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J. Sep. Sci.

2014, 37, 2331–2339 2331

Saı̈da Chideh1,2 Research Article


Serge Pilard3
Jacques Attoumbré4
Robert Saguez1 5-O-Caffeoylshikimic acid from Solanum
Alshaimaa Hassan-Abdallah5
Dominique Cailleu3 somalense leaves: Advantage of centrifugal
Anne Wadouachi6
Sylvie Baltora-Rosset1
partition chromatography over conventional
1 EDYSAN
column chromatography
FRE 3498
CNRS-Université de Picardie
Jules Verne, UFR de Pharmacie, Solanum somalense leaves, used in Djibouti for their medicinal properties, were extracted by
Amiens Cedex, France MeOH. Because of the high polyphenol and flavonoid contents of the extract, respectively,
2 Centre de Recherche, Université
determined at 80.80 ± 2.13 mg gallic acid equivalent/g dry weight and 24.4 ± 1.01 mg
de Djibouti, Avenue Georges
quercetin equivalent/g dry weight, the isolation and purification of the main polyphenols
Clémenceau, Djibouti
3 Plate-Forme Analytique, UFR were carried out by silica gel column chromatography and centrifugal partition chromatog-
des Sciences, Bâtiment raphy. Column chromatography led to 11 enriched fractions requiring further purification,
Serres-Transfert, Amiens Cedex, while centrifugal partition chromatography allowed the easy recovery of the main compound
France
4 S.I.P.R.E – Comité Nord, of the extract. In a solvent system composed of CHCl3 /MeOH/H2 O (9.5:10:5), 21.8 mg of
Achicourt, France this compound at 97% purity was obtained leading to a yield of 2.63%. Its structure was
5 Institut de Recherches
established as 5-O-caffeoylshikimic acid by mass spectrometry and NMR spectroscopy. This
Medicinales, CERD, Djibouti work shows that S. somalense leaves contain very high level of 5-O-caffeoylshikimic acid
6 LG2A FRE 3517
CNRS-Université de Picardie (0.74% dry weight), making it a potential source of production of this secondary metabolite
Jules Verne, Institut de Chimie that is not commonly found in nature but could be partly responsible of the medicinal
de Picardie FR 3085, Amiens properties of S. somalense leaves.
Cedex, France

Keywords: Bioautography / Caffeoylshikimic acids / Dactylifric acid / Polyphenols


Received March 3, 2014 / Solanum somalense
Revised June 11, 2014 DOI 10.1002/jssc.201400226
Accepted June 12, 2014

1 Introduction Bioactive known compounds of plants are divided into


three main categories: (i) terpenes and terpenoids (approx-
Investigating the scientific basis for the use of medicinal imately 25 000 types), (ii) alkaloids (approximately 12 000
plants is of great interest for several reasons: (i) plants re- types), and (iii) phenolic compounds (approximately 8000
main the primary source of traditional medicine, (ii) a marked types) [4]. Given the difficulty of a comprehensive analysis
increase in the use of herbal medicines or botanical dietary of plant extracts, studies usually focus on a limited range of
supplements is observed in Western medicine, and (iii) plant- compound classes often highlighted by a preliminary rapid
derived drugs are the main sources for finding lead com- biological screening of the total extract. Criteria of chemotax-
pound structures that might reach the drug market [1, 2]. onomy and uses in traditional medicine may also guide the
Thousands of traditional herbal medicines have been widely choice of the class of compounds that will be targeted [4].
used and studied but Djiboutian ones have not received much The relationship between various diseases such as can-
attention up to now despite their significant contribution cer, hypertension, diabetes, cardiovascular, neurodegenera-
to the management of public healthcare in the country [3]. tive diseases, and oxidative damages has been extensively in-
Among the different medicinal plants used in the Randa re- vestigated and established [5, 6]. At the same time, the search
gion in Djibouti, Solanum somalense has various medicinal for antioxidants from natural sources to prevent these oxida-
uses but, as far as we know, has not been studied from a tive damages is currently under development. Among sec-
phytochemical and pharmacological point of view [3]. ondary metabolites, polyphenols generally exhibit good an-
tioxidant properties and are thus often sought in medicinal
plants but may cause false-positive results in enzymatic and
Correspondence: Dr. Sylvie Baltora-Rosset, EDYSAN FRE 3498 cellular screening procedures due to their protein-binding
CNRS-Université de Picardie Jules Verne, UFR de Pharmacie, 1 ability and their changes in cellular redox potential [7,8]. Var-
rue des Louvels, 80037 Amiens Cedex, France ious analytical methods have been reported for the analysis
E-mail: sylvie.baltora-rosset@u-picardie.fr and purification of bioactive natural compounds [4, 7, 9] and
Fax: +33-03-2282-7469
for the most abundant polyphenols in plants, phenolic acids,
Abbreviations: CPC, centrifugal partition chromatography; and flavonoids [10]. Numerous articles on their extraction,
DPPH, 2,2-diphenyl-1-picrylhydrazyl; DW, dry weight; GAE, separation, and detection methods have been published over
gallic acid equivalent; QE, quercetin equivalent the past two decades [11]. SPE methods are currently used


