1 , Vinj = 1 ␮L at 210 nm and Vinj = 20 ␮L at 430 nm
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

Fig. 5. UV Chromatogram of a CPC collected fraction of Kniphofia uvaria (30 000 ppm), column Viridis BEH 2EP (250 × 4.6 mm; 5 ␮m), Mobile phase (A) CO2 , (B) MeOH,
gradient elution: 0–5 min: 2% B, 5–14 min: 2–30%B, 14–19 min: 30% B, 19–21 min: 30–2% B, 3 mL min−1 , Vinj = 20 ␮L, detection: UV = 430 nm.
 J. Duval et al. / J. Chromatogr. A 1447 (2016) 26–38
Fig. 6. Distribution coefficient of TAG (LLL) according to different Arizona Systems.
Fig. 7. Representation of two injection modes tested to inject the oily extract in CPC.
For the analysis of triacylglycerides and other lipids, the sep-
aration was performed at 210 nm with 3 Kinetex C18 columns
(150 × 4.6 mm, 2.6 ␮m) (Phenomenex, Le Pecq, France) coupled in
series (except by using UPC2 : only C18 Kinetex column was used).
The mobile phase was composed of CO2 /Methanol (90:10) (v: v) in
isocratic elution condition. The flow rate with this column was of
1.6 mL min−1 . The back pressure regulator was fixed at 10 MPa and
several temperatures were tested: 15 ◦ C, 25 ◦ C and 35 ◦ C to optimize
the analysis. The injected volume was 1 ␮L.
2.3. Super/subcritical fluid extraction of seeds
2.3.1. Non-selective extraction
This extract was provided by LVMH-Recherche. K. uvaria seeds
were collected for LVMH-Recherche. 300 g seeds were grinded with
a Braun blender of 1 L during 30 s. Samples were extracted in Sepa-
rex SFE-500 apparatus in extraction conditions of 29 MPa, 60 ◦ C and
with pure CO2 . The extraction time was defined by the CO2 mass
eluted through the extraction cell. When the CO2 mass reached
3 kg, the extraction was stopped. In the end, the oil was covered
and dried under vacuum with some millilitres of ethanol in order
to help the drying. The oil extract was stored at 4 ◦ C under nitrogen.
2.3.2. Selective extraction of anthraquinones by SFE
K. uvaria seeds were collected and grinded as previously. The
SFE apparatus was a MV-10ASFE provided by Waters (Guyancourt,
France). This one was equipped with a pumping system which
allows adding modifier to CO2 , up to six different ones. 10 stainless
steel cylindrical extraction cells located in the oven with a 5 mL-
capacity were used for the SFE extraction. The extraction cell was
fully filled with cotton (white area with blue ray; Fig. 2) and sand
(pink area; Fig. 2) on both ends to prevent preferential pathway
(Fig. 2). Experiments were performed with the same batch of seeds
in order to have homogeneous samples. A grinded seeds sample of
0.5 g was weighed and used for extraction (green area; Fig 2). The
effect of different temperatures, 30 and 60 ◦ C, back pressures of 10
and 20 MPa, and co-solvent percentages of ethanol and ethanol:
water (80:20) (v: v), 0, 5, 30%, to the extraction amount has been
investigated (Table 2). The extraction process lasted 1 h, with a flow
rate of 3 mL min−1 . The final mass of collected oil was weighted and
31
the yield was calculated. The yield corresponds to the mass of the
final extract compared to the mass of seeds used for the extraction.
Then, the extract collected was dissolved in the required volume of
methanol: methylene chloride (1:1) to achieve a concentration of
30 000 ppm prior to injection in the SFC system. This solution was
analyzed by SFC, either at 210 nm for TAG (Kinetex C18 system) or
at 430 nm (polar phase system) for anthraquinones.
2.4. Centrifugal partial chromatography
2.4.1. CPC device
The fractionation step was performed with a FCPC (Fast CPC)
from Rousselet Robatel Kromaton (Annonay, France) using a rotor
of circular disks made of partition cells linked by capillary ducts
with a column capacity of 50 mL. The mobile phase was pumped by
an Agilent 1100 infinity pump (American). The oil (obtained from
non-selective SFE) was introduced in the column using a 6-port
medium pressure injection valve (Upchurch Scientific, Oak Harbor,
WA, United States) equipped with a 10 mL loop. The CPC instrument
was coupled with a UV detector Spectroflow 783 model from Kon-
tron Instruments (Montigny Le Bretonneux, France) set at 210 nm
or 430 nm and an evaporative light scattering detector SEDEX 55
(ELSD) purchased from SEDERE (Alfortville, France) used in the fol-
lowing conditions: temperature of nebulizer was 50 ◦ C, pressure of
nebulizer was 0.2 MPa and gain was 6. CPC effluent was all sent in
the UV detector but not all directed through the ELSD detector: it
was split using a variable flow splitter (VFS) from Rheodyne (Rohn-
ert Park, CA, USA). The auxiliary stream was supplied by a one-way
LC-10 AS pump (Shimadzu, Japan) delivering an EtOH solution at a
flow rate of 0.3 mL min−1 . The part not switched through the detec-
tion device was collected in 25 mL collecting tubes, and each tube
was then subjected to SFC analysis in order to characterize the
minor bioactive fraction of compounds in the K. uvaria oil.
2.4.2. Selection of solvent system
The suitable solvent systems for enrichment by CPC were
selected in a series of Arizona solvent systems [19]. This series
contains 23 mixtures (from A to Z, except E, I and O), of four sol-
vents (water/methanol for the polar phase, 50% of the mixture,
and heptane/ethyl acetate for the non-polar one, representing the
remaining 50% of the mixture). In each phase (aqueous or organic),
the proportions of solvents are varied, to cover a wide array of polar-
ity. When going from A to Z system, the methanol portion increases
with regards to the water portion, and the heptane portion
increases with regards to the ethyl acetate portion. The selection
of the suited system was achieved as follows: about 200 ␮L of oily
32 J. Duval et al. / J. Chromatogr. A 1447 (2016) 26–38

