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Sa¨ıda Chideh1,2 Serge Pilard3 Jacques Attoumbre´ 4 Robert Saguez1 Alshaimaa Hassan-Abdallah5 Dominique Cailleu3 Anne Wadouachi6 Sylvie Baltora-Rosset1 1 EDYSAN
FRE 3498 CNRS-Universite´ de Picardie Jules Verne, UFR de Pharmacie, Amiens Cedex, France 2 Centre de Recherche, Universite ´ de Djibouti, Avenue Georges ´ Clemenceau, Djibouti 3 Plate-Forme Analytique, UFR ˆ des Sciences, Batiment Serres-Transfert, Amiens Cedex, France 4 S.I.P.R.E – Comite ´ Nord, Achicourt, France 5 Institut de Recherches Medicinales, CERD, Djibouti 6 LG2A FRE 3517 CNRS-Universite´ de Picardie Jules Verne, Institut de Chimie de Picardie FR 3085, Amiens Cedex, France Received March 3, 2014 Revised June 11, 2014 Accepted June 12, 2014
Research Article
5-O-Caffeoylshikimic acid from Solanum somalense leaves: Advantage of centrifugal partition chromatography over conventional column chromatography Solanum somalense leaves, used in Djibouti for their medicinal properties, were extracted by MeOH. Because of the high polyphenol and flavonoid contents of the extract, respectively, determined at 80.80 ± 2.13 mg gallic acid equivalent/g dry weight and 24.4 ± 1.01 mg quercetin equivalent/g dry weight, the isolation and purification of the main polyphenols were carried out by silica gel column chromatography and centrifugal partition chromatography. Column chromatography led to 11 enriched fractions requiring further purification, while centrifugal partition chromatography allowed the easy recovery of the main compound of the extract. In a solvent system composed of CHCl3 /MeOH/H2 O (9.5:10:5), 21.8 mg of this compound at 97% purity was obtained leading to a yield of 2.63%. Its structure was established as 5-O-caffeoylshikimic acid by mass spectrometry and NMR spectroscopy. This work shows that S. somalense leaves contain very high level of 5-O-caffeoylshikimic acid (0.74% dry weight), making it a potential source of production of this secondary metabolite that is not commonly found in nature but could be partly responsible of the medicinal properties of S. somalense leaves. Keywords: Bioautography / Caffeoylshikimic acids / Dactylifric acid / Polyphenols / Solanum somalense DOI 10.1002/jssc.201400226
1 Introduction Investigating the scientific basis for the use of medicinal plants is of great interest for several reasons: (i) plants remain the primary source of traditional medicine, (ii) a marked increase in the use of herbal medicines or botanical dietary supplements is observed in Western medicine, and (iii) plantderived drugs are the main sources for finding lead compound structures that might reach the drug market [1, 2]. Thousands of traditional herbal medicines have been widely used and studied but Djiboutian ones have not received much attention up to now despite their significant contribution to the management of public healthcare in the country [3]. Among the different medicinal plants used in the Randa region in Djibouti, Solanum somalense has various medicinal uses but, as far as we know, has not been studied from a phytochemical and pharmacological point of view [3].
Correspondence: Dr. Sylvie Baltora-Rosset, EDYSAN FRE 3498 CNRS-Universite´ de Picardie Jules Verne, UFR de Pharmacie, 1 rue des Louvels, 80037 Amiens Cedex, France E-mail: [email protected] Fax: +33-03-2282-7469
Abbreviations: CPC, centrifugal partition chromatography; DPPH, 2,2-diphenyl-1-picrylhydrazyl; DW, dry weight; GAE, gallic acid equivalent; QE, quercetin equivalent
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Bioactive known compounds of plants are divided into three main categories: (i) terpenes and terpenoids (approximately 25 000 types), (ii) alkaloids (approximately 12 000 types), and (iii) phenolic compounds (approximately 8000 types) [4]. Given the difficulty of a comprehensive analysis of plant extracts, studies usually focus on a limited range of compound classes often highlighted by a preliminary rapid biological screening of the total extract. Criteria of chemotaxonomy and uses in traditional medicine may also guide the choice of the class of compounds that will be targeted [4]. The relationship between various diseases such as cancer, hypertension, diabetes, cardiovascular, neurodegenerative diseases, and oxidative damages has been extensively investigated and established [5, 6]. At the same time, the search for antioxidants from natural sources to prevent these oxidative damages is currently under development. Among secondary metabolites, polyphenols generally exhibit good antioxidant properties and are thus often sought in medicinal plants but may cause false-positive results in enzymatic and cellular screening procedures due to their protein-binding ability and their changes in cellular redox potential [7,8]. Various analytical methods have been reported for the analysis and purification of bioactive natural compounds [4, 7, 9] and for the most abundant polyphenols in plants, phenolic acids, and flavonoids [10]. Numerous articles on their extraction, separation, and detection methods have been published over the past two decades [11]. SPE methods are currently used
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to recover prefractionated samples or to remove polyphenols from plant extracts because SPE consists of simple elution and concentration via polymeric resins. In practice, SPE often suffers from low resolution and efficiency [7]. However, SPE is frequently used for cleanup procedures, mainly because it offers the possibility of combining on-line extraction with chromatographic methods such as HPLC [11]. The main methods described for the determination of phenolic acids and flavonoids are chromatographic techniques such as silica gel or sephadex column chromatography, TLC, GC, HPLC, or LC–ESI-MS [4, 11]. Because the chemical structures of polyphenols have various degrees and patterns of hydroxylation, methylation or glycosylation, their analysis by GC requires a preliminary chemical derivatization. Actually, their fractionation from natural sources is usually performed using a combination of methods, mainly column chromatography followed by HPLC [12]. To overcome the use of multiple steps, a new method, countercurrent separation, based solely on differences in the solubility of natural products was developed in the 1970s [3, 13, 14]. CCC is a chromatographic technique that has some advantages when compared with LC techniques: (i) no nonspecific adsorption to a solid support, (ii) higher selectivity, (iii) higher sample loading capacity, (iv) reduction of solvent quantity, and (v) shorter separation 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 compounds mainly found in the Palmae family [16]. Up to now, the largest industrial use of shikimic acids has been the proR or other antiviral drugs for use against duction of Tamiflu avian flu, and the demand for shikimic acid and its derivatives is expected to increase dramatically in the event of a pandemic flu outbreak. Zeng et al. [17] also demonstrated the antiproliferative activity of 5-O-caffeoylshikimic acid against different cancer cell lines. Very few plant sources of these acids have been described. Elaeis guineensis (oil palm) and Phoenix dactylifera (date palm) are the only sources where caffeoyl shikimic acids are the major polyphenols [16,18–20]. 5-O-Caffeoylshikimic acid was also obtained in small quantities from Vaccinium corymbosum (10 mg for 32 g of extract) together with 21 phenolics and showed the potential for nutraceutical applications [21]. The compound was also isolated from Livistona chinensis through a multistep procedure leading to a very low yield: 12 mg for 90.21 g of crude extract. The aim of this work was to develop an efficient bioassayguided method for the fractionation of the methanolic extract of S. somalense. The preliminary screening of the antioxidant properties of the extract, as well TLC analysis with selective polyphenol developers, suggested that the methanolic extract had a high polyphenol content. As described above, a review of the literature indicates that no single method is regarded as standard for extracting polyphenols from plants. In addition, to our knowledge, for caffeoylshikimic acids, only multistep methods including column chromatography and HPLC are described. Therefore, it seemed interesting to develop a new method for isolating these types of phenolic components by C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the use of centrifugal partition chromatography (CPC). As conventional chromatographic separation on silica gel is considered to be one of the reference methods for comparing the success of newly developed methodologies, the two methods were carried out in parallel. Our objective was fully achieved and led to the successful preparative isolation of highly pure major phenolic component from the S. somalense leaves. 5O-Caffeoylshikimic acid was identified by ESI-MS and NMR spectroscopy and will be used for further biological studies. This paper is the first report on the separation of bioactive compounds of S. somalense by CPC.