C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
2332 S. Chideh et al. J. Sep. Sci. 2014, 37, 2331–2339

to recover prefractionated samples or to remove polyphenols the use of centrifugal partition chromatography (CPC). As
from plant extracts because SPE consists of simple elution conventional chromatographic separation on silica gel is con-
and concentration via polymeric resins. In practice, SPE of- sidered to be one of the reference methods for comparing the
ten suffers from low resolution and efficiency [7]. However, success of newly developed methodologies, the two methods
SPE is frequently used for cleanup procedures, mainly be- were carried out in parallel. Our objective was fully achieved
cause it offers the possibility of combining on-line extrac- and led to the successful preparative isolation of highly pure
tion with chromatographic methods such as HPLC [11]. The major phenolic component from the S. somalense leaves. 5-
main methods described for the determination of phenolic O-Caffeoylshikimic acid was identified by ESI-MS and NMR
acids and flavonoids are chromatographic techniques such spectroscopy and will be used for further biological studies.
as silica gel or sephadex column chromatography, TLC, GC, This paper is the first report on the separation of bioactive
HPLC, or LC–ESI-MS [4, 11]. Because the chemical struc- compounds of S. somalense by CPC.
tures of polyphenols have various degrees and patterns of
hydroxylation, methylation or glycosylation, their analysis by
GC requires a preliminary chemical derivatization. Actually,
2 Materials and methods
their fractionation from natural sources is usually performed
using a combination of methods, mainly column chromatog-
2.1 Materials
raphy followed by HPLC [12]. To overcome the use of multiple
steps, a new method, countercurrent separation, based solely
Solanum somalense leaves were collected from Tadjourah Dis-
on differences in the solubility of natural products was de-
trict of Randa Region in north Djibouti. Plant identification
veloped in the 1970s [3, 13, 14]. CCC is a chromatographic
was carried out at the National Herbarium (ETH), Addis
technique that has some advantages when compared with LC
Ababa University (AAU) and voucher specimens were de-
techniques: (i) no nonspecific adsorption to a solid support,
posited in the Herbarium at Djibouti. All organic solvents
(ii) higher selectivity, (iii) higher sample loading capacity,
were of analytical grade and purchased from VWR. Reagents
(iv) reduction of solvent quantity, and (v) shorter separation
were purchased from Sigma–Aldrich.
time. Therefore, CCC has been successfully applied to the
analysis and separation of various natural products including
polyphenols [9, 11–13, 15].
Caffeoylshikimic acids are characteristic phenolic com- 2.2 Biological evaluation
pounds mainly found in the Palmae family [16]. Up to now,
the largest industrial use of shikimic acids has been the pro- Determination of total polyphenols. Total phenolic contents
duction of Tamiflu R
or other antiviral drugs for use against were determined according to the literature [19]. A total of
avian flu, and the demand for shikimic acid and its deriva- 0.50 mL of the sample diluted in MeOH was added to 2.5 mL
tives is expected to increase dramatically in the event of a of 1:10 diluted Folin–Ciocalteu reagent. After 4 min, 2 mL
pandemic flu outbreak. Zeng et al. [17] also demonstrated the of saturated sodium carbonate solution (about 75 g/L) was
antiproliferative activity of 5-O-caffeoylshikimic acid against added. After 2 h of incubation at room temperature, the ab-
different cancer cell lines. Very few plant sources of these sorbance of the reaction mixture was measured at 760 nm.
acids have been described. Elaeis guineensis (oil palm) and Gallic acid was used as a reference standard and the re-
Phoenix dactylifera (date palm) are the only sources where sults were expressed as milligram gallic acid equivalent (mg
caffeoyl shikimic acids are the major polyphenols [16,18–20]. GAE)/(g dry weight (DW)) of plant material. Gallic acid con-
5-O-Caffeoylshikimic acid was also obtained in small quanti- centrations ranging from 0 to 100 ␮g/mL were prepared, and
ties from Vaccinium corymbosum (10 mg for 32 g of extract) the standard calibration curve was obtained using a linear fit
together with 21 phenolics and showed the potential for nu- (r2 = 0.9972). The samples were analyzed in triplicate.
traceutical applications [21]. The compound was also isolated Determination of total flavonoids. Total flavonoids were an-
from Livistona chinensis through a multistep procedure lead- alyzed according to the aluminum chloride method described
ing to a very low yield: 12 mg for 90.21 g of crude extract. in Ref. [8]. A total of 0.5 mL of each sample and 300 ␮L of
The aim of this work was to develop an efficient bioassay- NaNO2 (1:20 w/v) were pipetted into a test tube. The con-
guided method for the fractionation of the methanolic extract tents were vortexed for 10 s and left at room temperature for
of S. somalense. The preliminary screening of the antioxidant 5 min. Three hundred microliters of AlCl3 (1:10 w/v), 2 mL
properties of the extract, as well TLC analysis with selective of 1 M NaOH, and 1.9 mL of distilled water were then added
polyphenol developers, suggested that the methanolic extract to the mixture. After 10 s of vortexing, the absorbance for
had a high polyphenol content. As described above, a review each sample was measured at 510 nm. Quercetin concentra-
of the literature indicates that no single method is regarded as tions ranging from 0 to 120 ␮g/mL were prepared, and the
standard for extracting polyphenols from plants. In addition, standard calibration curve was obtained using a linear fit (r2
to our knowledge, for caffeoylshikimic acids, only multistep = 0.9831). Quercetin was used as a reference standard and
methods including column chromatography and HPLC are the results were expressed as (mg QE)/(g DW) (where QE
described. Therefore, it seemed interesting to develop a new is quercetin equivalent) of plant material. The samples were
method for isolating these types of phenolic components by analyzed in triplicate.