Fig. 8. UV Chromatograms of CPC elution fractions with loss of stationary phase for the collection 7 (A) and without loss of stationary phase for the collection 7 (B) CO2 /MeOH
(90/10), T = 25 ◦ C, pout = 15 MPa, flow rate = 1.6 mL min−1 , vinj = 1 ␮L at 210 nm, viridis BEH 2-Ethylpyridine (250 × 4.6 mm; 5 ␮m).

sample was added to 4-mL test tubes, and a total mixture of 2 mL
solvents was added. Each test tube was rigorously shaken for sev-
eral minutes and left to set at room temperature until equilibrium
was reached. Then 500 ␮L of organic phase (mainly heptane/ethyl
acetate) and aqueous phase (mainly water/methanol) were taken
out the vial to be evaporated under nitrogen stream and dis-
solved again with 500 ␮L methanol: methylene chloride (50:50)
(v: v). Then, 5 ␮L of the organic and aqueous phases were ana-
lyzed by SFC both for the analysis of anthraquinones at 430 nm
and triglycerides at 210 nm. The partition coefficient (Kd = Cu /Cl ) of
each compound family between the two phases of the CPC system is
defined by the ratio Aupper /Alower , where Aupper and Alower were the
SFC-UV peak areas of interest compounds in the upper and lower
phases.

Fig. 9. Calculation of weight yield of aloe emodin and rhein in CPC elution and extrusion fractions obtained with Arizona system Q (1 mL min−1 , descending mode, 1 mL
crude oil injected)—analysis of fractions of Kniphofia uvaria (30 000 ppm) by SFC-UV, column Viridis BEH 2EP (250 × 4.6 mm; 5 ␮m), mobile phase (A) CO2 , (B) MeOH, gradient
elution: 0–5 min: 2% B, 5–14 min: 2–30% B, 14–19 min: 30% B, 19–21 min: 30–2% B, 3 mL/min, Vinj = 20 ␮L, detection: UV = 430 nm.
 J. Duval et al. / J. Chromatogr. A 1447 (2016) 26–38 33

Table 4
Identification of anthraquinones in the extract of Kniphofia uvaria.

Structure Trivial name Luna Hilic Retention Viridis BEH 2-EP BEH-Xbridge amide Detected in the extract
time (min) Retention time (min) Retention time (min)