2 Materials and methods 2.1 Materials Solanum somalense leaves were collected from Tadjourah District of Randa Region in north Djibouti. Plant identification was carried out at the National Herbarium (ETH), Addis Ababa University (AAU) and voucher specimens were deposited in the Herbarium at Djibouti. All organic solvents were of analytical grade and purchased from VWR. Reagents were purchased from Sigma–Aldrich.
2.2 Biological evaluation Determination of total polyphenols. Total phenolic contents were determined according to the literature [19]. A total of 0.50 mL of the sample diluted in MeOH was added to 2.5 mL of 1:10 diluted Folin–Ciocalteu reagent. After 4 min, 2 mL of saturated sodium carbonate solution (about 75 g/L) was added. After 2 h of incubation at room temperature, the absorbance of the reaction mixture was measured at 760 nm. Gallic acid was used as a reference standard and the results were expressed as milligram gallic acid equivalent (mg GAE)/(g dry weight (DW)) of plant material. Gallic acid concentrations ranging from 0 to 100 g/mL were prepared, and the standard calibration curve was obtained using a linear fit (r2 = 0.9972). The samples were analyzed in triplicate. Determination of total flavonoids. Total flavonoids were analyzed according to the aluminum chloride method described in Ref. [8]. A total of 0.5 mL of each sample and 300 L of NaNO2 (1:20 w/v) were pipetted into a test tube. The contents were vortexed for 10 s and left at room temperature for 5 min. Three hundred microliters of AlCl3 (1:10 w/v), 2 mL of 1 M NaOH, and 1.9 mL of distilled water were then added to the mixture. After 10 s of vortexing, the absorbance for each sample was measured at 510 nm. Quercetin concentrations ranging from 0 to 120 g/mL were prepared, and the standard calibration curve was obtained using a linear fit (r2 = 0.9831). Quercetin was used as a reference standard and the results were expressed as (mg QE)/(g DW) (where QE is quercetin equivalent) of plant material. The samples were analyzed in triplicate. www.jss-journal.com
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2.3 Apparatus The CPC instrument used in this study is a SPOT CPC 100 Light (Armen Instrument) fitted with a rotor of ten circular partition disks (1000 partition cells: 0.1 mL per cell; total column capacity of 100 mL). The rotation speed can be chosen from 0 to 4000 rpm. The effluent was monitored by a Lash 06 DAD detector (ECOM, Prague) equipped with a preparative flow cell operating at 254 nm and collected by a LS 5600 (Armen) fraction collector. The HPLC used was a Shimadzu HPLC System including a LC-20AT pump and SPD-M20A diode-array detector. LC–ESI-MS spectra were obtained on a UFLC Prominence (Shimadzu) system coupled with a Q-TOF Ultima Global high-resolution hybrid quadrupole TOF instrument (Waters-Micromass), equipped with a pneumatically assisted electrospray ionization source (Z-spray) and an additional sprayer (lock spray) for the reference compound, allowing accurate mass measurements of ions and their elemental compositions determination. NMR spectra were recorded at 300 K on a Bruker (Wissembourg, France) DRX-500 spectrometer equipped with a broadband inverse probe. DMSOd6 was used as the solvent. Proton resonance assignments and structural elucidation were performed by 1D (1 H and 13 C NMR) and 2D homo- and heteronuclear experiments (correlated spectroscopy, heteronuclear single quantum coherence, and heteronuclear multiple-bond correlation). 2.4 Preparation of crude extract Fifty grams of dried leaves of S. somalense were powdered and treated three times with 500 mL of cyclohexane. After filtration through Whatman filter paper, the plant material was subjected to three extractions with AcOEt (500 mL each time, room temperature, 24 h). The same procedure was applied with MeOH and with H2 O. The extracts from the same solvent were combined and the solvent was removed under the reduced pressure to give the crude extracts.
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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. Twenty microliters was used for injection, which was repeated three times. 2.5.3 LC–ESI-MS The crude extract and the purified fractions were loaded (2 L) on a 100 × 2 mm, 2.7 m Nucleoshell RP18 column (Macherey Nagel) and maintained at 30⬚C. The elution was performed using a 0.25 mL/min mobile phase gradient programmed from water (A), acetonitrile (B), both containing 0.1% v/v HCOOH, as follows (A/B): 95:5 (t = 0 min), 95:5 (t = 5 min), 5:95 (t = 30 min), 5:95 (t = 40 min), 95:5 (t = 45 min), and 95:5 (t = 50 min). The effluent was flow-split via a polyether ether ketone tee with one-third of the flow directed toward the electrospray source of the Q-TOF and the residual two-third directed toward an UV detector (Shimadzu SPD-20A) set to 254 nm. ESI-MS data were recorded in the positive- and negative-ion modes with capillary voltage of ±2.5 kV and cone voltage of 50 V. The source and desolvation temperatures were 120 and 250⬚C, respectively. Nitrogen was used as a drying and nebulizing gas at flow rates of 450 and 100 L/h, respectively. Scanning was performed in the range 50–1550 Da at a scan rate of 1 s/scan and spectra were collected in the profile mode at a resolution of 10 000 (full width at half maximum). Lock mass correction, using appropriate cluster ions of an orthophosphoric acid solution (0.1% in H2 O/CH3 CN 50:50, v/v), was applied for accurate mass measurements. For MS/MS experiments, argon was used as collision gas and the collision energy was set to 20 V. Data acquisition and processing were performed with MassLynx 4.0 SP4 software.