C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
J. Sep. Sci. 2014, 37, 2331–2339 Liquid Chromatography 2333

2.3 Apparatus binary gradient of H2 O (solvent A) and MeCN (solvent B) both


containing 0.1% v/v HCOOH, with a flow rate of 1 mL/min:
The CPC instrument used in this study is a SPOT CPC 100 5–95% B to 95–5% B in 45 min. Twenty microliters was used
Light (Armen Instrument) fitted with a rotor of ten circular for injection, which was repeated three times.
partition disks (1000 partition cells: 0.1 mL per cell; total col-
umn capacity of 100 mL). The rotation speed can be chosen 2.5.3 LC–ESI-MS
from 0 to 4000 rpm. The effluent was monitored by a Lash
06 DAD detector (ECOM, Prague) equipped with a prepara- The crude extract and the purified fractions were loaded
tive flow cell operating at 254 nm and collected by a LS 5600 (2 ␮L) on a 100 × 2 mm, 2.7 ␮m Nucleoshell RP18
(Armen) fraction collector. The HPLC used was a Shimadzu column (Macherey Nagel) and maintained at 30⬚C. The elu-
HPLC System including a LC-20AT pump and SPD-M20A tion was performed using a 0.25 mL/min mobile phase gra-
diode-array detector. LC–ESI-MS spectra were obtained on a dient programmed from water (A), acetonitrile (B), both con-
UFLC Prominence (Shimadzu) system coupled with a Q-TOF taining 0.1% v/v HCOOH, as follows (A/B): 95:5 (t = 0 min),
Ultima Global high-resolution hybrid quadrupole TOF instru- 95:5 (t = 5 min), 5:95 (t = 30 min), 5:95 (t = 40 min), 95:5 (t
ment (Waters-Micromass), equipped with a pneumatically = 45 min), and 95:5 (t = 50 min). The effluent was flow-split
assisted electrospray ionization source (Z-spray) and an addi- via a polyether ether ketone tee with one-third of the flow
tional sprayer (lock spray) for the reference compound, allow- directed toward the electrospray source of the Q-TOF and
ing accurate mass measurements of ions and their elemental the residual two-third directed toward an UV detector (Shi-
compositions determination. NMR spectra were recorded at madzu SPD-20A) set to 254 nm. ESI-MS data were recorded
300 K on a Bruker (Wissembourg, France) DRX-500 spec- in the positive- and negative-ion modes with capillary voltage
trometer equipped with a broadband inverse probe. DMSO- of ±2.5 kV and cone voltage of 50 V. The source and desolva-
d6 was used as the solvent. Proton resonance assignments tion temperatures were 120 and 250⬚C, respectively. Nitrogen
and structural elucidation were performed by 1D (1 H and 13 C was used as a drying and nebulizing gas at flow rates of 450
NMR) and 2D homo- and heteronuclear experiments (corre- and 100 L/h, respectively. Scanning was performed in the
lated spectroscopy, heteronuclear single quantum coherence, range 50–1550 Da at a scan rate of 1 s/scan and spectra were
and heteronuclear multiple-bond correlation). collected in the profile mode at a resolution of 10 000 (full
width at half maximum). Lock mass correction, using appro-
priate cluster ions of an orthophosphoric acid solution (0.1%
2.4 Preparation of crude extract
in H2 O/CH3 CN 50:50, v/v), was applied for accurate mass
measurements. For MS/MS experiments, argon was used as
Fifty grams of dried leaves of S. somalense were powdered
collision gas and the collision energy was set to 20 V. Data
and treated three times with 500 mL of cyclohexane. After
acquisition and processing were performed with MassLynx
filtration through Whatman filter paper, the plant material
4.0 SP4 software.
was subjected to three extractions with AcOEt (500 mL each
time, room temperature, 24 h). The same procedure was ap-
plied with MeOH and with H2 O. The extracts from the same
solvent were combined and the solvent was removed under 2.6 Separation methods
the reduced pressure to give the crude extracts.
2.6.1 Silica gel column chromatography separation