Chrysophanol 1.66 2.03 1.71 x

Physcion 2.25 2.3 2.1 x

Aloe emodin 9.28 9.71 10.53 x

Emodin 9.46 10.73 10.05

Rhein 10.78 14.30 11.58 x

Juglon 1.53 2.01 1.71

Plumbagin 1.46 1.52 1.55

Fig. 10. Influence of pressure, temperature and nature of modifier on the extraction of TAG (A and B), aloe emodin (C and D) and rhein (E and F).
34 J. Duval et al. / J. Chromatogr. A 1447 (2016) 26–38
2.4.3. Centrifugal partition chromatography procedure
The elution-extrusion method was used for CPC. Elution of com-
pounds was performed in descending mode (the organic phase
was used as stationary phase while aqueous phase was used as
mobile phase). The multilayer coiled column was first entirely filled
with the upper organic stationary phase (50 mL). The lower aque-
ous mobile phase was then pumped into the head end of the inlet
column at a flow-rate of 1 mL min−1 , with a revolution speed of
2000 rpm. The pressure of the system was 2.8 MPa. A fraction of
30 mL of the stationary phase was displaced out of the column by
the mobile phase before to achieve the system equilibrium. Conse-
quently, the ratio of stationary phase to mobile phase is 2/3 (v/v).
After the equilibrium was reached, as indicated by a constant pres-
sure and a clear mobile phase eluting at the tail outlet, the sample
solution (either pure or dissolved oil) was injected thanks to the
manual sample port. After fractionation and collection of numerous
elution fractions of 20 mL of the mobile phase, the whole content
of CPC column was extruded by pumping the organic phase in
descending mode at 3 mL min−1 and 500 rpm.
3. Results and discussions
3.1. Development of a chromatographic method for analysis of
anthraquinones
The seed extracts contain numerous FFA and glycerides (DAG
and TAG) and minor compounds such as the anthraquinones.
A previous study achieved in SFC with a set of coupled C18
columns showed the great ability of C18 bonded phases to separate
the TAG species having few structural differences [20]. However,
anthraquinones were eluted at the beginning of the chromatogram,
and coeluted with the FFA. Consequently, to quantify the effect
of the studied parameters of selective SFE or CPC conditions on
the anthraquinone content of varied collected fractions, we devel-
oped a new chromatographic method in SFC using polar stationary
phases.
The columns are all silica-based, most of them have identi-
cal dimensions, apart from the TSK-gel Amide 80 and the three
core–shell phases (Kinetex HILIC, Ascentis Express OH5 and Accu-
core HILIC) that were shorter columns than the others. All phases
were 5 ␮m particles, apart from the three superficially porous
columns (2.6 and 2.7 ␮m), XBridge BEH Amide (3.5 ␮m) and TSK-gel
amide 80 column (3 ␮m).
Two columns were based on bare silica (Viridis Silica and
Atlantis HILIC). The XBridge HILIC is an organic/inorganic hybrid
silica. Two columns were alkyl-amide bonded silica, but the exact
nature of the bonded ligand is undisclosed: TSK-gel Amide 80 and
XBridge BEH Amide. Four columns were alcohol-type stationary
phases: one a poly(vinyl alcohol) phase (YMC-Pack PVA-Sil NP),
where the polymer (of undisclosed nature) is coated on silica, the
second column is a cross-linked diol (Luna HILIC) and the third
is a poly(ethylene glycol) ligand deposited on silica (Discovery
HS PEG). The last alcohol-type column is a penta-hydroxyl group
but the nature of the bonded ligand is not well-known (Ascen-
tis Express OH5). One column had a unique 1H-1,2,4-Triazole
bonded group (Cosmosil HILIC). Another column was a nitrophenyl
bonded group (Nucleosil 100-5 NO2). Another column was a unique
BEH-2-Ethylpyridine bonded group (Viridis BEH 2EP). One was a
cyanoalkyl where the exact nature of the ligand is undisclosed
(ACE CN-ES). Another column was a butyl group bonded on silica
(ACE 5C4). Finally, there was one amine stationary phase, YMC-
Pack Polyamine II. The column is a polyamine phase containing
only secondary and tertiary amine groups.
On the chromatographic profiles (Fig. 3), four clusters of
columns appear:
A YMC polyamine II, TSK-gel amide 80, XBridge BEH Amide and
Cosmosil HILIC columns.
B Luna HILIC, Viridis silica, Atlantis HILIC, XBridge HILIC, Discovery
HS PEG, ACE CN-ES, Nucleosil 100-5 NO2 columns.
C Ascentis Express OH5, YMC pack PVA-SIL NP, Accucore HILIC,
Kinetex HILIC, Viridis BEH 2EP columns.
D ACE-5-C4 column.
According to the chromatographic profile, the first cluster
(Fig. 3A) includes the two amide phases (XBridge BEH Amide and
TSK-gel amide 80), one amine phase (YMC polyamine II) and one tri-
azole phase (Cosmosil HILIC). The chromatographic profile is shared
in 3 groups of chromatographic peaks, one peak around 1.2 min, a
second group of two main peaks around 1.5 min and several minor
peaks around 3.5 min. Using polar stationary phases such as these,
the retention order for glyceride compounds is first the TAG, then
DAG and FFA. Anthraquinones are included in the last eluted group.
The second cluster (Fig. 3B) comprises silica phases (Atlantis
