Chemical composition and antimicrobial activity of Boswellia serrata oleo-gum-resin essential oil extracted by superheated steam

Abstract Oleo-gum-resin is a complex mixture of essential oils, polysaccharides, and resin acids. The objectives of the present study were to evaluate the variation in chemical components and antimicrobial activity of essential oils extracted by superheated steam at various temperatures. The optimum essential oil yield was obtained at the highest superheated steam temperature (210 °C). In total, twenty-one compounds were quantified by GC-MS with α-pinene as the major compound, followed by α-thujene, trans-verbenol, β-thujone, p-cymene, m-cymene, and sabinene. Antimicrobial activity was performed by disc diffusion, resazurin microtitre-plate and micro-dilution broth susceptibility assays in which essential oil extracted at 150 °C and 180 °C revealed the highest antibacterial and antifungal activity, respectively. It is concluded that superheated steam is an effective method for the isolation of essential oil from oleo-gum-resin that improves the recovery of essential oil as well as antimicrobial activity. Graphical Abstract


Introduction
Boswellia serrata Roxb. is an oleo-gum-resin producing plant, found in dry areas of Pakistan, India, Oman, Yemen, Saudi Arabia, and Nigeria. There are forty-three different species in the genus the Boswellia, among these species B. serrata has commercial importance due to its industrial and pharmacological properties. Essential oil (EO) is the commercially important, lower molecular weight, volatile, secondary metabolites of aromatic plants and has been used in food, flavor, and perfume industries (Mothana 2012;Soumya et al. 2019). In traditional Unani and Ayurveda medicinal systems, oleo-gum-resin and EO have been utilized for the treatment of cough, cold, asthma, stomach, dysentery, diarrhea, and pulmonary diseases (Aman and Balu 2009;Manjunath et al. 2018). It has been reported that Boswellia species EO and isolated compounds exhibit strong immunomodulatory, antibacterial, anti-inflammatory, cytotoxicity, anticancer activity and several other important pharmacological activities (Shamraiz et al. 2020;Bini Araba et al. 2021;Tegasne et al. 2021;Wang et al. 2021). Oleo-gum-resins are plant excretions made up of polysaccharides, EO, and resin acids (Sarup et al. 2015). Steam distillation, supercritical fluid CO 2 extraction, and hydrodistillation techniques have been frequently used for the isolation of EO from oleo-gumresins (Ayub et al. 2018). These techniques have some major disadvantages such as high fuel consumption, equipment cost, low extraction efficiency, oxidation, and degradation of compounds (Zeng et al. 2016;Ayub et al. 2018). Superheated steam is a type of steam that has a temperature higher than normal steam. It is obtained by providing extra heat to the normal steam. It carries a lot of usable thermodynamic energy in the form of the latent heat of vaporization and comes with several advantages such as high temperatures at normal pressure, extremely high thermal conductivity, high extraction capability and low oxygen conditions (preventing oxidation of components being extracted). Previously, superheated steam has been used for drying and baking food products (Barbieri et al. 2004;Rouatbi et al. 2007). A keen review of already published literature reports show that superheated steam is the first time being used for isolation of EO from oleo-gum-resins. The main objectives of the present study were to study the effect of superheated steam temperatures on yield, chemical composition, and antimicrobial activity of B. serrata oleo-gum-resins EO.

