Chemical Composition of the Oleogum Resin Essential Oils of Boswellia dalzielii from Burkina Faso

Frankincense, the oleogum resin from members of Boswellia, has been used as medicine and incense for thousands of years, and essential oils derived from frankincense are important articles of commerce today. A new source of frankincense resin, Boswellia dalzielii from West Africa has been presented as a new, alternative source of frankincense. In this work, the oleogum resins from 20 different Boswellia dalzielii trees growing in Burkina Faso, West Africa were collected. Hydrodistillation of the resins gave essential oils that were analyzed by GC-MS and GC-FID. The essential oils were dominated by α-pinene (21.0%–56.0%), followed by carvone (2.1%–5.4%) and α-copaene (1.8%–5.0%). Interestingly, there was one individual tree that, although rich in α-pinene (21.0%), also had a substantial concentration of myrcene (19.2%) and α-thujene (9.8%). In conclusion, the oleogum resin essential oil compositions of B. dalzielii, rich in α-pinene, are comparable in composition to other frankincense essential oils, including B. sacra, B. carteri, and B. frereana. Additionally, the differences in composition between samples from Burkina Faso and those from Nigeria are very slight. There is, however, a rare chemotype of B. dalzielii that is dominated by myrcene, found both in Burkina Faso as well as Nigeria.