HILIC, Viridis silica, XBridge HILIC), the poly(ethylene glycol) phase
(Discovery HS PEG), the cyanoalkyl phase (ACE CN ES), the p-
nitrophenyl phase (Nucleosil 100-5 NO2), the cross-linked diol
phase (Luna HILIC) and the last hydroxyl phase (Ascentis Express
OH5). The chromatographic profile (Fig. 3B) shows varied selec-
tivities of groups 2 and 3, and a lower retention time for group
3.
The third cluster (Fig. 3C) contains the two core-shell phases
(Accucore HILIC and Kinetex HILIC), the poly(vinyl alcohol) phase
(YMC pack PVA-SIL NP) and the 2-Ethylpyridine phase (Viridis BEH
2EP). It presents an intermediate chromatographic profile between
the first two clusters.
The last cluster (Fig. 3D) is composed of only the butyl station-
ary phase (ACE 5C4). The last chromatographic profile has shown a
lower separation of the three groups.
Finally, one column from each of the three first clusters was
retained from the final optimization: XBridge BEH Amide, Luna
HILIC and Viridis BEH 2EP. The selected columns all have iden-
tical dimensions except for the size of particles. The stationary
phases of Luna HILIC and Viridis BEH 2EP were 5 ␮m particles and
XBridge BEH Amide was 3.5 ␮m. Luna HILIC was a hydroxyl-type
phase, Viridis BEH 2EP was 2-Ethylpyridine bonded hybrid silica
and XBridge BEH Amide was a trifunctional amide group bonded
on hybrid silica.
In the goal to provide a greater resolution between groups of
peaks, and in-between each group of peaks, a methanol gradient
was applied on these three phases, beginning with lower modi-
fier content (2%). An additional wavelength 430 nm (a secondary
absorption peak of anthraquinone) was also used, for a better detec-
tion of compounds of interest.
From the comparison of the superposition of two chro-
matograms obtained at 210 nm and 430 nm for the three
chromatographic columns (Fig. 4), it appears that the XBridge
BEH Amide and Viridis BEH 2-Ethylpyridine columns did separate
interest compounds (full lines) from glyceride families and FFA
(dashed lines), whereas with the Luna HILIC column, the first peak
of anthraquinones coelutes with glycerides.
The two major peaks observed at 430 nm were selected to
calculate the resolution (Table 3). The resolution of these two
chromatographic peaks is larger with the column Viridis BEH 2EP
compared to Luna HILIC or XBridge BEH Amide columns.
To optimize chromatographic resolution, different conditions
were studied. A second gradient, reaching 30% methanol instead
of 20% was applied to reduce the analysis duration. Column tem-
perature was set at 35, 25 or 15 ◦ C. The resolution between the
 J. Duval et al. / J. Chromatogr. A 1447 (2016) 26–38
two main anthraquinone peaks increased at low temperatures, and
the analysis duration was reduced. So 15 ◦ C was retained for the
chromatographic method on a polar stationary phase.
To achieve a better identification of these compounds, the anal-
ysis of standard compounds on the 3 columns was carried out,
as shown in Table 4. The retention times of juglone and chryso-
phanol on XBridge BEH Amide column were the same. Thus, no
identification between these two compounds is possible with this
column when only UV detection is employed. However, with the
Luna HILIC and Viridis BEH 2EP columns, the discrimination was
possible. Moreover, with the XBridge BEH Amide, the elution order
of aloe emodin and emodin was switched compared to the 2 other
columns. Focusing on Luna HILIC, a coelution between emodin and
rhein is also noted.
Although of change of elution order was observed for two com-
pounds with the XBridge BEH Amide, these compounds eluted
according to their polarity as can be observed with the structures
in Table 4. Indeed, chrysophanol and physcion eluted first. Aloe
emodin and rhein eluted later because they have an additional polar
group. The differentiation between aloe emodin and rhein is the
presence of the carboxylic group on rhein, the acidic group induc-
ing stronger interactions with the basic stationary phase (Viridis
BEH 2-Ethylpyridine). Finally, the compounds of interest in the K.
uvaria oil extract (Fig. 5) are: chrysophanol (1), physcion (2), aloe
emodin (3) and rhein (4). The two latter are the major ones whereas
the two former are very minor compounds.
To summarize the above, the screening of seventeen columns
has thus permitted to select a set of 3 chromatographic columns,
allowing both the separation of anthraquinone from glycerides
and the identification and quantification of anthraquinone species.
Thanks to this set, a comparison in terms of retention of standards
was performed to reveal four well-known anthraquinones (poten-
tial bioactive compounds) in the extract of the seeds of K. uvaria:
physcion, chrysophanol, aloe emodin and rhein. The final choice
of Viridis BEH 2-Ethylpyriridine and of the analysis conditions of
the extract of K. uvaria was based on the high resolution between
anthraquinone species, and on the satisfactory separation between
anthraquinone and glyceride families.
3.2. Centrifugal partition chromatography (CPC)
