**3. Discussion**

Data presented in Table 2 clearly indicate that there are major di fferences in the composition of the essential oils of three studied cultivars. All three grades of resin sample gave the highest value for α-pinene, similar findings reported by already mentioned authors [11] and a comparable range for α-pinene was noted in di fferent fractions of the essential oil of *B. sacra* [13]. The percentage of monoterpenes and sesquiterpenes reported for the Grade 1 essential oil of Najdi showed similar values as reported for *Boswellia serrata* [14]. However, there is a major di fference in the values recorded for α-pinene in this study, which was 79.59%, while only 5.3% was reported for *B. serrata* [15].

The increased time of extraction through hydrodistillation in three cultivars has resulted in a reduction of monoterpenes, while at the same time the content in % of sesquiterpenes is increased. Moreover, on the basis of data collected, it can be stated that the same species of plants behave di fferently in terms of the yield of active components of essential oils under di fferent environments. The Najdi sample showed a higher percentage for monoterpenes in all three grades compared to the other two frankincense samples.

Concerning the antimicrobial activity, it was interesting to note that both *S. aureus* and *P. aeruginosa* tested strains were susceptible to most of the essential oils obtained from the three different cultivars. As there are not many antimicrobials concurrently active against the above-mentioned pathogens, these results are relevant because there is a pressing demand for effective antimicrobials towards these microorganisms present on the WHO/OMS priority bacteria list. Therefore, tested essential oils could be a source for new anti-staphylococcal and anti-pseudomonal molecules. Grade 2 of Sahli showed a MIC at 0.264 mg/mL for *P. acnes*, an important pathogen of dermatologic relevance. The activity could be explained by a higher concentration of sesquiterpenes in the extracts obtained by subsequent hydrodistillation of 4 h.

A previous article on four essential oils from different *Boswellia* species showed a significant activity against *Candida tropicalis* and *C. albicans* [16]. In our study we found that all tested samples showed an interesting antifungal activity against *C. albicans* and *M. furfur* with MIC values ranging from 27.28 to 0.240 mg/mL. A high percentage of α-pinene in all Grade 1 essential oils (and in general in monoterpenes) could explain the activity against tested fungal pathogens. Our findings could enable the use of frankincense oil blends in many fields, such as starting products for dermatological bacterial and fungal infections treatments.

In conclusion, GC-MS analysis of *B. sacra* essential oil indicates α-pinene as its primary component. α-Pinene has been found to be one of the integral components of many other antimicrobial essential oils, and thus can be inferred as one of the main players in the antimicrobial activity of *B. sacra* essential oils. Indeed, many of the monoterpenoids putatively identified in our study have been previously reported to have a potent broad antimicrobial spectrum activity. Moreover, the occurrence of drug resistance towards an essential oil in a microorganism is very rare because of its multicomponent nature that necessitates modification of numerous targets [17].

#### **4. Materials and Methods**

## *4.1. Sample Collection*

The different *B. sacra* gum resins were purchased from local market, collected over a period from March to May 2016, and compared with authenticated samples preserved in the Herbarium Center of Nizwa University, Oman, under Voucher number UC29.

The sample named Najdi was obtained from a plateau behind the Dhofar Mountains, where a rapid decline in rainfall and moisture was noted in this region. At the same time, temperature variances rise and desert weather changes the climate of the plain, including the southern slopes. Though annual rainfall in the Jebel region ranges from 500–750 mm, precipitation in the Nejd is recorded only in traces. The sample named Sahli was obtained from valleys (Shabi) of the Dhofar region. The normal annual precipitation is around 110 mm but can be from 70 to 360 mm. July to August is usually the rainy period. The sample Houjri is from Jebel Samhan of the Dhofar region. The Jebel hill range forms a distinct agro-climatic region. Thick fogs are detained back through ranges from inner desert areas. Precipitation is predominantly high and it can be from 600 to 700 mm, the highest in the country, which supports an enduring flora cover.

