1604 Original Papers Isolation of Guttiferones from Renewable Parts of Symphonia globulifera by Centrifugal Partition Chromatography Authors Kevin Cottet1, Yann Fromentin 1, Marina Kritsanida 1, Raphaël Grougnet 1, Guillaume Odonne 2, Christophe Duplais3, Sylvie Michel1, Marie-Christine Lallemand 1 Affiliations The affiliations are listed at the end of the article Key words Abstract one A and proceeding to its quantification, a cen- l" Symphonia globulifera ! trifugal partition chromatography methodology l" Clusiaceae The aim of this study was to investigate the spe- using a two-phase solvent system of cyclohex- l" centrifugal partition cies Symphonia globulifera, a source of polycyclic ane/ethyl acetate/methanol/water (20:1:20 :1, chromatography polyprenylated acyl phloroglucinols such as gutti- v/v/v/v) was applied to each extract. In conclu- l" countercurrent chromatography ferone A, which is known to exhibit a variety of bi- sion, a centrifugal partition chromatography sys- guttiferones ological activities including noticeable antileish- tem has been developed to ensure a fast, reliable, l" PPAPs manial properties. Our goal was the identification and scalable way to isolate, with a high level of and the quantification of guttiferone A in differ- purity, guttiferone A from five renewable parts of ent renewable parts of S. globulifera and its pre- S. globulifera. Moreover, this methodology can be parative isolation. To the best of our knowledge, extended to the isolation of other polycyclic there is no data concerning its mechanism of ac- polyprenylated acyl phloroglucinols such as gutti- tion. Consequently, it is particularly interesting ferones B, C, and D. to isolate it in gram quantities in order to estab- lish structure activity relationship studies. After Supporting information available online at performing high-performance liquid chromatog- http://www.thieme-connect.de/products raphy profiles detecting the presence of guttifer- Introduction activity (IC50 = 2.93 µg/mL) [9]. Only GA has been Downloaded by: Deakin University. Copyrighted material. received October 14, 2014 ! evaluated against Leishmania donovani,even revised April 28, 2015 Symphonia globulifera L. f. (Clusiaceae), a medium though 17 PPAPs have been isolated and charac- accepted June 12, 2015 to tall tree with characteristic yellow latex, spread terized from S. globulifera [1,11–13]. Owing to Bibliography over tropical areas, is a rich source of natural the therapeutic potential of guttiferones and its DOI http://dx.doi.org/ products called guttiferones [1]. These com- analogs [10], it would be pertinent to obtain some 10.1055/s-0035-1557773 pounds are members of the polycyclic poly- of them selectively and in gram quantities for Published online September 21, prenylated acyl phloroglucinols (PPAPs) type B semi-synthesis and further pharmacological and 2015 family [2–6]. They have a bicyclo[3.3.1]nonane- structure activity relationship (SAR) studies. Planta Med 2015; 81: 1604–1608 © Georg Thieme trione core structure, bearing prenyl or geranyl High-performance liquid chromatography (HPLC) Verlag KG Stuttgart · New York · side chains and an additional acyl group, exempli- profiles were performed to detect the presence of ISSN 0032‑0943 fied by guttiferones A (GA), B (GB), C (GC), and D GA in five renewable parts of S. globulifera. All the l" Correspondence (GD) ( Fig. 1). PPAPs characterized in S. globulifera have been Prof. Dr. Marie-Christine Over the recent years, numerous biological activ- isolated from roots, so far; however, GA has also Lallemand ities, including anti-parasite, antiviral, and antitu- been isolated from its leaves and seeds [1,9, 11, Laboratoire de Pharmacognosie UMR CNRS 8638 COMETE moral properties, have been established for PPAPs 12]. To our knowledge, no other organs have been Université Paris Descartes [7–12]. In particular, an antileishmanial property investigated. For availability and sustainability Sorbonne Paris Cité has been described for GA. The increasing resis- reasons, we focused on renewable parts, such as 4 avenue de lʼObservatoire 75006 Paris tance of Leishmania parasites, the toxicity of the latex, pericarps, seeds, leaves, and flowers France current therapy, and the lack of a human vaccine (l" Fig. 2). Up to now, PPAPs have been isolated us- Phone: + 33153739693 generate an urgent need to discover effective ing classical chromatography methods [13], Fax: + 331 40469658 marie-christine.lallemand@ new-targeted drugs [7,8]. In this goal, GA appears which require silica and time consumption. In parisdescartes.fr as a potential lead compound, showing noticeable contrast, centrifugal partition chromatography Cottet K et al. Isolation of Guttiferones… Planta Med 2015; 81: 1604–1608 Original Papers 1605 Fig. 1 Chemical structures of guttiferones A (1), B (2), C (3), and D (4). Fig. 2 Plant parts of S. globulifera are represented in the following series: seeds, pericarps, latex, leaves, and flowers. Copyright