Improved antimicrobial activities of Boswellia sacra essential oils nanoencapsulated into hydroxypropyl-beta-cyclodextrins

Natural antimicrobials have recently gained increasing interest over synthetic antimicrobials to overcome foodborne pathogens and food microbial contamination. Essential oils (EOs) obtained from Boswellia sacra resins (BO) were utilized for respiratory disorders, rheumatoid arthritis, malignant tumors, and viral infections. Like other EOs, the therapeutic potential of BO is hindered by its low solubility and bioavailability, poor stability, and high volatility. Several studies have shown excellent physicochemical properties and outstanding therapeutic capabilities of EOs encapsulated into various nanocarriers. This study extracted BO from B. sacra resins via hydrodistillation and encapsulated it into hydroxypropyl-beta-cyclodextrins (HPβCD) using the freeze-drying method. The developed inclusion complexes of BO (BO-ICs) had high encapsulation efficiency (96.79 ± 1.17%) and a polydispersity index of 0.1045 ± 0.0006. BO-ICs showed presumably spherical vesicles (38.5 to 59.9 nm) forming multiple agglomerations (136.9 to 336.8 nm), as determined by UHR-TEM. Also, the formation and stability of BO-ICs were investigated using DSC, FTIR, FE-SEM, UHR-TEM, 1H NMR, and 2D HNMR (NOESY). BO-ICs showed greater thermal stability (362.7 °C). Moreover, compared to free BO, a remarkable enhancement in the antimicrobial activities of BO-ICs was shown against three different bacteria: Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. BO-ICs displayed significant antibacterial activity against Pseudomonas aeruginosa with an MIC90 of 3.93 mg mL−1 and an MIC50 of 0.57 mg mL−1. Also, BO-ICs showed an increase in BO activity against Escherichia coli with an MIC95 of 3.97 mg mL−1, compared to free BO, which failed to show an MIC95. Additionally, BO-ICs showed a more significant activity against Staphylococcus aureus with an MIC95 of 3.92 mg mL−1. BO encapsulation showed significantly improved antimicrobial activities owing to the better stability, bioavailability, and penetration ability imparted by encapsulation into HPβCD.