Despite being one of the most wide-ranging species, the oleoresin essential oil of Boswellia dalzielii has only recently been examined [21]. Boswellia dalzielii (see Figure 1) inhabits wooded to open savannahs from Chad to Mali; the most significant populations appear to be in Burkina Faso, Nigeria, and Mali. The trees are 4-13 m tall, typically with papery or scaly bark, fragrant white flowers, serrated compound leaves, and aromatic resin [22]. Although resin harvesting has only recently Although there have been a number of studies on the compounds in the bark, there has been little work on the volatile compounds in this species. Two studies have examined the hydrodistilled leaf essential oils of B. dalzielii; Kohoude et al. found that the oil was dominated by δ-3-carene (27.7%) and α-pinene (15.2%) with smaller amounts of p-cymene (9.5%), β-phellandrene (8.5%), isolongifolene (6.2%), and myrcene (5.7%) [29], while Kubmarawa et al. found oils dominated by α-pinene (45.7%) and α-terpinene (11.5%) [30]. Recent work by DeCarlo et al. provided the first study on B. dalzielii oleoresin essential oil, examining single-tree oleoresin samples from northern Nigeria [21]. They found that the majority of essential oils were dominated by α-pinene (21.7%-76.6%), some with lower levels of αthujene (2.0%-17.6%) and p-cymene (0.3%-15.6%); a second, much rarer chemotype was rich in myrcene (up to 35.2%), sometimes with a significant level of limonene (up to 32.9%). These samples were found to be rich in monterpenes but almost devoid of sesquiterpenes.
Along with Nigeria, Burkina Faso hosts one of the largest populations of B. dalzielii. The species is widespread across three of the four phytogeographic zones of the country (from 500 to 900 mm annual rainfall range). Populations are gregarious and colonize more often rocky hills and glacis. Mean density of trees in natural stands ranges between 7 and 10 trees per 1000 m 2 according to phytogeographic zones in Burkina Faso [31]. The regeneration is very poor due to human disturbances (bushfire, pasture, agriculture) and climate pejoration (drought, rain fluctuations) [32,33].
The prior work on B. dalzielii oleoresin essential oil from Nigeria found multiple chemotypes as well as intrachemotypic compositional variation [21], and several other Boswellia species are known to display multiple chemotypes [1,5,11,19] (unpublished results from our laboratory). Given the extensive geographical range of B. dalzielii, it's likely that additional chemical diversity is present beyond that captured in the study from Nigeria. Therefore, in this study, we examine the essential oils from oleoresins taken directly from individual trees in Burkina Faso to determine if additional chemical variation is present. Each oleoresin sample was hydrodistilled using the same apparatus (Clevenger) and analyzed by GC-MS and GC-FID by the same operators under the same conditions. Although there have been a number of studies on the compounds in the bark, there has been little work on the volatile compounds in this species. Two studies have examined the hydrodistilled leaf essential oils of B. dalzielii; Kohoude et al. found that the oil was dominated by δ-3-carene (27.7%) and α-pinene (15.2%) with smaller amounts of p-cymene (9.5%), β-phellandrene (8.5%), isolongifolene (6.2%), and myrcene (5.7%) [29], while Kubmarawa et al. found oils dominated by α-pinene (45.7%) and α-terpinene (11.5%) [30]. Recent work by DeCarlo et al. provided the first study on B. dalzielii oleoresin essential oil, examining single-tree oleoresin samples from northern Nigeria [21]. They found that the majority of essential oils were dominated by α-pinene (21.7%-76.6%), some with lower levels of α-thujene (2.0%-17.6%) and p-cymene (0.3%-15.6%); a second, much rarer chemotype was rich in myrcene (up to 35.2%), sometimes with a significant level of limonene (up to 32.9%). These samples were found to be rich in monterpenes but almost devoid of sesquiterpenes.
Along with Nigeria, Burkina Faso hosts one of the largest populations of B. dalzielii. The species is widespread across three of the four phytogeographic zones of the country (from 500 to 900 mm annual rainfall range). Populations are gregarious and colonize more often rocky hills and glacis. Mean density of trees in natural stands ranges between 7 and 10 trees per 1000 m 2 according to phytogeographic zones in Burkina Faso [31]. The regeneration is very poor due to human disturbances (bushfire, pasture, agriculture) and climate pejoration (drought, rain fluctuations) [32,33].
The prior work on B. dalzielii oleoresin essential oil from Nigeria found multiple chemotypes as well as intrachemotypic compositional variation [21], and several other Boswellia species are known to display multiple chemotypes [1,5,11,19] (unpublished results from our laboratory). Given the extensive geographical range of B. dalzielii, it's likely that additional chemical diversity is present beyond that captured in the study from Nigeria. Therefore, in this study, we examine the essential oils from oleoresins taken directly from individual trees in Burkina Faso to determine if additional chemical variation is present. Each oleoresin sample was hydrodistilled using the same apparatus (Clevenger) and analyzed by GC-MS and GC-FID by the same operators under the same conditions.
A hierarchical cluster analysis of the essential oil compositions revealed two major groups ( Figure 2): One dominated by high α-pinene, and one rich in myrcene. Although unusual, the myrcene sample is only moderately dissimilar to the predominant α-pinene samples; we therefore conclude that there is a single, α-pinene dominant chemotype with a rare subchemotype rich in myrcene. The chemical compositions do not appear to correlate with the geographical locations from which they were taken, either within central Burkina Faso or between central and western Burkina Faso.        tr  tr  ---tr  tr  tr  tr  1416 1417 -1579  1582  Caryophyllene oxide  ------tr  tr  -tr  1942 1944  tr  tr  ---tr  tr  tr  tr  2130 2138    1119  a RI: Retention Index determined in reference to a homologous series of n-alkanes on a ZB-5ms column. b RI: Retention Index from the databases. c Entries in boldface were used in the cluster analysis. d Percentages are average of three runs (±standard deviations). e tr = "trace" (<0.05%).

Discussion
The oleogum resin essential oil of Boswellia dalzielii has only been described once previously, to our knowledge, in oleoresins taken from trees in northern Nigeria [21]. Our results are largely consistent with the findings of that study: In both areas, the essential oils are most commonly dominated by α-pinene, with a small number of samples showing high levels of myrcene. Levels of α-pinene were generally higher in Nigeria (42.6%-72.1%) compared to Burkina Faso (21.0%-56.0%) and α-thujene and p-cymene were less prevalent in this study. Additionally, in contrast to the samples from Nigeria, the samples from this study were found to contain an appreciable percentage of sesquiterpenes, particularly α-copaene.
Further work will be necessary to determine if the essential oils from Burkina Faso versus Nigerian B. dalzielii oleoresins show differential biological activities. In general, the reason for the diversity of terpenes and especially the importance of minor constituents is not clear [42]. The differential profiles likely convey some ecological benefits, though, as even essential oils from the same species, showing only modest chemical differences, do vary significantly in the degree to which they inhibit different strains of microbial pathogens [34]. This may indicate that the variation is related to variation in pathogenic threats.
The myrcene-dominated resins are intriguing as they represent only a small number of samples and differ greatly from the dominant α-pinene chemotype in both Nigeria and Burkina Faso. Environmental conditions can influence the chemical composition of plant volatiles [43][44][45]; although there was no geographic pattern observed here with regard to chemical variation, the myrcene chemotype samples taken in Nigeria were from the same geographic location. However, biotic factors also play a role: in a study of the heartwood essential oil of Santalum insulare in the Marquesas Islands, different chemotypes were observed in trees only a few meters apart, implying differences based on genetics or possibly pathogen attack history [46]; our laboratory has also observed different oleoresin chemical compositions in Boswellia carteri trees only a few meters apart in Somaliland (unpublished results from our laboratory). Further work is thus necessary to elucidate the reasons for the observed chemotypic differences in B. dalzielii.