The non-selective extract of K. uvaria seeds contains mainly
lipids (TAG, DAG and FFA) whereas the bioactive compounds
(anthraquinones) are minor. For a cosmetic use, it could be interest-
ing to obtain samples with a higher concentration of the bioactive
compounds, so the fractionation between these compounds and
lipids was needed. One method to concentrate minor compounds
has been the use of CPC on the non-selective extract.
Firstly, we developed one method to separate the TAG, the major
compounds of the oily extract, from the rest of the matrix. For
this purpose, it was necessary to investigate the possible injection
modes with a highly viscous sample. Then, we tried to improve
the fractionation selectivity, to separate anthraquinones from other
lipids (DAG, FFA).
3.2.1. TAG fractionation
3.2.1.1. Selection of a CPC system for triacylglycerides. The Arizona
system was used to define the best solvent system to separate TAG
from the oily extract. Because TAG are non-polar molecules, the 5
biphasic systems tested were T, V, X, Y, Z, containing the highest
proportions of methanol and heptane (Table 5).
The value of Kd of TAG was calculated based on SFC analysis
performed at 210 nm with 3 Kinetex C18 columns (125 × 4.6 mm;
2.7 um), as described in Section 2.4.2. From the Kd values of different
biphasic systems used for the isolation of TAG (Fig. 6), Arizona sys-
tem X (heptane, ethyl acetate, methanol and water, 9/1/9/1, v/v/v/v)
35
Table 5
Proportion of solvents for the studied Arizona systems (from T to Z systems).
Arizona system Water MeOH EtOAc Heptane
T 1 3 1 3
V 1 5 1 5
X 1 9 1 9
Y 1 19 1 19
Z 0 1 0 1
was selected. Indeed, TAG partition coefficient Kd was the high-
est among the five values obtained. This value indicates that TAG
are present in the stationary phase (mainly organic phase) for the
greater part. So, thanks to the significant Kd value, TAG could be
isolated from the rest of the oil.
3.2.1.2. Injection conditions for CPC. The injection of a lipid sam-
ple can lead to difficulties in the establishment of the equilibrium
between the two liquid phases. Because the solubility of lipids is
higher in the organic phase (stationary), the injection of a great
volume of sample can saturate this phase, disturbing the hydrody-
namic equilibrium. Moreover, when injecting a large sample mass,
the stationary phase can overflow, leading to a loss of this sta-
tionary phase. This phenomenon is called “flooding” and has been
described elsewhere [21].
Two kinds of injection mode have been tested by using 5 mL of
oil: a standard and a sandwich injection mode (Fig. 7). The standard
mode is defined by the sample introduction after equilibration of
the mobile and stationary phases. Unfortunately, and probably due
to the high viscosity of the oily extract introduced, both mobile
and stationary phase were eluted out of the column. The second
injection tested is by so-called sandwich technique, in which the
sample was injected after filling the column with stationary phase
but before mobile-phase elution. A first separation and fractiona-
tion were performed with 5 mL of an oily extract injected with the
sandwich mode. No elution of the phases was observed and this
mode was selected. Thanks to this injection mode, a probable com-
plete mixing of the sample in the stationary phase occurs, strongly
reducing the flooding phenomenon. Indeed, no TAG were obtained
in the elution fraction of mobile phase except in one elution frac-
tion showing a small loss of stationary phase in the elution phase
(Fig. 8A). This chromatogram corresponds to the elution fraction 7
and was obtained after the elution of 100 mL of mobile phase.
To avoid this remaining drawback, i.e. a contamination of the
mobile phase by the stationary phase, the injection volume was
reduced down to 1 mL. Thanks to the decrease of volume injected,
no loss of stationary phase was noted. Thus, the amount injected
in the CPC column is significant because an increase of the amount
injected leads to increase the sample mass load and trains dramatic
effects as the loss of the stationary phase [21]. Thus, it is possible to
fractionate the TAG from highly viscous crude oil samples without
needing to dilute the sample.
The influence of the dilution factor was also investigated. 1 mL of
crude oil and 1 mL diluted in 4 mL of stationary phase solvent were
both tested. The same separation was observed and no flooding or
loss of stationary phase occurred in the elution fraction 7 (Fig. 8B).
The dilution of oil injected has no influence on the recovery of the
oil.
Whatever the oily sample, the key factors for a CPC separation
are the injection mode and the quantity injected in order not to
alter the liquid-liquid equilibrium in the column.
The TAG remained in the stationary phase, whereas the DAG,
the FFA and the anthraquinone compounds were eluted with the
mobile phase. Nevertheless, a higher purification of anthraquinone
should be achieved by CPC.
36 J. Duval et al. / J. Chromatogr. A 1447 (2016) 26–38

