Separation of neutral and acidic triterpenes from Mastic gum using Centrifugal Partition Chromatography (CPC) and Supercritical Fluid Chromatography (SFC-CO2)

M Hamzaoui; A Angelis; V Laskari; A Termentzi; J Hubert; N Fokialakis; N Aligiannis; JH Renault; AL Skaltsounis

doi:10.1055/s-0035-1565686

Separation of neutral and acidic triterpenes from Mastic gum using Centrifugal Partition Chromatography (CPC) and Supercritical Fluid Chromatography (SFC-CO2)

M Hamzaoui ¹, A Angelis ², V Laskari ¹, A Termentzi ¹, J Hubert ², N Fokialakis ¹, N Aligiannis ¹, JH Renault ², AL Skaltsounis ¹

¹Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, University of Athens, Panepistimiopolis Zografou, Athens, Greece
²UMR CNRS 7312, Université de Reims Champagne-Ardenne, Bât. 18, Moulin de la Housse, BP 1039, Cedex 2, 51687, Reims, France

Abstract

Pistacia lentiscus L. var. chia is a native shrub in southern Chios (Greece) and is cultivated for the production of Mastic gum, a natural resin obtained after “hurting” the trunk and branches. This resin is collected for more than 2,500 years and is used in traditional medicine for various gastrointestinal disorders such as gastralgia, dyspepsia, and peptic ulcer [1]. Moreover this natural resin is used in perfumery, dentistry and the production of chewing gum, as well as a spice and flavoring in Mediterranean cuisine. The therapeutic properties of mastic gum are due to their bioactive triterpenes that constitute about 75% of the resin [2]. The present work aims to develop a rapid and effective process for the fractionation of mastic gum leading to the isolation of the main triterpenes (neutral and acidic) in pure form. The first step of the procedure was the separation of triterpenic fraction from the polymeric part by liquid-liquid extraction method. The obtained triterpenic fraction was then elaborated by centrifugal partition chromatography (CPC). Initially the CPC technique was used in the pH-zone refining displacement mode. 7 g of the initial crude extract without polymer were successfully separated into fractions selectively enriched inionizable triterpenes isomers (masticadienolic acid and isomasticadienolic acid, masticadienonic acid and isomasticadienonic acid, oleanonic acid and moronic acid). Neutral triterpenes were, subsequently fractionated by step-gradient elution CPC method resulting to a good separation of the major constituents (oleanolic aldehyde, tirucallol, dammaradienone). In order to isolate pure isomers in sufficient quantities from the enriched acidic triterpenes CPC fractions, an original preparative technique, Supercritical fluid Chromatography SFC-CO₂, was used. The method was successfully developed to separate the major acidic triterpenes including the production of pure isomers using a chiral column. Identification and structure elucidation of the isolated compounds was achieved by LC-HRMS, GC-MS and NMR analysis. Since that mastic gum was recently added to the European Union's products with a Protected Designation of Origin (PDO) all these isolated compounds could be used for the chemical profiling and quality control of this row material and their products.

Acknowledgement: This work was supported by a Marie Curie Industry-Academia Partnerships and Pathways (IAPP) Fellowship within the 7th European Community Framework (286287).

References:

[1] Al Said M, Ageel AM, Parmar NS, Tariq M. Evaluation of mastic, a crude drug obtained from Pistacia lentiscus for gastric and duodenal anti-ulcer activity. J Ethnopharmacol 1986; 15: 271 – 278

[2] Paraschos S, Magiatis P, Mitaku S, Petraki K, Kaliaropoulos A, Maragoudakis P et al. In vitro and in vivo activity of chios mastic gum extracts and constituents against Helicobacter pylori. Antimicrob Agents Ch 2007; 51: 551 – 559