Results and discussion
The yield of essential oils (EOs) extracted from B. serrata oleo-gum-resin by superheated steam at different temperatures are given in Figure S1. It was observed that the yield of EO increased with increasing the steam temperature. The highest EO yield (10.7%) was obtained at 210 C, while the lowest EO yield (8.5%) was obtained at 120 C. The higher energy contents and more penetration power of superheated steam at higher temperature may enhance the vaporization of EO from oleo-gum-resin which ultimately increased the recovery of EO. The results of the chemical composition of EOs extracted by superheated steam distillation at different temperatures are given in supplementary material Table S1. Overall, twenty one compounds were quantified with a-pinene (89.07 to 77.95%) as the main compound, followed by a-thujene (4.49 to 3.72%), trans-verbenol (2.48 to 0.68%), b-thujone (2.21 to 0.86%), p-cymene (2.11 to 0.93%), m-cymene (1.89 to 0.89%) and sabinene (1.66 to 0.93%). The results are in good agreement with the previous study in which B. serrata oleo-gum-resin EO isolated by supercritical fluid CO 2 reveals a-pinene (76.63%) as a major compound, followed by a-thujene (2.06%), verbenene (1.48%), thujone (1.12%), trans-Pinocarveo (5.31%) and Terpinene-4-ol (2.28%) (Ayub et al. 2018). The chemical composition of the present study is in good agreement with already published literature report in which Boswellia serrata oleo-gum-resin EO extracted by steam distillation from Indian origin contained a-pinene as a major compound, followed by a-thujene (Verghese et al. 1987). In another report, a-pinene was found as a major component of Boswellia serrata bark EO (Kasali et al. 2002). Monoterpenes (90.30 to 97.11%) and oxygenated monoterpenes (2.87 to 9.06%) were the major classes of terpenes present in B. serrata oleo-gum-resin EO. In monoterpenes, a-pinene and a-thujene were the major components, while the b-thujone and trans-verbenol were the major oxygenated monoterpenes. The components that varied with superheated steam temperatures were a-pinene (highest 89.07% in superheated steam EO of 120 C temperature), camphene, sabinene, b-pinene, m-cymene (highest in 150 C temperature EO: 1.48, 1.66, 1.11 and 1.89%, respectively), a-thujene, p-cymene, b-thujone (highest 180 C temperature EO: 4.64, 2.11, and 2.21% respectively) linalool, trans-verbenol and isopinocamphene (0.64, 3.48, and 0.39% respectively, highest in 210 C temperature superheated steam EO). In short, the superheated steam extraction temperatures significantly improved the EO yield and also affected the chemical components of B. serrata ole-gum-resin EO. It has been reported that the high extraction temperature may cause degradation and artifacts formation in EOs (Venditti 2020). Notably, Low extraction temperature (120 C) was favorable for higher percentage of major component (a-pinene) and high temperature (210 C) was more suitable for oxygenated monoterpenes (linolool, trans-verbenol, Isopinocamphone and myrtenol). Antimicrobial activity results of EOs against four pathogenic bacterial and fungal strains are given in supplementary material Table  S2. The inhibition zone and MICs values of EOs extracted at different superheated steam temperatures were in range of 12.08 ± 0.03 to 28.80 ± 0.06 mm and 28.14 ± 1.72 to 281.46 ± 7.02 mg/mL, respectively, and the values for position controls (Amoxicillin and Fluconazole) were 21.11 ± 0.02 to 28.53 ± 0.05 mm and 2.92 ± 0.84 to 13.33 ± 2.17 mg/mL, respectively. It was noticed that superheated steam EO of 150 C revealed higher antibacterial potential with greater inhibition zones (21.54 ± 0.07 to 18.70 ± 0.09 mm) and smaller MIC values (84.44 ± 1.68 to 112.58 ± 1.98 mg/mL), while the EO extracted at 210 C revealed least antibacterial activity with smaller inhibition zones (11.47 ± 0.07 to 20.27 ± 0.02 mm) and larger MIC values (77.23 ± 1.54 to 281.46 ± 5.62 mg/mL). Notably, EOs exhibited higher antibacterial activity with smaller MIC values (63.33 ± 1.26 to 281.46 ± 5.62 mg/mL), larger inhibition zones (11.47 ± 0.07 to 21.54 ± 0.07 mm) against gram negative bacterial strains (P. multocida and E. coli) and lower antibacterial activity with higher MIC values (105.14 ± 1.98 to 281.47 ± 2.76 mg/ mL) and smaller inhibition zones (11.25 ± 0.02 to 15.35 ± 0.09 mm) against gram positive bacterial strains (S. aureus and B. subtilis).