Introduction
][3] Several industries in the European Union employ EOs in several sectors, including food (additives/avorings), pharmaceuticals, and perfumes.Also, individual compounds extracted from EOs were used in food avorings. 4,5Besides their promising antibacterial and other therapeutic properties, most EOs are identied as Generally Recognized as Safe (GRAS), making them highly important and interesting as food additives and preservatives. 6,7herefore, many commercial products utilize the antimicrobial activities of different EOs, such as using Melaleuca alternifolia EOs (extracted from tea tree oil) as antiseptics owing to their broad-spectrum antimicrobial activities. 8EOs of citrus, sage, and rosemary in the 'DMC Base Natural' product, commercially available in Spain, are used as food preservatives. 9ther EOs, not identied by the manufacturer, were used in the 'Protecta One' and 'Protecta Two' products commercially available in the US and used as food additives (identied as safe additives according to GRAS). 10 Finally, carvone, isolated from the EOs obtained from Caraway seeds, is used in the 'Talent' product (The Netherlands) as a sprout inhibitor for potatoes and for ghting several fungal strains and diseases of stored potatoes. 11oswellia sacra belongs to the Burseraceae family which mainly grows in Yemen, Oman, and Somalia. 12The resins of B. sacra have been utilized for several therapeutic purposes such as in chronic respiratory tract disorders, rheumatoid arthritis, malignant tumors, urinary tract disorders, and viral infections. 12,13The unique therapeutic properties of the B. sacra resins are mainly attributed to their EOs (BO), which were reported to have a rich content of different bioactive compounds such as polyphenols, terpenes, sesquiterpenes, and nonoxygenated terpenes. 14,156][17][18] The BO safety was established on normal (non-cancerous) cells such as normal breast cells (MCF10-2A) and human embryonic kidney cells (HEK-293). 15imilar to other EOs, the therapeutic properties of BO are hindered by their low solubility and bioavailability, poor stability, and high volatility.0][21][22] BO was encapsulated inside PLGA-PCL nanoparticles and showed greater antiproliferative and apoptotic efficiency against breast cancer. 15yclodextrins, a group of cyclic oligosaccharides, are composed of several glucopyranosyl units connected by a- (1,4)  linkages.The most widely used cyclodextrins are a-, b-, and gcyclodextrins.The number of glucopyranose units incorporated in the chemical structures of cyclodextrins is used to differentiate them. 23,24Owing to their unique structures, composed of a hydrophilic surface and a hydrophobic cavity, cyclodextrins can host various molecules to develop their corresponding inclusion complexes (ICs) eventually.The formation of ICs with cyclodextrins is reported to improve the bioavailability, stability, release prole, physicochemical characteristics, and therapeutic efficacy of the encapsulated molecules. 24,25The attachments of hydroxypropyl functional groups to the surfaces of b-cyclodextrins can develop hydroxypropyl-beta-cyclodextrins (HPbCD), well-established with greater safety and solubility. 26urthermore, the Food and Drug Administration (FDA) has approved using HPbCD as excipients in oral and intravenous solutions. 27,28Also, the FDA approved the use of a-cyclodextrins, b-cyclodextrins, and g-cyclodextrins as food additives in 2000-2004 and were classied as safe (GRAS). 29,30In Europe, the use of b-cyclodextrins, in particular, was approved as a food additive (E 459) with an acceptable daily intake of 5 mg kg −1 day −1 .The FDA's list of Inactive Pharmaceutical Ingredients includes sulfobutylether-b-cyclodextrins (SBE-bCDs) and HPbCD. 29,30yclodextrins can be found in a variety of pharmaceutical products due to their wide range of applications.For instance, they are employed in eye drops and aqueous parenteral solutions, nasal sprays, and tablets.In the European market, several medicinal uses of cyclodextrins and their derivatives are available, including the use of g-cyclodextrins in Minoxidil solutions, b-cyclodextrins in Cisapride suppositories and Cetirizine tablets, randomlymethylated-b-cyclodextrins in nasal sprays (for hormonal replacement therapy), HPbCD in Itraconazole, and SBE-bCDs in parenteral antimycotic Voriconazole.Infusion preparations comprising alprostadil (prostaglandin E1) with a-cyclodextrins are also available in Japanese and German markets. 29,302][33][34][35][36] Numerous studies have documented the successful encapsulation of various EOs into HPbCD, demonstrating exceptional antibacterial efficacy.For example, encapsulating EOs of guava leaves improved their antibacterial activity against Escherichia coli (E.coli) by 2 fold and against Staphylococcus aureus (S. aureus) by 4 fold. 37][40] Inspired by this context, this study sought the extraction of BO from B. sacra resins via hydrodistillation, formation of the ICs of BO with HPbCD (BO-ICs) by freeze-drying, characterization of BO-IC physicochemical properties, and the assessment of their antimicrobial activities against Pseudomonas aeruginosa (P.aeruginosa), E. coli, and S. aureus.S. aureus and E. coli have been commonly associated with food contamination and foodborne pathogens, 41 whereas P. aeruginosa could infrequently be linked to food illnesses although it represents an opportunistic bacterium that has been isolated from soil, vegetables, drinking water, and food resulting in serious illnesses. 42Also, targeting P. aeruginosa with conventional disinfectants and antiseptics is more challenging since it usually colonizes on common surfaces developing more resisting biolms. 43Hence, in light of the FDA-approval of cyclodextrins use as food excipients and their safety proles, [27][28][29][30] BO encapsulation into HPbCD can enhance their antimicrobial activity and might further endorse their use as food preservatives and/or in food-packaging for controlling food contamination and food-borne pathogens.

Microorganisms
The P. aeruginosa, E. coli, and S. aureus bacterial strains (ATCC numbers 27853, 25922, and 25923, respectively) were provided by Nawah Scientic Inc. (Cairo, Egypt).Bacteria were allowed to cultivate in nutrient broth and were incubated for 24 h at 37 °C.Bacteria were further maintained in glycerol (15% v/v), at −20 °C.

BO extraction from the resins of B. sacra
BO was extracted from the resins of B. sacra using hydrodistillation. 15Briey, the hydrodistillation process was carried out on the resins in an oven (Milestone ETHOS X, Milestone, Italy).The oven had been equipped with an IR temperature sensor and two magnetrons (950 W).The resins were rst soaked in distilled water at a 1 : 5 (w/v) ratio, and the mixture was heated to 100 °C.The mixture could be blended well with the help of an electromechanical agitator.Other parameters were set at 2.3 bar for the pressure applied and 8 °C for the cooling system utilized, using a chiller (Smart H150-2100 chiller, LabTech, MA).The chiller was connected to a condenser to ensure a constant cooling temperature of 8 °C provided within the condenser.Several extraction cycles were delivered, where each cycle could be set at a specic time interval (45 min).Finally, the obtained extract at the end of each cycle was collected, and its corresponding volume was recorded. 152.BO compositional analysis using GC-MS BO composition was investigated using GC-MS analysis (Agilent Technologies gas chromatography (7890B) coupled with a mass spectrometer detector (5977B)), as previously reported.15

BO encapsulation into HPbCD
BO encapsulation into HPbCD was conducted by the freezedrying technique to eventually develop their corresponding BO-ICs. 44,45Briey, an aqueous solution of HPbCD was rst prepared, adding 5 g of HPbCD to 25 mL of distilled water.Then, the prepared solution received 0.5 g of BO which was slowly added, and the nal mixture was placed on a stirrer in the dark (200 rpm for 24 h, at 24 °C) to allow the development of BO-ICs.Consequently, the unencapsulated particles were removed from the obtained suspension by ltering it using 0.45 mm PTFE lters.As a result, the resulting solution, which now includes the BO-ICs only, was le in a freezer for 18 h, at −20 °C, before being lyophilized using a freeze-dryer for 72 h, allowing the sublimation of all the moisture contained (TOPT-10C freezedryer, Toption Group Co. Ltd., Xi'an, China).Finally, the lyophilized powders were kept in sealed containers inside a desiccator.