#### *4.2. Extraction of Essential Oils*

The essential oils from different cultivars of *B. sacra* were obtained through a hydrodistillation method using Clevenger-type apparatus. A total of 250 g of finely grounded frankincense oleogum resin was added to 2500 mL distilled water in a 5000 mL bottom flask placed in a heating mantle. Hydrodistillation was performed under atmospheric pressure at boiling temperature (about 100 ◦C) using a closed steam jacket. Following two hours of hydrodistillation, a Grade 1 essential oil of each oleogum resin was collected, whereas Grade 2 and Grade 3 essential oils were collected after 4 and 6 h of successive distillation of the same resin sample. Therefore, a total of nine gum resin essential oils

were obtained. The resulted essential oils were collected, dried with anhydrous sodium sulfate, and kept in a dark glass bottle at 4◦ C until analyzed by gas chromatography–mass spectrometry (GC-MS).

#### *4.3. GC*/*MS Analysis*

GC/MS analysis of obtained essential oils was carried out by using a Thermo Scientific DSQ II single quadrupole system of an EI (electron ionization) type, employed in full scan. The temperatures of the injector and ion source were 265 ◦C and 260 ◦C, respectively. The capillary column was a ZB-WAX (30 m x 0.25 mm i.e., film breadth 0.25 μm, (Phenomenex, Italy)). The temperature of the oven was set and the column temperature started at 50 ◦C, was raised at 3 ◦C/min to 250 ◦C, and after that a second gradient was used to 300 ◦C at 40 ◦C per minute, which was then seized for 3 min under isothermal conditions. The flow rate of helium, the carrier gas, was 1 mL/min. A sample of 1μL was inoculated with a split ratio of 1:100. The temperature of the ion source was 260 ◦C, the temperature of the MSn transfer line was 265 ◦C and the temperature of the injector was 270 ◦C. Ionization voltage was 70 eV and the mass range scanned was 35–550 *<sup>m</sup>*/*<sup>z</sup>*. Each oil component was identified on the basis of its retention index, relative to C8-C36 n-alkanes, from MS library searches using the NIST and Wiley GC-MS databases, and by comparison with mass spectral data in the literature.

The percent composition of the essential oil was computed by a standardization process from the GC peak areas, supposing identical mass response factor for all components. Triplicate analyses were prepared for each oil sample.

#### *4.4. Antimicrobial Activity, Minimum Inhibitory Concentrations (MICs) Determination*

The MICs of essential oils for seven reference bacterial strains (two strains of *Staphylococcus aureus* (ATCC 25923; ATCC 6538), two reference strains of *Pseudomonas aeruginosa* (ATCC 15442; ATCC 9027) and *Staphylococcus epidermidis* ATCC 12228, *Staphylococcus hominis* ATCC 27844, and *Propionibacterium acnes* ATCC 11827) were defined by means of an already described technique for a dilution antimicrobial susceptibility test for bacteria that grow aerobically (CLSI), with few amendments [18]. Concisely, 0.1 mL of essential oil was mixed with 0.1mL of culture medium (Mueller Hinton broth, Sigma-Aldrich, Milan, Italy) present in a well of a sterile 96-wells plate, and a 1:2 dilution series with broth medium was performed. A growth control and a negative sterile well control was included for each plate. A bacterial suspension (10 μL) from a suspension in NaCl 0.9% of isolated clusters, preferred from a 18 to 24 h agar plate and attuned to ge<sup>t</sup> a turbidity equivalent to a 0.5 McFarland standard, was added into the wells and the final test concentration of bacteria was approximately 5 × 10<sup>6</sup> CFU/mL bacterial. The 96-well plates were incubated at 37 ◦C for 24 h and MICs were read by a microplate spectrophotometer (GloMax®-Multi Detection System, Promega Italia s.r.l, Milan, Italy) as the lowest concentration of the sample that fully suppresses bacterial growth in the microdilution wells and whose optical density (OD) at 570 nm is comparable to OD values of negative control wells (only medium, without inoculum).

Antifungal activity against *Candida albicans* (ATCC 10231) and *Malassezia furfur* (ATCC 14521) were evaluated in terms of MICs by using a similar micro-method but by employing Sabouraud broth (Sigma-Aldrich, Milan, Italy) or Sabouraud broth with added 2% olive oil in the case of *M. furfur*, as growth media, and with an incubation time of 48 h.