Yann Fromentin, with kind permission. (CPC) allows for the fast isolation of pure secondary metabolites As GA was our main compound of interest, we performed its without solid support and large amounts of toxic solvents, in quantification in each part using calibration curves from 0.1 mg/ agreement with some of the green chemistry principles [14]. This mL to 0.5 mg/mL. " 2 paper describes the characterization and quantification of GA l Table 1 shows the amount of GA in each part and the r was Downloaded by: Deakin University. Copyrighted material. from five S. globulifera extracts via HPLC profiles and its first iso- taken as a measure of precision. High amounts (1.8 to 5.4% w/w) lation by an appropriate CPC system. were quantified in all of the extracts except for the leaves extract, prompting us to develop a reliable isolation method. After assessment of the GA presence in the selected extracts, we Results and Discussion focused on providing an effective CPC purification method. This ! technique uses a two-phase solvent system made from a combi- Before starting the isolation work, we wanted to ensure the pres- nation of mutually immiscible solvents. ence of GA in the different extracts studied and proceed to its Considering the polarity and physicochemical properties of these quantification. In this goal, HPLC profiles were realized for five PPAPs, we decided to use a suitable system adapted from ARIZO- renewable parts of S. globulifera. The chromatograms of ethyl NA (heptane/ethyl acetate/methanol/water) and to replace hep- acetate fractions obtained from the partition of the methanolic tane with cyclohexane for cost reasons. We started our investiga- extracts are represented in l" Fig. 3. tion, as recommended for solvent consumption, using a system The plant parts showed different HPLC profiles. The presence of leading to a GA partition coefficient K close to one (19:1:15:5; four guttiferones was identified by comparison with controls ob- cyclohexane/ethyl acetate/methanol/water) already developed in tained during previous works [10] and isolation through classical our laboratory [14]. Unfortunately, the purities of the desired methods. These compounds were detected at the average reten- product were not sufficient to our criterion. Further investiga- tion times of 28.4 min, 44.7 min, 44.7 min, and 47.5 min for GA, tions with systems leading to a higher K led to a higher GA purity. GB, GC, and GD, respectively. GA was detected in all samples. In K ratios of the four characterized PPAPs were calculated to opti- fact, GA appears as a major PPAP compound in the latex, flower, mize the system. pericarp, and seed extracts. However, it is only detectable as Finally, the system 20:1:20:1 permitted the separation of gutti- traces in the leaves extract, where GC is the principal PPAP. ferones from other compounds. Even if the K of GA appears quite high in comparison with general CPC standards (0.5 < K < 2), it al- Cottet K et al. Isolation of Guttiferones… Planta Med 2015; 81: 1604–1608 1606 Original Papers Fig. 3 HPLC chromatograms of the five extracts at 254 nm. Plant parts Pericarps Seeds Latex Flowers Leaves Table 1 HPLC quantification of Quantification by HPLC 5.40% 3.30% 12.17% 1.80% 4.20 × 10−4 % GA in each plant part expressed as r² 0.94 0.90 0.88 0.91 0.96 a%(mGA/mdried mat). lows significant retention of all PPAPs in the stationary phase, as latex and pericarps, encouraging us to obtain gram quantities while almost all other apolar compounds come out. Meanwhile, of this compound for semi-synthesis and SAR studies [10,15]. K differences between these PPAPs were sufficient enough in this This led us to establish a scale-up procedure on the 1 L rotor. Up system to isolate GB, GA, and GC sequentially. to 12 g of extract were injected representing a threefold higher By using the system 20:1:20:1, we were able to perform a PPAPs concentration than those used for the 250 mL rotor. The quality selective isolation without overlap between products of interest, of the separation was similar and confirmed the feasibility and Downloaded by: Deakin University. Copyrighted material. with the exception of GD, which was eluted in a mixture with GC linearity of the scale-up procedure and the possibility to obtain (fractograms are available as Supporting Information). For the gram quantities of pure GA. isolated compounds, 1D and 2D NMR spectroscopy data and αD In conclusion, we have developed a CPC-based method leading to matched with data reported in the literature [11]. HPLC of these the fast, reliable, and scalable isolation of GA from S. globulifera compounds was performed in order to control purity; chromato- methanolic extracts. This technique could be applied for the iso- grams are represented in l" Fig. 4. lation and identification of other and eventually new PPAPs. l" Fig. 5 shows the results obtained by the CPC separation. The analysis of the seed extract confirmed the high concentration in different guttiferones.