2.6 Separation methods 2.6.1 Silica gel column chromatography separation
2.5 Detection by TLC, HPLC, and LC–MS 2.5.1 TLC Fractionation and separation were monitored by TLC carried out on Silica Gel 60 F254 (Merck) plates developed with EtOAc/HCOOH/HOAc/H2 O (100:11:11:20) visualized by UV light (254 and 365 nm) and/or with vanillin-H2 SO4 reagent, Neu reagent (1% diphenylboric acid ethanolamine complex in EtOH) and 2,2-diphenyl-1-picrylhydrazyl (DPPH solution, 2 mg/mL MeOH). 2.5.2 HPLC HPLC analyses of the crude extract and of the CPC peak fractions were conducted at 254 and 365 nm on a 250 × 4.6 mm, 5 m, Prevail RP C18 column (Grace) using a linear C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Half of the methanolic crude extract (3.9 g) was subjected to silica gel (Merck 70–230 m) column chromatography eluted 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 fractions that were checked for their composition by TLC with four types of visualizers: UV254 , vanillin sulfuric, Neu reagent, and DPPH test. This combination of visualization allowed the gathering of the initial fractions into 11 fractions (F1–F11), which were weighed and submitted to further analyses. 2.6.2 CPC separation 2.6.2.1 Selection of the two-phase solvent system The solvent system was selected according to the distribution constant KD of the six main peaks of the unknown compounds (X) (called A–F) visualized on HPLC chromatogram of the crude methanolic extract at 365 nm. The KD value was www.jss-journal.com
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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. Methanolic crude extract was dissolved in the tested solvent system and vortexed for 30 s. After separation and evaporation under reduced pressure, the residue of each layer was dissolved in 500 L of methanol for HPLC analysis. The KD values were calculated according to the ratio: concentration of compound X in the stationary phase/concentration of compound X in the mobile phase [20]. 2.6.2.2 Collection and analysis of fractions The solvent system used for separation was chloroform/methanol/water in the ratio 9.5:10:5 v/v/v. The biphasic system was prepared just before use by thoroughly mixing volumes of solvent in the above ratio. After the equilibration was established, the stationary phase (upper phase in the descending mode) was pumped into the column at a flow rate of 30 mL/min while the apparatus was run at 500 rpm according to the equilibration mode of the apparatus. After injection of the sample (0.83 g of crude extract in 10 mL of a mixture 50:50 of the two phases), the mobile phase was perfused at 2500 rpm at a flow rate of 4 mL/min for 60 min and then to 8 mL/min under 35 b during the run. The eluent was monitored at 254 nm and 36 fractions (fi ) of 15 mL were collected and analyzed by HPLC. The volume of the stationary phase displaced by the mobile phase was measured and used to determine VM . The stationary phase volume (VS ) was calculated according to the relationship VC = VM + VS since the column volume (VC ) is known. The stationary phase retention Sf was expressed as VS /VC (70% in the experiment). The selectivity (separation factor between two compounds X and X ) was calculated as follows: ␣ = KD(X) /KD(X ) [20].
3 Results and discussion 3.1 Biological evaluation of the extract Leaves of S. somalense, which are mainly used for the preparation of traditional medicines, were extracted with solvents of different polarity: cyclohexane, AcOEt, MeOH, and water. The 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 extracts had the highest content of secondary metabolites and showed quite similar TLC profiles, but the water residue was quickly subjected to microbial contamination, which is one C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
of the main problems with the use of water plant extracts or decoctions [21]. Under these conditions, only the methanolic extract was further analyzed by TLC in different detection conditions. The main phytochemicals were detected by UV, vanillin sulfuric acid, and Neu reagent while the antioxidant activity was evaluated using DPPH radical scavenging assay according to Zhang et al. [8]. The results of revelation by Neu reagent and DPPH (Fig. 1A and B, respectively) showed that polyphenols were present in large quantities in the methanolic extract from S. somalense leaves and also exhibited a high antioxidant total activity due to a DPPH radical scavenging capacity of almost all the compounds present in the extract. Therefore, the total contents of polyphenol and flavonoid were quantitatively evaluated and assessed at 80.80 ± 2.13 mg GAE/g DW and 24.4 ± 1.01 mg QE/g DW, respectively. In their study of the antioxidant properties of methanol extracts of 45 whole medicinal herbs, Li et al. [22] showed that their polyphenols contents could range from 1 to 52 mg GAE/g DW. In this context, the value obtained for S. somalense is very high. Furthermore, it was shown by the same authors and others [8, 22, 23] that a high correlation existed between high levels of polyphenols and the antioxidant capacities of the studied extracts. Finally, the effects of plants on health are often attributed to their polyphenolic components [10].
3.2 Analytical study of the extract While keeping in mind that it is necessary to perform more than one type of antioxidant capacity measurement to take into account the various mechanisms of antioxidant actions of plant extracts [24], we decided to study the major polyphenols present in the extract to evaluate their potential healthpromoting benefits identified by the ethnological surveys in Djibouti [3]. The analysis of the crude extract by HPLC and LC–ESI-MS (Fig. 2A) indicated the presence of six main peaks, which were characterized by their retention times (Rt), their UV absorption maxima, and their molecular weight (Table 1).
3.3 Separation and determination of polyphenols To separate and identify these major polyphenols, a conventional method such as activity-guided fractionation on silica www.jss-journal.com
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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 us to obtain 11 fractions Fi (Fig. 3). As shown by TLC (Fig. 1A), gel column chromatography did not allow an efficient separation of polyphenols: only the major compound of the crude extract (peak B) was obtained with a yield of 1.08% but its purity was not sufficient (87%) (Figs. 1A and 2B) to consider further biological evaluations because the minor compound in the fraction also showed an antioxidant activity. Nevertheless, this step was very useful to roughly characterize the six main components of the extract detectable by HPLC at 365 nm and
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to identify the structure of the major compound B (Table 1). The LC–ESI-MS spectra (Fig. 4A and B) revealed [M+H]+ and [M−H]− ions at m/z 337.09 and 335.07, respectively. A molecular formula of C16 H16 O8 was determined using accurate mass measurements, [M+H]+ : found 337.0935 for 337.0923 calculated and [M−H]− : found 335.0777 for 335.0767 calculated. In a positive mode (Fig. 4A), an abundant ion at m/z 163.03 (C9 H7 O3 ) seems to indicate the presence of a caffeoyl moiety. Moreover UV spectrum of B showed absorption maxima at 211 and 316 nm suggesting a phenolic acid structure.
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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 B C D E F
25.9 29.1 35.4 36.3 38.1 38.8
210, 235, 317 211, 316 208, 255, 354 197, 255, 282, 355 199, 250, 346 199, 265, 347
354 336 610 594 464 594
0.53 0.90 0.43 nd nd 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 50.0 g
Methanolic extract 7.8 g Silica-gel column Chromatography 3.9 g 11 fractions Total fraction weight: 2.14 g F1 weight: 42.4 mg Purity 87 %
Centrifugal Partition Chromatography 0.83 g 38 fractions f19 (7.1 mg 86%) f20 (7.2 mg 91%) f21-f23 (21.8 mg 97%) f24 (5.8 mg 85%)
Figure 3. Processing flowchart for the preparative extraction of the methanolic crude extract of S. somalense leaves by conventional column chromatography and by CPC. The purity of each fraction was determined by analytical HPLC. 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.