Table 6 could be explained by the presence of carboxylic acid functional

Proportion of solvents for the studied Arizona systems (from K to Q systems).
group on rhein. With the quaternary solvent system composed of
Arizona System Water MeOH EtOAc Heptane water, methanol, ethyl acetate and heptane, no data about the ion-
K 2 1 2 1 ization state of rhein is described in the literature. Consequently,
L 3 2 3 2 different interaction with protic molecules could occur. This expla-
M 6 5 6 5 nation is supported by several previous studies with varied CPC
N 1 1 1 1 systems [24,25].
Q 3 2 3 2

3.3. Enrichment in anthraquinones by SFE

3.2.2. Anthraquinone fractionation
A specific CPC method was then performed to isolate
anthraquinones in a selective way from the oil. Each elution and
extrusion fraction was analyzed by SFC-UV.
3.2.2.1. Selection of a CPC system for aloe emodin and rhein. After
the first attempts of fractionation of the TAG from the oily extract,
a finer separation of target compounds like rhein and aloe emodin
from the non-selective extract was desired. This required a care-
ful selection of a two-phase solvent system that would provide an
appropriate distribution coefficient (KD ) between 0.5 and 5. Sev-
eral Arizona systems were tested and KD values were calculated to
choose the final system. Rhein and aloe emodin have Log P values of
4.6 and 3.4. Compared to TAG (Log P > 20), these anthraquinones are
more polar. 7 medium Arizona systems were thus tested (K–N, P–R)
containing higher water and ethyl acetate percentages compared
to the previously used system X, by using the method described
in Section 2.4.2 (Table 6). However, two of them (Arizona systems
R and P) yielded emulsions causing a third phase, so they were
removed from this list. The calculation of KD was performed for
TAG, DAG, fatty acids and anthraquinones in order to isolate and
concentrate anthraquinones from all fatty compounds present in
the matrix. As presented in Table 7, system Q was the best to isolate
anthraquinone derivatives. Indeed, the value of KD for fatty acids,
DAG and TAG were superior to 1, indicating that the major part
of these compounds will remain in the organic (stationary) phase.
On the opposite, aloe emodin and rhein, having Kd values inferior
to 1, will be better extracted by the aqueous phase. This CPC sys-
tem composition is in accordance with other studies reporting the
fractionation of other anthraquinones [22,23].
The second method to concentrate anthraquinones was the
super/subcritical fluid extraction. The aim was to find extrac-
tion conditions to obtain a selective extraction for anthraquinones
directly from the seeds, thus avoiding the CPC fractionation.
As described in the Section 2 (Table 2), numerous extraction
conditions were studied. The results are presented in Table 8. To
evaluate repeatability, one extraction was realized in the same con-
ditions three times and the relative standard deviation (R.S.D) of
yield (ratio of the extracted mass to the mass of seeds) was 1.4%,
indicating excellent repeatability.
To ensure the right choice of conditions allowing the extraction
of a high amount of anthraquinone together with a low amount of
glycerides, the EF factor (Enrichment factor) was calculated in func-
tion of the normalized area of TAG, aloe emodin and rhein according
to the formulae:
1
EF =
Normalized area of Triglycerides
×Normalized area of Aloeemod in × Normalized area of Rhein
The normalized area is calculated as the ratio of the area of com-
pounds in the extract to the most important area of compounds of
all extracts. This calculation means that EF is greater when low TAG
amounts were extracted and/or higher anthraquinone amounts. EF
is thus indicative of the extraction selectivity to favour the recov-
ery of purified anthraquinones in the extract. As shown in Table 8,
it is then easy to classify extraction conditions according to the EF
factor.

