Elsevier

Phytochemistry

Volume 66, Issue 12, June 2005, Pages 1499-1514
Phytochemistry

A chemical investigation by headspace SPME and GC–MS of volatile and semi-volatile terpenes in various olibanum samples

https://doi.org/10.1016/j.phytochem.2005.04.025Get rights and content

Abstract

Six different olibanum samples with certified botanical origin were analyzed by headspace SPME–GC/MS in order to define their mono-, sesqui- and diterpenic composition, as pertinent criteria of identification. Boswellia carteri and Boswellia sacra olibanum have quite similar chemical composition, with isoincensole acetate as the main diterpenic biomarker. Although Boswellia serrata olibanum also exhibits this biomarker, the presence of methylchavicol, methyleugenol and an unidentified oxygenated sesquiterpene distinguishes B. serrata olibanum from the two other species. The characteristic chemical compounds of Boswellia papyrifera are the diterpenic biomarkers incensole and its oxide and acetate derivatives, n-octanol and n-octyl acetate. Boswellia frereana olibanum is devoid of diterpenes of the incensole family but contains a high amount of many dimers of α-phellandrene. The chemical composition of olibanum, which is demonstrated to be different for each Boswellia species allowed the determination of the taxonomic origin of frankincense samples purchased on various markets in East Africa, in the Near East and in Yemen. Moreover, terpenic fingerprints allowed the botanical origin of olibanum used in traditional incense mixtures to be identified. Furthermore, this study gave us the opportunity to assign a botanical origin to an archaeological frankincense sample.

Graphical abstract

Six different olibanum samples with certified botanical origin (two Boswellia carteri, and Boswellia sacra, Boswellia frereana, Boswellia papyrifera, Boswellia serrata) were analyzed by headspace SPME–GC/MS in order to define their mono-, sesqui- and diterpenic composition, as pertinent criteria of identification. The chemical composition of olibanum, which is demonstrated to be different for each Boswellia species allowed the determination of the taxonomic origin of frankincense samples purchased on various markets, of olibanum used in traditional incense mixtures and of an archaeological frankincense sample.

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Introduction

One of the research axes of our laboratory is the study of mummification balms and unguents of the Ancient Egypt. According to ancient papyrus and hieroglyphs on the walls inside temples, the recipe of these balms is elaborate and needs many constituents with their own ritual significance: vegetable oils, animal fats, pitches, waxes, honey, gums, resins, gum resins (Tchapla et al., 1999). Among the gum resins, olibanum (also called frankincense), used alone or in mixture, represents the universe of the Punt country from where the Gods of Egypt originate. Egyptologists think that olibanum as well as myrrh, another precious gum resin, was essentially used either in perfumes, or in fumigation during embalming ceremonies or possibly as a component of mummification balms for Pharaoh, persons of rank, or priests (at least from the 18th Dynasty). Incense burners have been found in graves since the Fifth Dynasty (Lucas and Harris, 1989). As incense was a generic term in Ancient Egypt, and frankincense could be obtained from different Boswellia trees (Baum, 1999), it is of fundamental importance to develop an analytical method in order to discriminate olibanum from other substances used as incense and to identify its botanical origin.

Our aim was to determine the possible occurrence of olibanum in mummification balms and unguents by headspace SPME. Among the methods of sample pre-treatment for GC–MS analysis, headspace SPME offers certain advantages when dealing with archaeological samples. Firstly, it is a sample non-destructive method. Secondly, it concentrates volatile compounds allowing their detection even at trace level and so requires only small amount of sample. Thirdly, non-volatile compounds such as fats, waxes and polysaccharides, often dominant in mummification balms, are not extracted by such a method, thus avoiding the fastidious sample pre-treatment required for classical GC–MS analysis. Previously, we have successfully applied headspace SPME to a wide range of resins and gum resins and to archaeological samples (Hamm et al., 2004).