The results of combined NMR experiments established the structure of B unambiguously as 5-O-caffeoylshikimic acid as described by Fukuoka [25] (data not shown). This was confirmed by MS/MS of the [M−H]− ion (Fig. 4C) showing the caffeoyl fragment (m/z 179.04, C9 H7 O4 ) and its characteristic loss of H2 O (m/z 161.03, C9 H5 O3 ) and CO2 (m/z 135.05, C8 H7 O2 ) [26]. Solanum species contain abundant caffeic acid derivatives that have implications in human health. Numerous studies have shown that efficacy and potency of these derivatives are very much dependant on their structure and this strong structure–function relationship make them good candidates for the development of new functional foods or pharmaceuticals [27]. Caffeic acid most often esterified with quinic acids such as in chlorogenic acids, is widely found in plants [28], whereas a careful examination of literature showed that esters with shikimic acid are not commonly found in nature despite valuable bioactivities [29]. We have recently shown that CPC is an attractive method to isolate bioactive compounds from Solanum species [30,31]. Hence, it was decided to implement this technique to the fractionation of S. somalense leaves that are a rich source of caffeic acid derivatives with potential interest. The key points critical C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
for performing separation by CPC are good solubility of the sample in the solvent system and the determination of a suitable two-phase system allowing a good partition behaviour of the target compounds. The efficiency of the partition is evaluated using the KD parameter. KD of the six main compounds was determined by HPLC in several solvent systems (Table 2). In the case of natural product studies by CPC, the sample complexity and the unknown nature of target compounds demand the careful consideration of the different KD values and to depart from the usual rules of choice of solvent system. Indeed, generally speaking, compounds of interest should be almost equally distributed between the two phases (KD values in the range 0.5–2) and the separation factor should be >1.5 to ensure the most successful conditions of separation [32]. At this stage of work, our attention was focused on the isolation of the main component of the extract (peak B, i.e. 5-O-caffeoylshikimic acid) and the KD values were examined according to this aim. Of all the possible combinations of solvents, we first chose two families of ternary solvent systems, EtOAc/MeOH/H2 O and CHCl3 /MeOH/H2 O, which accounted for >60% of the separation of glycosylated flavonoids in the literature [33], although we knew that other types of compounds were present in the extract. Moreover, despite the difficulty of using BuOH solvent due to its weak volatility, this solvent was also tested because of its ability to be a major organic phase modifier. Based on the data collected (Table 2), we chose to perform the experiments on the basis of the following requirements: (i) for all the peaks (A–F), KD values had to be in the smallest possible range to allow the separation of all the compounds within a reasonable time, (ii) a greater part of the values should be close to the interval 0.5–2 [34], and (iii) KD values f the six different compounds should lead to separation factors close to 1.5 [34]. With these constraints and with the objective to target the major compound B, the system CHCl3 /MeOH/H2 O (9.5:10:5) (KD values of 10.00, 3.34, 4.62, 4.86, 1.37, 0.16, respectively, for peaks A–F; separation factor ␣ between compound B and compound C of 1.4) seemed to be promising for achieving the isolation of the compound B by CPC. Crude S. somalense extract (0.82 g) was fractionated and despite the rather uncertain nature of this initial choice, the www.jss-journal.com
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163.03
A
%
100
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337.09
0 50
100
150
200
250
300
350
400
450
500
335.07
B
%
100
m/z 550
179.04
0 50
100
150
200
250
300
350
400
450
500
179.04
100
m/z 550
C O
C9H7O4m/z 179.04 - H2O m/z 161.03 - CO2 m/z 135.05
OH
O
%
HO
O OH OH C9H7O3+ m/z 163.03
135.05
OH
161.03 335.08
0
100
150
200
250
300
operation proved to be successful. Each fraction collected during the separation process was weighed and analyzed by HPLC (Fig. 3) showing that the fractions f19 –f24 contained almost solely the compound B with purity varying from 86 to 97%. A total of 21.8 mg of 5-O-caffeoylshikimic acid was obtained from the three fractions of highest purity, f21 –f23 (97% purity), leading to a yield of 2.63%. Its structure was confirmed by NMR spectroscopy. By taking into account the quantity of B in fractions f19 –f24 (39.3 mg), it was also possible to calculate the content of 5-O-caffeoylshikimic acid in S. somalense leaves that is 0.74% of DW. Studies to isolate secondary metabolites from plant material are often unique studies and it is therefore difficult to have reference points to compare and evaluate the performance of natural product isolation approaches. However our results are quite satisfactory within the context of the isolation of natural products. The use of CPC to isolate the major compound of C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
350
m/z
Figure 4. LC–ESI-MS spectra of compound B and fragmentation scheme (a) positive-ion mode (b) negative-ion mode, and (c) MS/MS of the [M−H]− ion at m/z 335.08.Conditions: Nucleoshell RP18 column (100 × 2 mm, 2.7 m) maintained at 30⬚C. The elution was performed using a 0.25 mL/min mobile phase gradient programmed from water (A), acetonitrile (B), both containing 0.1% v/v HCOOH, as follows (A/B): 95:5 (t = 0 min), 95:5 (t = 5 min), 5:95 (t = 30 min), 5:95 (t = 40 min), 95:5 (t = 45 min), and 95:5 (t = 50 min).
S. somalense leaves has enabled us to show the great advantage of this technology compared to the separation on a silica column, which is a more conventional technique overwhelmingly used for the fractionation of natural products. Indeed, our results may be analyzed according to the two primary dimensions highlighted by Pauli et al. [9] in their meta-analysis of natural products purification schemes. The first criterion disclosed in their study is the number of purification steps. On this point, our study helps to show the superiority of CPC compared to silica gel column: a large amount (21.8 mg) of 5-O-caffeoylshikimic acid was obtained in only a single CPC step, leading to a yield of 2.63% compared to 1.08% for silica gel chromatography (Fig. 3). Moreover, a major widely known disadvantage of silica gel purification was also noted in our study: 45% of the extract was irreversibly absorbed on the column, while almost all the samples were recovered by CPC. The second dimension described by Pauli et al. [9], is the www.jss-journal.com
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Table 2. KD (distribution coefficient) of six targets peaks measured in different solvent systems
Solvent system
Composition
A
B
CHCl3 /MeOH/H2 O
04:03:02 06:07:04 08:10:05 9.5:10:5 11:10:05 13:10:05 15:10:05 12:10:05 42:32:5:21 42:30:8:20 45:30:5:20 40:30:10:20 37:37:4:22 38:30:12:20 40:20:40 50:00:50 47:06:47 44:11:44 40:20:40 36:18:36 50:00:50 47.5:05:47.5 45:10:45 42.5:15:42.5 45:10:05:40
98.37 39.26 26.20 10.00 8.00 12.75 41.51 49.73 22.81 13.61 24.62 11.11 17.20 10.95 0.04 0.02 0.03 0.02 0.04 0.06 0.03 0.05 0.12 0.21 0.10
17.00 10.25 6.08 3.34 2.57 3.44 6.86 7.19 3.36 2.49 3.84 2.21 4.20 2.23 0.51 0.88 0.83 0.77 0.54 0.34 0.53 0.88 1.42 1.49 1.06
CHCl3 /MeOH/BuOH/H2 O
EtOAc/MeOH/H2 O
EtOAc/BuOH/H2 O
AcOEt/MeOH/BuOH/H2 O
chromatographic methodology: CCC methods are used only sporadically (average 0.9%) and despite recent developments of this technology, its proportional use in natural product studies has actually decreased from 1.7 to 0.3% during the period of the survey. Through this work, we have attempted to promote the use of CPC by providing some elements to rationalize the solvent selection point that often restricts the development of CPC in isolation approaches. In addition, we showed that a KD value of 3.3 (different from 1) allowed us to carry out the isolation of 5-O-caffeoylshikimic acid in
J. Sep. Sci. 2014, 37, 2331–2339
Sa¨ıda Chideh1,2 Serge Pilard3 Jacques Attoumbre´ 4 Robert Saguez1 Alshaimaa Hassan-Abdallah5 Dominique Cailleu3 Anne Wadouachi6 Sylvie Baltora-Rosset1 1 EDYSAN