3.2.2.2. CPC isolation of aloe emodin and rhein. The Arizona sys-
tem Q was selected and CPC separation was performed. 12 elution
fractions and 9 extrusion fractions were analyzed by SFC-UV to
determine peak areas of target compounds in each fraction. The
results are presented in Fig. 9. On this figure the normalized area is
plotted for different fractions. The normalized area (in%) is defined
as the ratio of Afraction /Atotal , where Afraction and Atotal were the peak
areas of compound in the fraction and the peak area of compound
in the extract. As can be seen on this figure, anthraquinones were
totally isolated from TAG, DAG in the fractions collected.
Only one part of fatty acids was collected with rhein. Moreover,
rhein showed a singular elution with system Q. As a matter of fact,
rhein eluted in the two first elution fractions with aloe emodin. In
the third elution fraction, only aloe emodin is present. From the
elution fraction 4–6, rhein eluted again. This surprising behaviour
3.3.1. Influence of the pressure and the temperature with pure
CO2
As appears in Table 8, when using pure carbon dioxide, pressure
had an impact on the extraction yield. Indeed, at 20 MPa, the total
yield (mass of oil extracted) was superior to the yield obtained at
10 MPa for both temperature (30 and 60 ◦ C).
The change of pressure modifies the density of the
super/subcritical fluid and, consequently, its solvent power:
increasing the pressure from 10 to 20 MPa causes an increase in
solvent density allowing to solubilize a larger quantity of com-
pounds in the super/subcritical fluid (extraction yield increases
from 7.4 to 22.5% at 30 ◦ C, and 3.2–17.0 at 60 ◦ C). These changes
are correlated by the variations of the TAG amount (normalized
values), when both the temperature rise and the pressure decrease
reduce the amount of extracted TAG. According to previous studies,

Table 7
Comparison of distribution coefficients of different compounds according to Arizona systems.

System KD - Aloe emodin KD - Rhein KD -Linoleic acid KD -oleoyl-linoleoyl-sn-glycerol (OL) KD -1,2,3-Trilinoleoylglycerol (LLL)

System K 15.4 14 10.7 5.1 8.9

System L 10.1 6.4 20.5 10 11.1
System M 6.2 6.1 19.9 0 0
System N 2.4 2.2 5.3 0 0
System Q 0.9 0.7 2.5 30.7 31.5
System X 9.8 2.1 0.1 0.9 190
 J. Duval et al. / J. Chromatogr. A 1447 (2016) 26–38 37

Table 8
SC CO2 extraction results with 0.5 g powdered Kniphofia uvaria seeds.

No. Extraction Extraction Extraction Yield of oil (%) Area normalized Area normalized Area normalized EF
Temperature (◦ C) Solvent (%) (v/v) Pressure (MPa) of Aloe emodine of Rhein (%) of TAG (%)
(%)

1 30 CO2 10 7,4 15 0 48 0
2 30 CO2 20 22,5 17 0 100 0
3 60 CO2 10 3,2 6 0 46 0
4 60 CO2 20 17,0 19 0 62 0
5 30 CO2 /EtOH (95/5) 10 21,8 54 5 92 3
6 30 CO2 /EtOH (95/5) 20 19,9 45 10 92 5
7 60 CO2 /EtOH (95/5) 10 22,9 75 7 20 26
8 60 CO2 /EtOH (95/5) 20 22,7 62 15 54 17
9 30 CO2 /EtOH (70/30) 10 28,3 90 74 31 218
10 30 CO2 /EtOH (70/30) 20 23,3 46 59 61 44
11 60 CO2 /EtOH (70/30) 10 26,1 94 88 47 174
12 60 CO2 /EtOH (70/30) 20 30,4 90 100 37 241
13 30 CO2 /EtOH/H2 O (95/4/1) 10 21,9 89 25 16 139
14 30 CO2 /EtOH/H2 O (95/4/1) 20 19,9 77 49 32 118
15 60 CO2 /EtOH/H2 O (95/4/1) 10 19,8 72 11 10 83
16 60 CO2 /EtOH/H2 O (95/4/1) 20 20,6 100 19 29 66
17 30 CO2 /EtOH/H2 O (70/24/6) 10 27,1 63 12 14 53
18 30 CO2 /EtOH/H2 O (70/24/6) 20 27,1 67 26 27 66
19 60 CO2 /EtOH/H2 O (70/24/6) 10 44,1 36 30 13 82
20 60 CO2 /EtOH/H2 O (70/24/6) 20 29,1 71 28 40 49