2.5 Detection by TLC, HPLC, and LC–MS Half of the methanolic crude extract (3.9 g) was subjected to
silica gel (Merck 70–230 ␮m) column chromatography eluted
2.5.1 TLC with AcOEt/MeOH/H2 O (100:0:0, 90:10:1, 80:20:1, 70:30:1,
60:40:1, 50:50:1, and 40:60:1). This separation resulted in 64
Fractionation and separation were monitored by TLC car- fractions that were checked for their composition by TLC with
ried out on Silica Gel 60 F254 (Merck) plates developed four types of visualizers: UV254 , vanillin sulfuric, Neu reagent,
with EtOAc/HCOOH/HOAc/H2 O (100:11:11:20) visualized and DPPH test. This combination of visualization allowed the
by UV light (254 and 365 nm) and/or with vanillin-H2 SO4 gathering of the initial fractions into 11 fractions (F1–F11),
reagent, Neu reagent (1% diphenylboric acid ethanolamine which were weighed and submitted to further analyses.
complex in EtOH) and 2,2-diphenyl-1-picrylhydrazyl (DPPH
solution, 2 mg/mL MeOH). 2.6.2 CPC separation

2.5.2 HPLC 2.6.2.1 Selection of the two-phase solvent system


The solvent system was selected according to the distribution
HPLC analyses of the crude extract and of the CPC peak constant KD of the six main peaks of the unknown com-
fractions were conducted at 254 and 365 nm on a 250 × pounds (X) (called A–F) visualized on HPLC chromatogram
4.6 mm, 5 ␮m, Prevail RP C18 column (Grace) using a linear of the crude methanolic extract at 365 nm. The KD value was


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2334 S. Chideh et al. J. Sep. Sci. 2014, 37, 2331–2339

Figure 1. TLC plates developed


with EtOAc/HCOOH/HOAc/H2 O (100:
11:11:20) and stained with (A) Neu
reagent visualized at 365 nm, (B) DPPH
solution in MeOH. Dots (100 ␮g) are
of (a) methanolic crude extract of S.
somalense (b) F1 (c) F5 (d) F8 (e) F10
(f) F11 (silica gel column fractions) (g)
compound B isolated by CPC.