FRE 3498 CNRS-Universite´ de Picardie Jules Verne, UFR de Pharmacie, Amiens Cedex, France 2 Centre de Recherche, Universite ´ de Djibouti, Avenue Georges ´ Clemenceau, Djibouti 3 Plate-Forme Analytique, UFR ˆ des Sciences, Batiment Serres-Transfert, Amiens Cedex, France 4 S.I.P.R.E – Comite ´ Nord, Achicourt, France 5 Institut de Recherches Medicinales, CERD, Djibouti 6 LG2A FRE 3517 CNRS-Universite´ de Picardie Jules Verne, Institut de Chimie de Picardie FR 3085, Amiens Cedex, France Received March 3, 2014 Revised June 11, 2014 Accepted June 12, 2014
Research Article
5-O-Caffeoylshikimic acid from Solanum somalense leaves: Advantage of centrifugal partition chromatography over conventional column chromatography Solanum somalense leaves, used in Djibouti for their medicinal properties, were extracted by MeOH. Because of the high polyphenol and flavonoid contents of the extract, respectively, determined at 80.80 ± 2.13 mg gallic acid equivalent/g dry weight and 24.4 ± 1.01 mg quercetin equivalent/g dry weight, the isolation and purification of the main polyphenols were carried out by silica gel column chromatography and centrifugal partition chromatography. Column chromatography led to 11 enriched fractions requiring further purification, while centrifugal partition chromatography allowed the easy recovery of the main compound of the extract. In a solvent system composed of CHCl3 /MeOH/H2 O (9.5:10:5), 21.8 mg of this compound at 97% purity was obtained leading to a yield of 2.63%. Its structure was established as 5-O-caffeoylshikimic acid by mass spectrometry and NMR spectroscopy. This work shows that S. somalense leaves contain very high level of 5-O-caffeoylshikimic acid (0.74% dry weight), making it a potential source of production of this secondary metabolite that is not commonly found in nature but could be partly responsible of the medicinal properties of S. somalense leaves. Keywords: Bioautography / Caffeoylshikimic acids / Dactylifric acid / Polyphenols / Solanum somalense DOI 10.1002/jssc.201400226
1 Introduction Investigating the scientific basis for the use of medicinal plants is of great interest for several reasons: (i) plants remain the primary source of traditional medicine, (ii) a marked increase in the use of herbal medicines or botanical dietary supplements is observed in Western medicine, and (iii) plantderived drugs are the main sources for finding lead compound structures that might reach the drug market [1, 2]. Thousands of traditional herbal medicines have been widely used and studied but Djiboutian ones have not received much attention up to now despite their significant contribution to the management of public healthcare in the country [3]. Among the different medicinal plants used in the Randa region in Djibouti, Solanum somalense has various medicinal uses but, as far as we know, has not been studied from a phytochemical and pharmacological point of view [3].
Correspondence: Dr. Sylvie Baltora-Rosset, EDYSAN FRE 3498 CNRS-Universite´ de Picardie Jules Verne, UFR de Pharmacie, 1 rue des Louvels, 80037 Amiens Cedex, France E-mail: [email protected] Fax: +33-03-2282-7469
Abbreviations: CPC, centrifugal partition chromatography; DPPH, 2,2-diphenyl-1-picrylhydrazyl; DW, dry weight; GAE, gallic acid equivalent; QE, quercetin equivalent
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Bioactive known compounds of plants are divided into three main categories: (i) terpenes and terpenoids (approximately 25 000 types), (ii) alkaloids (approximately 12 000 types), and (iii) phenolic compounds (approximately 8000 types) [4]. Given the difficulty of a comprehensive analysis of plant extracts, studies usually focus on a limited range of compound classes often highlighted by a preliminary rapid biological screening of the total extract. Criteria of chemotaxonomy and uses in traditional medicine may also guide the choice of the class of compounds that will be targeted [4]. The relationship between various diseases such as cancer, hypertension, diabetes, cardiovascular, neurodegenerative diseases, and oxidative damages has been extensively investigated and established [5, 6]. At the same time, the search for antioxidants from natural sources to prevent these oxidative damages is currently under development. Among secondary metabolites, polyphenols generally exhibit good antioxidant properties and are thus often sought in medicinal plants but may cause false-positive results in enzymatic and cellular screening procedures due to their protein-binding ability and their changes in cellular redox potential [7,8]. Various analytical methods have been reported for the analysis and purification of bioactive natural compounds [4, 7, 9] and for the most abundant polyphenols in plants, phenolic acids, and flavonoids [10]. Numerous articles on their extraction, separation, and detection methods have been published over the past two decades [11]. SPE methods are currently used
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to recover prefractionated samples or to remove polyphenols from plant extracts because SPE consists of simple elution and concentration via polymeric resins. In practice, SPE often suffers from low resolution and efficiency [7]. However, SPE is frequently used for cleanup procedures, mainly because it offers the possibility of combining on-line extraction with chromatographic methods such as HPLC [11]. The main methods described for the determination of phenolic acids and flavonoids are chromatographic techniques such as silica gel or sephadex column chromatography, TLC, GC, HPLC, or LC–ESI-MS [4, 11]. Because the chemical structures of polyphenols have various degrees and patterns of hydroxylation, methylation or glycosylation, their analysis by GC requires a preliminary chemical derivatization. Actually, their fractionation from natural sources is usually performed using a combination of methods, mainly column chromatography followed by HPLC [12]. To overcome the use of multiple steps, a new method, countercurrent separation, based solely on differences in the solubility of natural products was developed in the 1970s [3, 13, 14]. CCC is a chromatographic technique that has some advantages when compared with LC techniques: (i) no nonspecific adsorption to a solid support, (ii) higher selectivity, (iii) higher sample loading capacity, (iv) reduction of solvent quantity, and (v) shorter separation 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 compounds mainly found in the Palmae family [16]. Up to now, the largest industrial use of shikimic acids has been the proR or other antiviral drugs for use against duction of Tamiflu avian flu, and the demand for shikimic acid and its derivatives is expected to increase dramatically in the event of a pandemic flu outbreak. Zeng et al. [17] also demonstrated the antiproliferative activity of 5-O-caffeoylshikimic acid against different cancer cell lines. Very few plant sources of these acids have been described. Elaeis guineensis (oil palm) and Phoenix dactylifera (date palm) are the only sources where caffeoyl shikimic acids are the major polyphenols [16,18–20]. 5-O-Caffeoylshikimic acid was also obtained in small quantities from Vaccinium corymbosum (10 mg for 32 g of extract) together with 21 phenolics and showed the potential for nutraceutical applications [21]. The compound was also isolated from Livistona chinensis through a multistep procedure leading to a very low yield: 12 mg for 90.21 g of crude extract. The aim of this work was to develop an efficient bioassayguided method for the fractionation of the methanolic extract of S. somalense. The preliminary screening of the antioxidant properties of the extract, as well TLC analysis with selective polyphenol developers, suggested that the methanolic extract had a high polyphenol content. As described above, a review of the literature indicates that no single method is regarded as standard for extracting polyphenols from plants. In addition, to our knowledge, for caffeoylshikimic acids, only multistep methods including column chromatography and HPLC are described. Therefore, it seemed interesting to develop a new method for isolating these types of phenolic components by C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the use of centrifugal partition chromatography (CPC). As conventional chromatographic separation on silica gel is considered to be one of the reference methods for comparing the success of newly developed methodologies, the two methods were carried out in parallel. Our objective was fully achieved and led to the successful preparative isolation of highly pure major phenolic component from the S. somalense leaves. 5O-Caffeoylshikimic acid was identified by ESI-MS and NMR spectroscopy and will be used for further biological studies. This paper is the first report on the separation of bioactive compounds of S. somalense by CPC.