The EO samples exhibited the highest antifungal activity against Fusarium solani with large inhibition zones (20.00 ± 0.05 to 28.80 ± 0.06 mm) and the smallest MIC values (28.14 ± 1.72 to 84.44 ± 2.48 mg/mL), while the EOs were least effective against Aspergillus niger with smaller inhibition zones (12.08 ± 0.03 to 17.80 ± 0.05 mm) and larger MIC values (112.58 ± 3.82 to 281.46 ± 7.02 mg/mL). Some previous studies show that B. serrata oleo-gum-resin EO isolated through normal steam distillation and hydrodistillation method exhibited excellent antimicrobial potential against different bacterial and fungal strains (Camarda et al. 2007;Gupta et al. 2017). EO is a complex mixture of up to 45 volatile compounds. Oxygenated hydrocarbons like alcohol, phenolic terpenes and some of the hydrocarbons showed antimicrobial activity (Bassol e and Juliani 2012) .The difference in antibacterial activity of EOs might be attributed to variations in chemical components of EOs. Previously, it has been reported that the extraction methods and conditions may affect the chemical components and antibacterial activity of EOs (Gli si c et al. 2007;Kokoska et al. 2008;Okoh et al. 2010). Essential oil is a complex mixture of several volatile compounds. Mostly, oxygenated hydrocarbons like alcohol, phenolic terpenes and some of the hydrocarbons show antimicrobial activity (Bassol e and Juliani 2012). The difference in antimicrobial activity of EOs might be attributed to variations in chemical components of EOs. Previously, it has been reported that the extraction methods and conditions may affect the chemical components and antibacterial activity of EOs (Gli si c et al. 2007;Kokoska et al. 2008;Okoh et al. 2010). GC-MS results showed the higher concentration of camphene, sabinene, b-pinene and m-cymene in superheated steam distilled EO at 150 C and a-thujene, pcymene, b-thujone in EO obtained at 180 C. These compounds might attribute for maximum antimicrobial activity of these oils (Supplementary material Table S1). This statement is further strengthened by already reported studies in which camphene, sabinene, b-pinene, a-thujene, p-cymene, b-thujone and m-cymene showed antimicrobial activity. It has been reported that sabinene, b-pinene showed dose-dependent antibacterial activity against drug-resistant bacterial strains (Utegenova et al. 2018). Similarly, in another study, camphene exhibited antimicrobial activity against different bacterial as well as yeast strains (Alma et al. 2004). m-cymene significantly inhibited the growth of gram-positive, gram-negative, food born and pathogenic bacterial as well as fungal strains (Kedia et al. 2014;Yang et al. 2014;Marchese et al. 2017). It has also been reported that a-thujene and p-cymene showed excellent antibacterial as well as antifungal activity against normal and drug-resistant acne, nail and scalp infection-causing pathogens (Sadhasivam et al. 2016). The least antibacterial activity of 210 C EO might be attributed to decomposition of major component a-pinene. It has been reported that the high temperature caused auto-oxidation of a-pinene and formation of myrtenol, verbenone and verbenol (Neuenschwander et al. 2010;Liu et al. 2021). The similar findings are observed in the present study that shows the gradual decrease in the concentration of a-pinene with increasing temperature and increased concentration of myrtenol, d-verbenone and trans-verbenol (Supplementary material  Table S1).

Experimental
See supplementary material.

Conclusions
It can be concluded from the result of the present study that the superheated steam temperatures significantly affected yield, chemical composition and antimicrobial activities of B. serrata oleo-gum-resin EO. Superheated steam EO extracted at 210 C temperature exhibited highest EO yield. In antimicrobial assays, superheated steam EO extracted at 150 C temperature showed optimum antibacterial activity and EO extracted at 180 C temperature revealed highest antifungal activity. The higher concentration of camphene, sabinene, b-pinene and m-cymene may be responsible for enhanced antimicrobial activity.

Disclosure statement
No potential conflict of interest was reported by the authors.