determined by HPLC analysis in the same conditions. of the main problems with the use of water plant extracts or
Methanolic crude extract was dissolved in the tested solvent decoctions [21]. Under these conditions, only the methano-
system and vortexed for 30 s. After separation and evapora- lic extract was further analyzed by TLC in different detection
tion under reduced pressure, the residue of each layer was conditions. The main phytochemicals were detected by UV,
dissolved in 500 ␮L of methanol for HPLC analysis. The KD vanillin sulfuric acid, and Neu reagent while the antioxidant
values were calculated according to the ratio: concentration of activity was evaluated using DPPH radical scavenging assay
compound X in the stationary phase/concentration of com- according to Zhang et al. [8]. The results of revelation by Neu
pound X in the mobile phase [20]. reagent and DPPH (Fig. 1A and B, respectively) showed that
polyphenols were present in large quantities in the methano-
2.6.2.2 Collection and analysis of fractions lic extract from S. somalense leaves and also exhibited a high
The solvent system used for separation was chloro- antioxidant total activity due to a DPPH radical scavenging
form/methanol/water in the ratio 9.5:10:5 v/v/v. The biphasic capacity of almost all the compounds present in the extract.
system was prepared just before use by thoroughly mixing Therefore, the total contents of polyphenol and flavonoid
volumes of solvent in the above ratio. After the equilibration were quantitatively evaluated and assessed at 80.80 ± 2.13 mg
was established, the stationary phase (upper phase in the de- GAE/g DW and 24.4 ± 1.01 mg QE/g DW, respectively. In
scending mode) was pumped into the column at a flow rate of their study of the antioxidant properties of methanol extracts
30 mL/min while the apparatus was run at 500 rpm accord- of 45 whole medicinal herbs, Li et al. [22] showed that their
ing to the equilibration mode of the apparatus. After injection polyphenols contents could range from 1 to 52 mg GAE/g
of the sample (0.83 g of crude extract in 10 mL of a mixture DW. In this context, the value obtained for S. somalense is
50:50 of the two phases), the mobile phase was perfused at very high. Furthermore, it was shown by the same authors
2500 rpm at a flow rate of 4 mL/min for 60 min and then to and others [8, 22, 23] that a high correlation existed between
8 mL/min under 35 b during the run. The eluent was moni- high levels of polyphenols and the antioxidant capacities of
tored at 254 nm and 36 fractions (fi ) of 15 mL were collected the studied extracts. Finally, the effects of plants on health
and analyzed by HPLC. The volume of the stationary phase are often attributed to their polyphenolic components [10].
displaced by the mobile phase was measured and used to de-
termine VM . The stationary phase volume (VS ) was calculated
according to the relationship VC = VM + VS since the column 3.2 Analytical study of the extract
volume (VC ) is known. The stationary phase retention Sf was
expressed as VS /VC (70% in the experiment). The selectivity While keeping in mind that it is necessary to perform more
(separation factor between two compounds X and X ) was than one type of antioxidant capacity measurement to take
calculated as follows: ␣ = KD(X) /KD(X ) [20]. into account the various mechanisms of antioxidant actions
of plant extracts [24], we decided to study the major polyphe-
3 Results and discussion nols present in the extract to evaluate their potential health-
promoting benefits identified by the ethnological surveys
3.1 Biological evaluation of the extract in Djibouti [3]. The analysis of the crude extract by HPLC
and LC–ESI-MS (Fig. 2A) indicated the presence of six main
Leaves of S. somalense, which are mainly used for the prepara- peaks, which were characterized by their retention times (Rt),
tion of traditional medicines, were extracted with solvents of their UV absorption maxima, and their molecular weight
different polarity: cyclohexane, AcOEt, MeOH, and water. The (Table 1).
residues obtained by sequential extraction were weighed and
analyzed by TLC (data not shown). The extraction yields were
1.8, 1.3, 14, and 20%, respectively. The methanolic and water 3.3 Separation and determination of polyphenols
extracts had the highest content of secondary metabolites and
showed quite similar TLC profiles, but the water residue was To separate and identify these major polyphenols, a conven-
quickly subjected to microbial contamination, which is one tional method such as activity-guided fractionation on silica


C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
J. Sep. Sci. 2014, 37, 2331–2339 Liquid Chromatography 2335

Figure 2. (A) LC chromatogram at 365 nm of methanolic crude extract of S. somalense, (B) LC chromatogram at 365 nm of fraction F1
obtained by silica gel column chromatography, and (C) LC chromatogram at 365 nm of fraction f23 obtained by CPC separation. Conditions:
Prevail RP C18 column (250 × 4.6 mm, 5 ␮m), using a linear binary gradient of H2 O (solvent A) and MeCN (solvent B) both containing 0.1%
v/v HCOOH, with a flow rate of 1 mL/min: 5–95% B to 95–5% B in 45 min.