2 Materials and methods 2.1 Materials Solanum somalense leaves were collected from Tadjourah District of Randa Region in north Djibouti. Plant identification was carried out at the National Herbarium (ETH), Addis Ababa University (AAU) and voucher specimens were deposited in the Herbarium at Djibouti. All organic solvents were of analytical grade and purchased from VWR. Reagents were purchased from Sigma–Aldrich.
2.2 Biological evaluation Determination of total polyphenols. Total phenolic contents were determined according to the literature [19]. A total of 0.50 mL of the sample diluted in MeOH was added to 2.5 mL of 1:10 diluted Folin–Ciocalteu reagent. After 4 min, 2 mL of saturated sodium carbonate solution (about 75 g/L) was added. After 2 h of incubation at room temperature, the absorbance of the reaction mixture was measured at 760 nm. Gallic acid was used as a reference standard and the results were expressed as milligram gallic acid equivalent (mg GAE)/(g dry weight (DW)) of plant material. Gallic acid concentrations ranging from 0 to 100 g/mL were prepared, and the standard calibration curve was obtained using a linear fit (r2 = 0.9972). The samples were analyzed in triplicate. Determination of total flavonoids. Total flavonoids were analyzed according to the aluminum chloride method described in Ref. [8]. A total of 0.5 mL of each sample and 300 L of NaNO2 (1:20 w/v) were pipetted into a test tube. The contents were vortexed for 10 s and left at room temperature for 5 min. Three hundred microliters of AlCl3 (1:10 w/v), 2 mL of 1 M NaOH, and 1.9 mL of distilled water were then added to the mixture. After 10 s of vortexing, the absorbance for each sample was measured at 510 nm. Quercetin concentrations ranging from 0 to 120 g/mL were prepared, and the standard calibration curve was obtained using a linear fit (r2 = 0.9831). Quercetin was used as a reference standard and the results were expressed as (mg QE)/(g DW) (where QE is quercetin equivalent) of plant material. The samples were analyzed in triplicate. www.jss-journal.com
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2.3 Apparatus The CPC instrument used in this study is a SPOT CPC 100 Light (Armen Instrument) fitted with a rotor of ten circular partition disks (1000 partition cells: 0.1 mL per cell; total column capacity of 100 mL). The rotation speed can be chosen from 0 to 4000 rpm. The effluent was monitored by a Lash 06 DAD detector (ECOM, Prague) equipped with a preparative flow cell operating at 254 nm and collected by a LS 5600 (Armen) fraction collector. The HPLC used was a Shimadzu HPLC System including a LC-20AT pump and SPD-M20A diode-array detector. LC–ESI-MS spectra were obtained on a UFLC Prominence (Shimadzu) system coupled with a Q-TOF Ultima Global high-resolution hybrid quadrupole TOF instrument (Waters-Micromass), equipped with a pneumatically assisted electrospray ionization source (Z-spray) and an additional sprayer (lock spray) for the reference compound, allowing accurate mass measurements of ions and their elemental compositions determination. NMR spectra were recorded at 300 K on a Bruker (Wissembourg, France) DRX-500 spectrometer equipped with a broadband inverse probe. DMSOd6 was used as the solvent. Proton resonance assignments and structural elucidation were performed by 1D (1 H and 13 C NMR) and 2D homo- and heteronuclear experiments (correlated spectroscopy, heteronuclear single quantum coherence, and heteronuclear multiple-bond correlation). 2.4 Preparation of crude extract Fifty grams of dried leaves of S. somalense were powdered and treated three times with 500 mL of cyclohexane. After filtration through Whatman filter paper, the plant material was subjected to three extractions with AcOEt (500 mL each time, room temperature, 24 h). The same procedure was applied with MeOH and with H2 O. The extracts from the same solvent were combined and the solvent was removed under the reduced pressure to give the crude extracts.
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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. Twenty microliters was used for injection, which was repeated three times. 2.5.3 LC–ESI-MS The crude extract and the purified fractions were loaded (2 L) on a 100 × 2 mm, 2.7 m Nucleoshell RP18 column (Macherey Nagel) and maintained at 30⬚C. The elution was performed using a 0.25 mL/min mobile phase gradient programmed from water (A), acetonitrile (B), both containing 0.1% v/v HCOOH, as follows (A/B): 95:5 (t = 0 min), 95:5 (t = 5 min), 5:95 (t = 30 min), 5:95 (t = 40 min), 95:5 (t = 45 min), and 95:5 (t = 50 min). The effluent was flow-split via a polyether ether ketone tee with one-third of the flow directed toward the electrospray source of the Q-TOF and the residual two-third directed toward an UV detector (Shimadzu SPD-20A) set to 254 nm. ESI-MS data were recorded in the positive- and negative-ion modes with capillary voltage of ±2.5 kV and cone voltage of 50 V. The source and desolvation temperatures were 120 and 250⬚C, respectively. Nitrogen was used as a drying and nebulizing gas at flow rates of 450 and 100 L/h, respectively. Scanning was performed in the range 50–1550 Da at a scan rate of 1 s/scan and spectra were collected in the profile mode at a resolution of 10 000 (full width at half maximum). Lock mass correction, using appropriate cluster ions of an orthophosphoric acid solution (0.1% in H2 O/CH3 CN 50:50, v/v), was applied for accurate mass measurements. For MS/MS experiments, argon was used as collision gas and the collision energy was set to 20 V. Data acquisition and processing were performed with MassLynx 4.0 SP4 software.