Olibanum, is a natural oleo-gum-resin that exudes from tappings in the bark of Boswellia trees. The genus Boswellia of the Burseraceae family includes approximately 23 species of small trees that grow mainly in Arabia, on the eastern coast of Africa and in India. Frankincense is a complex mixture composed of about 5–9% highly aromatic essential oil (mono- and sesquiterpenes), 65–85% alcohol-soluble resins (diterpenes, triterpenes), and the remaining water-soluble gums (polysaccharides) (Tucker, 1986, Khan and Farooqi, 1991). Mono- and sesquiterpenes are highly volatile compounds, diterpenes exhibit low volatility, triterpenes very low volatility and polysaccharides are not volatile. Mono- and sesquiterpenes can be easily trapped by headspace SPME, but they are rarely characteristic of olibanum, because of their frequent occurrence in the vegetable kingdom. However, considering compounds with low volatility, conclusions could be very different. Diterpenes like incensole or isoincensole, their oxide or acetate derivatives and triterpenes like the boswellic acids can be considered as biomarkers of olibanum. Because headspace SPME is not relevant to the analysis of compounds of very low volatility, such as triterpenes, the present work focuses on the semi-volatile diterpene biomarkers. A previous paper presents the optimization of experimental parameters for headspace SPME of the diterpenes of a commercial olibanum sample (Hamm et al., 2003). Several publications deal with the GC or GC–MS analysis of volatile components of olibanum extracts or essential oils (Baser et al., 2003, Basar et al., 2001, Baratta et al., 1998, Dekebo et al., 1999, Hayashi et al., 1998, Ammar et al., 1994, Lawrence, 1992, Chiavari et al., 1991, Ma et al., 1991, Vernin et al., 1990; etc.), but samples were often purchased from markets and so lacked certified botanical origin. Thus, their conclusions cannot be used for positive identification.

From an archaeological point of view, knowing the botanical origin of olibanum used in mummification balms would be key information, documenting traditional craftsmanship and possible trade routes from ancient Egypt. To fill this gap in knowledge, the present contribution focuses on possible ways of determining the botanical origin of olibanum samples, of both ancient and modern origin.

The first step of this work was to build a database of the volatile terpenes of each olibanum with certified botanical origin and to derive characteristic signatures associated with the botanical origin. A second step was to apply these signatures to define the species of samples purchased in markets from various countries, alone or mixed with other plant products. Finally, we tested our method on an archaeological frankincense sample, not only to find its botanical origin, but also to determine which compounds it could still contain after approximately 2000 years.

Section snippets

Olibanum of certified botanical origin

The main compounds detected by headspace SPME–GC/MS in the six olibanum samples of certified origin are collected in Table 1. We have verified that qualitative results obtained with headspace SPME were identical with those obtained when simply dissolving olibanum samples in dichloromethane, except for triterpenes.

The first observation is the similarity in chemical composition between the two Boswellia carteri samples and the sample of Boswellia sacra. For these three olibanum, we found α-pinene

Conclusion

The terpenic composition (particularly the diterpenic biomarkers) characteristic to five species of Boswellia (B. carteri, B. sacra, B. frereana, B. papyrifera and B. serrata) was successfully established by headspace SPME and GC–MS. The database developed during this study has allowed us to determine the botanical origin of olibanum samples purchased in souks in various countries as well as the botanical origin of frankincense used in traditional incense mixtures, and validates the use of

Samples

The six olibanum samples with certified botanical origin, i.e., B. carteri (Somalia), B. carteri (Aden), B. sacra (Oman), B. papyrifera (Ethiopia), B. serrata (India), B. frereana (Somalia), and Kyphi were purchased from Scents of Earth (Sun City, USA). The Kyphi is a re-creation by Scents of Earth of an ancient Egyptian recipe. The Greek incense was brought by A. Tchapla from a monastery at Mount Athos. It is a traditional incense, burned during religious ceremonies. Wolf society (15 rue de

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