higher pressure values are recommended for the TAG extraction
[26,27].
Moreover, the temperature rise from 30 to 60 ◦ C causes a
decrease of the density of the extracting solvent. As expected,
Table 8 shows that the extraction yield of the total oil decreases
with the temperature increase.
The increase in pressure also favours the aloe emodin recov-
ery, whereas for rhein, pressure and temperature changes have no
consequences on its solubility in the fluid: this compound is not
solvated and never extracted by pure CO2 . In conclusion, although
modifications of pressure and temperature were performed; the
factor EF is always equal to 0 because the rhein compound was not
extracted (Table 8).
3.3.2. Influence of the modifier: nature and percentage
Table 8 shows the results of global yield (%) for SFE with pure CO2
and CO2 + modifier (EtOH and EtOH/H2 O), for 5 and 30% of modi-
fier. Evaluating the results in this table, it can be verified that the
addition of a modifier in SFE process had mainly a positive influ-
ence on the extraction efficiency for low temperature and pressure
conditions (assessed by the global yield of each sample) and in the
selectivity of the extraction, except for the lower modifier content
(5%) (assessed by the EF factor) (Table 8). As expected, the amount
of extracted TAG seems rather decrease with the increase in the
polar modifier content.
Firstly, focusing on the results of TAG in Fig. 10A and B, the
extraction of these lipids depends on the polarity of the extract-
ing fluid. The extraction of TAG is favoured with pure CO2 and 5%
of ethanol (at 30 ◦ C). At the opposite, the use of a modifier mixture
of ethanol/water, or a high percentage of pure ethanol (30%) allows
obtaining the lower proportion of TAG in the final extract.
Secondly, the extraction of aloe emodin depends on the polarity
of the extracting phase. Indeed, compared to the use of pure carbon
dioxide, the addition of ethanol or ethanol/water yielded improved
extraction of this compound. Moreover, focusing on Fig. 10C and D,
it appears that the use of 5% of ethanol/water and 30% ethanol are
the best extraction conditions. The use of 30% ethanol/water is not
as effective as the use of 5% ethanol/water, probably because the
solubility of aloe emodin in the super/subcritical extracting fluid
decreases. Moreover, the optimal conditions to extract aloe emodin
with 30% ethanol are achieved with a pressure of 10 MPa whereas
with 5% ethanol/water, it is necessary to apply a higher pressure of
20 MPa. Similar observations were reported in the literature [28].
Finally, for the extraction of rhein, as shown in Fig. 10E and F, the
best conditions are obtained with 30% ethanol. In contrast with aloe
emodin, the use of an ethanol/water modifier is not an advantage.
As rhein is an acidic compound, it can be somewhat surprising that
it should be less soluble in the ethanol/water fluid than aloe emodin.
Besides, the use of a low temperature of 30 ◦ C is more convenient
to extract rhein.
Based on the EF values (Table 8), it is clear that the optimal con-
ditions to obtain an enriched extract of K. uvaria oil were with 30%
absolute ethanol at 60 ◦ C and 20 MPa. This condition does not pro-
vide an optimal yield of K. uvaria oil, but it provides a selective
extraction of anthraquinones. However, satisfactory EF values are
also obtained with 5% of ethanol/water, which could be an alterna-
tive condition if the use of a lower amount of modifier is desired.
4. Conclusion
The described methods present a good strategy to screen, con-
centrate and purify bioactive anthraquinone compounds from a
complex vegetable oil extract. The difficulty of these kinds of matri-
ces is due to the presence of major non active compounds, in which
the minor bioactive ones can be dissolved. The separation of the
minor compounds from the major part of the sample requires either
(i) a fractionation step after a first non-selective extraction, or (ii)
the direct use of a selective extraction whenever possible.
In the first case, CPC was successfully applied to the non-
selective oily extract, to fractionate the TAG from the matrix and to
enrich the anthraquinone part vs the numerous family of glyceride
compounds, by selecting adequate CPC systems from the Arizona
scale. For this kind of highly viscous samples, the injection mode
and injected volume are of prime importance to maintain a proper
equilibrium between the stationary and the mobile phase during
the chromatographic process. This method is the most selective
technique for the enrichment of anthraquinones. Nevertheless, fill-
ing and equilibration of the column are both time-consuming, and
the sample should be a liquid extract, thus requires a first extraction
step of the solid seeds.
Secondly, SFE allowed achieving a selective extraction of the
seeds of K. uvaria, i.e. an extraction that favoured the anthraquinone
recovery and that minimized the amount of co-extracted TAG. The
38 J. Duval et al. / J. Chromatogr. A 1447 (2016) 26–38
compounds solubility was strongly related to the modifier nature
and percentage, or for pure CO2 to pressure and temperature con-
ditions. The best selective extraction (higher enrichment factor) of
the two anthraquinones was obtained using 30% of pure ethanol at
60 ◦ C and 20 MPa. This green approach could be selected by the cos-
metic industry, which favours the use of safe solvents for most of
the processes. This method is fast, due to the dynamic process, and
green. It can be performed directly from the seeds. However, SFE is
less selective compared to the CCC with regards to the isolation of
anthraquinones.
Last but not least, two analysis methods were developed by
super/subcritical fluid chromatography, both with non-polar and
polar stationary phases, to identify and quantify the target com-
pounds and either due to their great quantity (TAG) or to their
bioactivity (anthraquinones). As discussed previously, the analy-
sis of minor bioactive compounds in a lipid matrix composed in
majority by TAG and DAG required a high-performance separation
method.
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