2.6 Separation methods 2.6.1 Silica gel column chromatography separation
2.5 Detection by TLC, HPLC, and LC–MS 2.5.1 TLC Fractionation and separation were monitored by TLC carried out on Silica Gel 60 F254 (Merck) plates developed with EtOAc/HCOOH/HOAc/H2 O (100:11:11:20) visualized by UV light (254 and 365 nm) and/or with vanillin-H2 SO4 reagent, Neu reagent (1% diphenylboric acid ethanolamine complex in EtOH) and 2,2-diphenyl-1-picrylhydrazyl (DPPH solution, 2 mg/mL MeOH). 2.5.2 HPLC HPLC analyses of the crude extract and of the CPC peak fractions were conducted at 254 and 365 nm on a 250 × 4.6 mm, 5 m, Prevail RP C18 column (Grace) using a linear C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Half of the methanolic crude extract (3.9 g) was subjected to silica gel (Merck 70–230 m) column chromatography eluted 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 fractions that were checked for their composition by TLC with four types of visualizers: UV254 , vanillin sulfuric, Neu reagent, and DPPH test. This combination of visualization allowed the gathering of the initial fractions into 11 fractions (F1–F11), which were weighed and submitted to further analyses. 2.6.2 CPC separation 2.6.2.1 Selection of the two-phase solvent system The solvent system was selected according to the distribution constant KD of the six main peaks of the unknown compounds (X) (called A–F) visualized on HPLC chromatogram of the crude methanolic extract at 365 nm. The KD value was www.jss-journal.com
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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. Methanolic crude extract was dissolved in the tested solvent system and vortexed for 30 s. After separation and evaporation under reduced pressure, the residue of each layer was dissolved in 500 L of methanol for HPLC analysis. The KD values were calculated according to the ratio: concentration of compound X in the stationary phase/concentration of compound X in the mobile phase [20]. 2.6.2.2 Collection and analysis of fractions The solvent system used for separation was chloroform/methanol/water in the ratio 9.5:10:5 v/v/v. The biphasic system was prepared just before use by thoroughly mixing volumes of solvent in the above ratio. After the equilibration was established, the stationary phase (upper phase in the descending mode) was pumped into the column at a flow rate of 30 mL/min while the apparatus was run at 500 rpm according to the equilibration mode of the apparatus. After injection of the sample (0.83 g of crude extract in 10 mL of a mixture 50:50 of the two phases), the mobile phase was perfused at 2500 rpm at a flow rate of 4 mL/min for 60 min and then to 8 mL/min under 35 b during the run. The eluent was monitored at 254 nm and 36 fractions (fi ) of 15 mL were collected and analyzed by HPLC. The volume of the stationary phase displaced by the mobile phase was measured and used to determine VM . The stationary phase volume (VS ) was calculated according to the relationship VC = VM + VS since the column volume (VC ) is known. The stationary phase retention Sf was expressed as VS /VC (70% in the experiment). The selectivity (separation factor between two compounds X and X ) was calculated as follows: ␣ = KD(X) /KD(X ) [20].
3 Results and discussion 3.1 Biological evaluation of the extract Leaves of S. somalense, which are mainly used for the preparation of traditional medicines, were extracted with solvents of different polarity: cyclohexane, AcOEt, MeOH, and water. The 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 extracts had the highest content of secondary metabolites and showed quite similar TLC profiles, but the water residue was quickly subjected to microbial contamination, which is one C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
of the main problems with the use of water plant extracts or decoctions [21]. Under these conditions, only the methanolic extract was further analyzed by TLC in different detection conditions. The main phytochemicals were detected by UV, vanillin sulfuric acid, and Neu reagent while the antioxidant activity was evaluated using DPPH radical scavenging assay according to Zhang et al. [8]. The results of revelation by Neu reagent and DPPH (Fig. 1A and B, respectively) showed that polyphenols were present in large quantities in the methanolic extract from S. somalense leaves and also exhibited a high antioxidant total activity due to a DPPH radical scavenging capacity of almost all the compounds present in the extract. Therefore, the total contents of polyphenol and flavonoid were quantitatively evaluated and assessed at 80.80 ± 2.13 mg GAE/g DW and 24.4 ± 1.01 mg QE/g DW, respectively. In their study of the antioxidant properties of methanol extracts of 45 whole medicinal herbs, Li et al. [22] showed that their polyphenols contents could range from 1 to 52 mg GAE/g DW. In this context, the value obtained for S. somalense is very high. Furthermore, it was shown by the same authors and others [8, 22, 23] that a high correlation existed between high levels of polyphenols and the antioxidant capacities of the studied extracts. Finally, the effects of plants on health are often attributed to their polyphenolic components [10].
3.2 Analytical study of the extract While keeping in mind that it is necessary to perform more than one type of antioxidant capacity measurement to take into account the various mechanisms of antioxidant actions of plant extracts [24], we decided to study the major polyphenols present in the extract to evaluate their potential healthpromoting benefits identified by the ethnological surveys in Djibouti [3]. The analysis of the crude extract by HPLC and LC–ESI-MS (Fig. 2A) indicated the presence of six main peaks, which were characterized by their retention times (Rt), their UV absorption maxima, and their molecular weight (Table 1).
3.3 Separation and determination of polyphenols To separate and identify these major polyphenols, a conventional method such as activity-guided fractionation on silica www.jss-journal.com
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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 us to obtain 11 fractions Fi (Fig. 3). As shown by TLC (Fig. 1A), gel column chromatography did not allow an efficient separation of polyphenols: only the major compound of the crude extract (peak B) was obtained with a yield of 1.08% but its purity was not sufficient (87%) (Figs. 1A and 2B) to consider further biological evaluations because the minor compound in the fraction also showed an antioxidant activity. Nevertheless, this step was very useful to roughly characterize the six main components of the extract detectable by HPLC at 365 nm and
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
to identify the structure of the major compound B (Table 1). The LC–ESI-MS spectra (Fig. 4A and B) revealed [M+H]+ and [M−H]− ions at m/z 337.09 and 335.07, respectively. A molecular formula of C16 H16 O8 was determined using accurate mass measurements, [M+H]+ : found 337.0935 for 337.0923 calculated and [M−H]− : found 335.0777 for 335.0767 calculated. In a positive mode (Fig. 4A), an abundant ion at m/z 163.03 (C9 H7 O3 ) seems to indicate the presence of a caffeoyl moiety. Moreover UV spectrum of B showed absorption maxima at 211 and 316 nm suggesting a phenolic acid structure.
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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 B C D E F
25.9 29.1 35.4 36.3 38.1 38.8
210, 235, 317 211, 316 208, 255, 354 197, 255, 282, 355 199, 250, 346 199, 265, 347
354 336 610 594 464 594
0.53 0.90 0.43 nd nd 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 50.0 g
Methanolic extract 7.8 g Silica-gel column Chromatography 3.9 g 11 fractions Total fraction weight: 2.14 g F1 weight: 42.4 mg Purity 87 %
Centrifugal Partition Chromatography 0.83 g 38 fractions f19 (7.1 mg 86%) f20 (7.2 mg 91%) f21-f23 (21.8 mg 97%) f24 (5.8 mg 85%)
Figure 3. Processing flowchart for the preparative extraction of the methanolic crude extract of S. somalense leaves by conventional column chromatography and by CPC. The purity of each fraction was determined by analytical HPLC. 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.