Collection of Oleogum Resins
Twenty Boswellia dalzielii oleoresin samples were collected directly from source trees in six locations across central and western Burkina Faso (Figure 3). Samples were collected during the dry season (February-March 2019), and each sample location was GPS tagged ( Table 2). All samples were taken from fresh resin exuding either naturally from tree branches or from wounds left by recent bark harvesting; consequently, age differences in the resin were not enough to significantly alter the resins chemical compositions. The trees were identified in the field by Anjanette DeCarlo and Stephen Johnson. A voucher specimen (Voucher number OUA6892) was deposited at the University of Ouagadougou herbarium and the identification confirmed by Amadé Ouédraogo, a botanist working there. chemical compositions. The trees were identified in the field by Anjanette DeCarlo and Stephen Johnson. A voucher specimen (Voucher number OUA6892) was deposited at the University of Ouagadougou herbarium and the identification confirmed by Amadé Ouédraogo, a botanist working there.

Hydrodistillation of Oleogum Resins
Hydrodistillations of the Boswellia dalzielii oleoresin samples were carried out in an all-glass Clevenger-type apparatus as previously described [21].

Hydrodistillation of Oleogum Resins
Hydrodistillations of the Boswellia dalzielii oleoresin samples were carried out in an all-glass Clevenger-type apparatus as previously described [21].

Gas-Chromatographic-Mass Spectral Analysis
Each of the B. dalzielii oleogum resin essential oils was analyzed by GC-MS as previously described [21]: Shimadzu GCMS-QP2010 Ultra (Shimadzu Scientific Instruments, Columbia, MD, USA), ZB-5ms capillary column (Phenomenex, Torrance, CA, USA). Identification of the essential oil components was based on their retention indices determined by reference to a homologous series of n-alkanes, and by comparison of their mass spectral fragmentation patterns with those reported in the literature [47], and our in-house library.

Gas Chromatographic-Flame Ionization Detection
Analysis of the B. dalzielii oleogum resin essential oils by GC-FID was carried out as previously described [21]: Shimadzu GC 2010 with flame ionization detector (Shimadzu Scientific Instruments, Columbia, MD, USA), ZB-5 capillary column (Phenomenex, Torrance, CA, USA). The percent compositions listed in Table 1 are averages from three separate runs of the essential oils were determined from peak areas and corrected using response factors for the different classes of chemical components [48].

Hierarchical Cluster Analysis
The chemical compositions of the B. dalzielii oleoresin essential oils were used in the hierarchical cluster analysis. The 20 essential oil compositions were treated as operational taxonomic units (OTUs), and the concentrations (percentages) of 23 major components were used to determine the chemical associations between these frankincense essential oils using agglomerative hierarchical cluster (AHC) analysis using XLSTAT Premium, version 2018.5.53172 (Addinsoft, Paris, France). Dissimilarity was determined using Euclidean distance, and clustering was defined using Ward's method.

Conclusions
Boswellia dalzielii oleoresin essential oils from Burkina Faso are similar to those from Nigeria in that they are generally dominated by α-pinene, but unlike the samples from Nigeria, those in the current study contained a significant percentage of sesquiterpenes. The reason for these differences is not clear, but it potentially points to different biological and ecological activity due to different pathogenic threats. A rare chemotype or subchemotype dominated by myrcene was observed both in this study and in samples from Nigeria; this is potentially also related to specific pathogenic threats that may exist in both locations, or possibly genetic factors. This species thus offers a promising option for future work to elucidate the drivers of intraspecific chemical variation.