gel column chromatography was first performed and allowed to identify the structure of the major compound B (Table 1).
us to obtain 11 fractions Fi (Fig. 3). As shown by TLC (Fig. 1A), The LC–ESI-MS spectra (Fig. 4A and B) revealed [M+H]+ and
gel column chromatography did not allow an efficient sepa- [M−H]− ions at m/z 337.09 and 335.07, respectively. A molec-
ration of polyphenols: only the major compound of the crude ular formula of C16 H16 O8 was determined using accurate
extract (peak B) was obtained with a yield of 1.08% but its pu- mass measurements, [M+H]+ : found 337.0935 for 337.0923
rity was not sufficient (87%) (Figs. 1A and 2B) to consider fur- calculated and [M−H]− : found 335.0777 for 335.0767 calcu-
ther biological evaluations because the minor compound in lated. In a positive mode (Fig. 4A), an abundant ion at m/z
the fraction also showed an antioxidant activity. Nevertheless, 163.03 (C9 H7 O3 ) seems to indicate the presence of a caffeoyl
this step was very useful to roughly characterize the six main moiety. Moreover UV spectrum of B showed absorption max-
components of the extract detectable by HPLC at 365 nm and ima at 211 and 316 nm suggesting a phenolic acid structure.


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2336 S. Chideh et al. J. Sep. Sci. 2014, 37, 2331–2339

Table 1. LC retention time, ␭max (UV nm), molecular weight, and TLC retention factor of compounds A–F

Peak Retention timea) UV ␭max (nm) Molecular weight Retention factorb)

A 25.9 210, 235, 317 354 0.53


B 29.1 211, 316 336 0.90
C 35.4 208, 255, 354 610 0.43
D 36.3 197, 255, 282, 355 594 nd
E 38.1 199, 250, 346 464 nd
F 38.8 199, 265, 347 594 nd

nd, not determined.


a) Conditions: analysis at 365 nm on a 250 × 4.6 mm, 5 ␮m, Prevail RP C18 column using a linear binary gradient of H2 O (solvent A) and
MeCN (solvent B) both containing 0.1% v/v HCOOH, with a flow rate of 1 mL/min: 5–95% B to 95–5% B in 45 min.
b) Conditions: Silica Gel 60 F254 (Merck) plates developed with EtOAc/HCOOH/HOAc/H2 O (100:11:11:20).