The results of combined NMR experiments established the structure of B unambiguously as 5-O-caffeoylshikimic acid as described by Fukuoka [25] (data not shown). This was confirmed by MS/MS of the [M−H]− ion (Fig. 4C) showing the caffeoyl fragment (m/z 179.04, C9 H7 O4 ) and its characteristic loss of H2 O (m/z 161.03, C9 H5 O3 ) and CO2 (m/z 135.05, C8 H7 O2 ) [26]. Solanum species contain abundant caffeic acid derivatives that have implications in human health. Numerous studies have shown that efficacy and potency of these derivatives are very much dependant on their structure and this strong structure–function relationship make them good candidates for the development of new functional foods or pharmaceuticals [27]. Caffeic acid most often esterified with quinic acids such as in chlorogenic acids, is widely found in plants [28], whereas a careful examination of literature showed that esters with shikimic acid are not commonly found in nature despite valuable bioactivities [29]. We have recently shown that CPC is an attractive method to isolate bioactive compounds from Solanum species [30,31]. Hence, it was decided to implement this technique to the fractionation of S. somalense leaves that are a rich source of caffeic acid derivatives with potential interest. The key points critical C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
for performing separation by CPC are good solubility of the sample in the solvent system and the determination of a suitable two-phase system allowing a good partition behaviour of the target compounds. The efficiency of the partition is evaluated using the KD parameter. KD of the six main compounds was determined by HPLC in several solvent systems (Table 2). In the case of natural product studies by CPC, the sample complexity and the unknown nature of target compounds demand the careful consideration of the different KD values and to depart from the usual rules of choice of solvent system. Indeed, generally speaking, compounds of interest should be almost equally distributed between the two phases (KD values in the range 0.5–2) and the separation factor should be >1.5 to ensure the most successful conditions of separation [32]. At this stage of work, our attention was focused on the isolation of the main component of the extract (peak B, i.e. 5-O-caffeoylshikimic acid) and the KD values were examined according to this aim. Of all the possible combinations of solvents, we first chose two families of ternary solvent systems, EtOAc/MeOH/H2 O and CHCl3 /MeOH/H2 O, which accounted for >60% of the separation of glycosylated flavonoids in the literature [33], although we knew that other types of compounds were present in the extract. Moreover, despite the difficulty of using BuOH solvent due to its weak volatility, this solvent was also tested because of its ability to be a major organic phase modifier. Based on the data collected (Table 2), we chose to perform the experiments on the basis of the following requirements: (i) for all the peaks (A–F), KD values had to be in the smallest possible range to allow the separation of all the compounds within a reasonable time, (ii) a greater part of the values should be close to the interval 0.5–2 [34], and (iii) KD values f the six different compounds should lead to separation factors close to 1.5 [34]. With these constraints and with the objective to target the major compound B, the system CHCl3 /MeOH/H2 O (9.5:10:5) (KD values of 10.00, 3.34, 4.62, 4.86, 1.37, 0.16, respectively, for peaks A–F; separation factor ␣ between compound B and compound C of 1.4) seemed to be promising for achieving the isolation of the compound B by CPC. Crude S. somalense extract (0.82 g) was fractionated and despite the rather uncertain nature of this initial choice, the www.jss-journal.com
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163.03
A
%
100
2337
337.09
0 50
100
150
200
250
300
350
400
450
500
335.07
B
%
100
m/z 550
179.04
0 50
100
150
200
250
300
350
400
450
500
179.04
100
m/z 550
C O
C9H7O4m/z 179.04 - H2O m/z 161.03 - CO2 m/z 135.05
OH
O
%
HO
O OH OH C9H7O3+ m/z 163.03
135.05
OH
161.03 335.08
0
100
150
200
250
300
operation proved to be successful. Each fraction collected during the separation process was weighed and analyzed by HPLC (Fig. 3) showing that the fractions f19 –f24 contained almost solely the compound B with purity varying from 86 to 97%. A total of 21.8 mg of 5-O-caffeoylshikimic acid was obtained from the three fractions of highest purity, f21 –f23 (97% purity), leading to a yield of 2.63%. Its structure was confirmed by NMR spectroscopy. By taking into account the quantity of B in fractions f19 –f24 (39.3 mg), it was also possible to calculate the content of 5-O-caffeoylshikimic acid in S. somalense leaves that is 0.74% of DW. Studies to isolate secondary metabolites from plant material are often unique studies and it is therefore difficult to have reference points to compare and evaluate the performance of natural product isolation approaches. However our results are quite satisfactory within the context of the isolation of natural products. The use of CPC to isolate the major compound of C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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m/z
Figure 4. LC–ESI-MS spectra of compound B and fragmentation scheme (a) positive-ion mode (b) negative-ion mode, and (c) MS/MS of the [M−H]− ion at m/z 335.08.Conditions: Nucleoshell RP18 column (100 × 2 mm, 2.7 m) maintained at 30⬚C. The elution was performed using a 0.25 mL/min mobile phase gradient programmed from water (A), acetonitrile (B), both containing 0.1% v/v HCOOH, as follows (A/B): 95:5 (t = 0 min), 95:5 (t = 5 min), 5:95 (t = 30 min), 5:95 (t = 40 min), 95:5 (t = 45 min), and 95:5 (t = 50 min).
S. somalense leaves has enabled us to show the great advantage of this technology compared to the separation on a silica column, which is a more conventional technique overwhelmingly used for the fractionation of natural products. Indeed, our results may be analyzed according to the two primary dimensions highlighted by Pauli et al. [9] in their meta-analysis of natural products purification schemes. The first criterion disclosed in their study is the number of purification steps. On this point, our study helps to show the superiority of CPC compared to silica gel column: a large amount (21.8 mg) of 5-O-caffeoylshikimic acid was obtained in only a single CPC step, leading to a yield of 2.63% compared to 1.08% for silica gel chromatography (Fig. 3). Moreover, a major widely known disadvantage of silica gel purification was also noted in our study: 45% of the extract was irreversibly absorbed on the column, while almost all the samples were recovered by CPC. The second dimension described by Pauli et al. [9], is the www.jss-journal.com
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Table 2. KD (distribution coefficient) of six targets peaks measured in different solvent systems
Solvent system
Composition
A
B
CHCl3 /MeOH/H2 O
04:03:02 06:07:04 08:10:05 9.5:10:5 11:10:05 13:10:05 15:10:05 12:10:05 42:32:5:21 42:30:8:20 45:30:5:20 40:30:10:20 37:37:4:22 38:30:12:20 40:20:40 50:00:50 47:06:47 44:11:44 40:20:40 36:18:36 50:00:50 47.5:05:47.5 45:10:45 42.5:15:42.5 45:10:05:40
98.37 39.26 26.20 10.00 8.00 12.75 41.51 49.73 22.81 13.61 24.62 11.11 17.20 10.95 0.04 0.02 0.03 0.02 0.04 0.06 0.03 0.05 0.12 0.21 0.10
17.00 10.25 6.08 3.34 2.57 3.44 6.86 7.19 3.36 2.49 3.84 2.21 4.20 2.23 0.51 0.88 0.83 0.77 0.54 0.34 0.53 0.88 1.42 1.49 1.06
CHCl3 /MeOH/BuOH/H2 O
EtOAc/MeOH/H2 O
EtOAc/BuOH/H2 O
AcOEt/MeOH/BuOH/H2 O
chromatographic methodology: CCC methods are used only sporadically (average 0.9%) and despite recent developments of this technology, its proportional use in natural product studies has actually decreased from 1.7 to 0.3% during the period of the survey. Through this work, we have attempted to promote the use of CPC by providing some elements to rationalize the solvent selection point that often restricts the development of CPC in isolation approaches. In addition, we showed that a KD value of 3.3 (different from 1) allowed us to carry out the isolation of 5-O-caffeoylshikimic acid in