Solanum somalense leaves for performing separation by CPC are good solubility of the
50.0 g sample in the solvent system and the determination of a suit-
able two-phase system allowing a good partition behaviour of
Methanolic extract the target compounds. The efficiency of the partition is evalu-
7.8 g ated using the KD parameter. KD of the six main compounds
was determined by HPLC in several solvent systems (Table 2).
Silica-gel column Centrifugal Partition In the case of natural product studies by CPC, the sample
Chromatography Chromatography
3.9 g 0.83 g complexity and the unknown nature of target compounds
11 fractions 38 fractions demand the careful consideration of the different KD values
Total fraction weight: 2.14 g f19 (7.1 mg 86%) f20 (7.2 mg 91%)
F1 weight: 42.4 mg f21-f23 (21.8 mg 97%) and to depart from the usual rules of choice of solvent system.
Purity 87 % f24 (5.8 mg 85%) Indeed, generally speaking, compounds of interest should be
almost equally distributed between the two phases (KD val-
Figure 3. Processing flowchart for the preparative extraction of
the methanolic crude extract of S. somalense leaves by conven-
ues in the range 0.5–2) and the separation factor should be
tional column chromatography and by CPC. The purity of each >1.5 to ensure the most successful conditions of separation
fraction was determined by analytical HPLC. Conditions: Prevail [32].
RP C18 column (250 × 4.6 mm, 5 ␮m), using a linear binary gra- At this stage of work, our attention was focused on the
dient of H2 O (solvent A) and MeCN (solvent B) both containing
isolation of the main component of the extract (peak B,
0.1% v/v HCOOH, with a flow rate of 1 mL/min: 5–95% B to 95–5%
B in 45 min. i.e. 5-O-caffeoylshikimic acid) and the KD values were ex-
amined according to this aim. Of all the possible combina-
tions of solvents, we first chose two families of ternary sol-
The results of combined NMR experiments established the vent systems, EtOAc/MeOH/H2 O and CHCl3 /MeOH/H2 O,
structure of B unambiguously as 5-O-caffeoylshikimic acid which accounted for >60% of the separation of glycosylated
as described by Fukuoka [25] (data not shown). This was flavonoids in the literature [33], although we knew that other
confirmed by MS/MS of the [M−H]− ion (Fig. 4C) showing types of compounds were present in the extract. Moreover,
the caffeoyl fragment (m/z 179.04, C9 H7 O4 ) and its char- despite the difficulty of using BuOH solvent due to its weak
acteristic loss of H2 O (m/z 161.03, C9 H5 O3 ) and CO2 (m/z volatility, this solvent was also tested because of its ability to be
135.05, C8 H7 O2 ) [26]. Solanum species contain abundant caf- a major organic phase modifier. Based on the data collected
feic acid derivatives that have implications in human health. (Table 2), we chose to perform the experiments on the basis
Numerous studies have shown that efficacy and potency of of the following requirements: (i) for all the peaks (A–F), KD
these derivatives are very much dependant on their struc- values had to be in the smallest possible range to allow the
ture and this strong structure–function relationship make separation of all the compounds within a reasonable time,
them good candidates for the development of new functional (ii) a greater part of the values should be close to the interval
foods or pharmaceuticals [27]. Caffeic acid most often esteri- 0.5–2 [34], and (iii) KD values f the six different compounds
fied with quinic acids such as in chlorogenic acids, is widely should lead to separation factors close to 1.5 [34].
found in plants [28], whereas a careful examination of litera- With these constraints and with the objective to tar-
ture showed that esters with shikimic acid are not commonly get the major compound B, the system CHCl3 /MeOH/H2 O
found in nature despite valuable bioactivities [29]. (9.5:10:5) (KD values of 10.00, 3.34, 4.62, 4.86, 1.37, 0.16,
We have recently shown that CPC is an attractive method respectively, for peaks A–F; separation factor ␣ between com-
to isolate bioactive compounds from Solanum species [30,31]. pound B and compound C of 1.4) seemed to be promising
Hence, it was decided to implement this technique to the frac- for achieving the isolation of the compound B by CPC.
tionation of S. somalense leaves that are a rich source of caffeic Crude S. somalense extract (0.82 g) was fractionated and
acid derivatives with potential interest. The key points critical despite the rather uncertain nature of this initial choice, the


C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
J. Sep. Sci. 2014, 37, 2331–2339 Liquid Chromatography 2337

163.03
100
A
%

337.09
0 m/z
50 100 150 200 250 300 350 400 450 500 550

335.07
100
B
%

179.04
0 m/z
50 100 150 200 250 300 350 400 450 500 550

179.04
100
C

O OH C9H7O4-
m/z 179.04
- H2O m/z 161.03
- CO2 m/z 135.05

HO O Figure 4. LC–ESI-MS spectra of com-


%

pound B and fragmentation scheme (a)


OH positive-ion mode (b) negative-ion mode,
OH and (c) MS/MS of the [M−H]− ion at m/z
335.08.Conditions: Nucleoshell RP18 col-
135.05 C9H7O3+
OH umn (100 × 2 mm, 2.7 ␮m) maintained
m/z 163.03
at 30⬚C. The elution was performed us-
161.03 ing a 0.25 mL/min mobile phase gradient
programmed from water (A), acetonitrile
335.08 (B), both containing 0.1% v/v HCOOH, as
follows (A/B): 95:5 (t = 0 min), 95:5 (t =
0 m/z 5 min), 5:95 (t = 30 min), 5:95 (t = 40 min),
100 150 200 250 300 350 95:5 (t = 45 min), and 95:5 (t = 50 min).

operation proved to be successful. Each fraction collected S. somalense leaves has enabled us to show the great advan-
during the separation process was weighed and analyzed by tage of this technology compared to the separation on a silica
HPLC (Fig. 3) showing that the fractions f19 –f24 contained column, which is a more conventional technique overwhelm-
almost solely the compound B with purity varying from 86 ingly used for the fractionation of natural products. Indeed,
to 97%. A total of 21.8 mg of 5-O-caffeoylshikimic acid was our results may be analyzed according to the two primary di-
obtained from the three fractions of highest purity, f21 –f23 mensions highlighted by Pauli et al. [9] in their meta-analysis
(97% purity), leading to a yield of 2.63%. Its structure was of natural products purification schemes. The first criterion
confirmed by NMR spectroscopy. By taking into account the disclosed in their study is the number of purification steps.
quantity of B in fractions f19 –f24 (39.3 mg), it was also pos- On this point, our study helps to show the superiority of CPC
sible to calculate the content of 5-O-caffeoylshikimic acid in compared to silica gel column: a large amount (21.8 mg) of
S. somalense leaves that is 0.74% of DW. 5-O-caffeoylshikimic acid was obtained in only a single CPC
Studies to isolate secondary metabolites from plant mate- step, leading to a yield of 2.63% compared to 1.08% for sil-
rial are often unique studies and it is therefore difficult to have ica gel chromatography (Fig. 3). Moreover, a major widely
reference points to compare and evaluate the performance of known disadvantage of silica gel purification was also noted
natural product isolation approaches. However our results are in our study: 45% of the extract was irreversibly absorbed on
quite satisfactory within the context of the isolation of natural the column, while almost all the samples were recovered by
products. The use of CPC to isolate the major compound of CPC. The second dimension described by Pauli et al. [9], is the


C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
2338 S. Chideh et al. J. Sep. Sci. 2014, 37, 2331–2339

Table 2. KD (distribution coefficient) of six targets peaks measured in different solvent systems

Solvent system Composition A B C D E F

CHCl3 /MeOH/H2 O 04:03:02 98.37 17.00 50.03 82.98 32.43 18.87


06:07:04 39.26 10.25 18.89 18.52 13.12 10.76
08:10:05 26.20 6.08 10.95 11.03 7.15 6.55
9.5:10:5 10.00 3.34 4.62 4.86 1.37 0.16
11:10:05 8.00 2.57 2.78 2.44 2.37 1.87
13:10:05 12.75 3.44 4.23 4.23 2.86 2.21
15:10:05 41.51 6.86 11.09 11.28 7.02 5.55
12:10:05 49.73 7.19 15.97 20.29 10.25 7.80
CHCl3 /MeOH/BuOH/H2 O 42:32:5:21 22.81 3.36 5.66 5.83 3.59 2.66
42:30:8:20 13.61 2.49 3.22 3.44 2.09 1.73
45:30:5:20 24.62 3.84 5.49 5.53 3.49 2.78
40:30:10:20 11.11 2.21 2.65 2.72 1.61 1.34
37:37:4:22 17.20 4.20 5.86 5.98 3.96 3.34
38:30:12:20 10.95 2.23 2.66 2.54 1.65 1.19
EtOAc/MeOH/H2 O 40:20:40 0.04 0.51 0.25 0.21 0.31 0.38
50:00:50 0.02 0.88 0.19 0.13 0.16 0.39
47:06:47 0.03 0.83 0.19 0.09 0.23 0.42
44:11:44 0.02 0.77 0.17 0.06 0.21 0.38
40:20:40 0.04 0.54 0.30 0.24 0.34 0.48
36:18:36 0.06 0.34 0.23 0.19 0.25 0.30
EtOAc/BuOH/H2 O 50:00:50 0.03 0.53 0.14 0.08 0.18 0.33
47.5:05:47.5 0.05 0.88 0.40 0.30 0.49 0.85
45:10:45 0.12 1.42 1.47 1.28 1.95 3.01
42.5:15:42.5 0.21 1.49 2.94 2.71 3.97 5.70
AcOEt/MeOH/BuOH/H2 O 45:10:05:40 0.10 1.06 0.96 0.86 1.28 1.82

chromatographic methodology: CCC methods are used only tracts and isolated compounds in different ways, antioxidant,
sporadically (average 0.9%) and despite recent developments microbiological, and others. In addition, taking into account
of this technology, its proportional use in natural product the work of Ziouti [35], the presence of 5-O-caffeoylshikimic
studies has actually decreased from 1.7 to 0.3% during the acid will be studied in other parts of the S. somalense plant.
period of the survey. Through this work, we have attempted All these studies will contribute to fully enhance and valorize
to promote the use of CPC by providing some elements to the medicinal resources of Djibouti.
rationalize the solvent selection point that often restricts the
development of CPC in isolation approaches. In addition, we The authors have declared no conflict of interest.
showed that a KD value of 3.3 (different from 1) allowed us to
carry out the isolation of 5-O-caffeoylshikimic acid in <3 h, by
taking into account all stages from the preparation of solvents
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