BO and BO-IC physicochemical characterization and bioassays
3.4.1.Average particle size (Z-average) and polydispersity index (PDI) determination.Using a Zetasizer, the Z-average and PDI of BO-ICs and free HPbCD were determined (Nano-Zetasizer employing a dynamic light scattering DLS, Malvern Instruments Ltd, Malvern, UK).To accomplish this, BO-ICs and HPbCD were separately suspended in distilled water (w/v) (3 : 1 ratio) and samples were measured at room temperature. 46.4.2.Morphology of BO-ICs and free HPbCD.A Field Emission Scanning Electron Microscope (FESEM, LEO Supra 55, Zeiss Inc., Oberkochen, Germany) was used to investigate the morphological features of BO-ICs and HPbCD.Aluminum stubs were used to xate the powder samples of BO-ICs and HPbCD, separately.Consequently, a thin layer of gold was sputtered (10 mA for 8 min) to coat the samples, before being observed at a magnication of 500×. 39Moreover, the encapsulation of BO and the morphology of BO-IC particles were examined using an Ultra-High Resolution Transmission Electron Microscopy (UHR-TEM), at 200 kV accelerating voltage (JEOL, JEM-2100 Plus, Tokyo, Japan).For this purpose, small amounts of BO-ICs and HPbCD were prepared in distilled water to form their corresponding suspensions.Samples were then sonicated (37 °C and 10 min).Aerwards, droplets of the nal suspensions were positioned on copper grids, covered with a 1 nm carbon lm, stained with uranyl acetate (2%), and le to dry using lter paper.Consequently, the samples were observed under 5k and 25k magnications. 44.4.3.Fourier transform infrared spectroscopy (FTIR) analysis.Within a range of 4000-400 cm −1 , the FTIR spectra of BO, HPbCD, and BO-ICs were obtained, using a Nicolet 380 FTIR (Thermo Scientic, Madison, WI).The spectra were examined for the presence of the characteristic peaks and the chemical structures of the recorded components and to ensure the BO encapsulation into HPbCD.BO-ICs and HPbCD were initially mixed with KBr (1 : 100) and then compressed into small discs utilizing a hydraulic press (15T manual press machine, China).For BO, a drop of oil was applied onto a piece of KBr and placed in front of an IR beam.40 3.4.4. B-IC encapsulation efficiency (% EE) and drug loading capacity (% DL).Using a UV visible double beam spectrophotometer, the concentration of BO encapsulated into BO-ICs was determined spectrophotometrically at 254 nm (Cary 3500 UV-Vis Engine, Agilent Technologies, Pty Ltd, Mulgrave, Australia).Briey, BO-ICs (5 mg) were suspended in acetonitrile (5 mL) and subsequently le in the dark at 24 °C, inside sealed containers while being mixed constantly for 72 h to facilitate the transfer of the BO entrapped to the solution.A calibration curve was developed for the BO, under the same conditions, in a range of concentrations of 3.125 to 200 mg mL −1 (BO: y = 0.0025x -0.0053 and r 2 = 0.9994).The % EE and % DL were then determined using the following eqn (1) and ( 2 3.4.5.H NMR spectra analyses.An NMR spectrometer (400 MHz, BRUKER BioSpin GmbH, D-76287 Rheinstetten, Germany) was used at 25 °C to obtain the 1 H NMR spectra of HPbCD, BO, and BO-ICs.Also, the 2D-HNMR spectrum (NOESY) of the BO-ICs was obtained.Each sample was initially dissolved in DMSO. 31anoscale Advances Paper 3.4.6.Stability investigations.The thermal behaviors of HPbCD, BO, BO-ICs, and the physical mixture of BO and HPbCD (1 : 10) were investigated by analyzing their corresponding DSC curves obtained using a differential scanning calorimeter (model: DSC-60 plus, Shimadzu, Kyoto, Japan).Briey, a constant rate of heat ow (10 °C min −1 ) was introduced, starting at 28 °C till reaching the end of the heat cycle at approximately 400 °C, under a nitrogen atmosphere. 44.4.7.Antimicrobial activity.HPbCD, BO, and BO-IC antimicrobial activities were examined against E. coli, S. aureus, and P. aeruginosa bacteria.For this purpose, direct broth microdilution assay was performed using 96-well plates, following previous studies with slight modications. 31,47The bacteria were initially prepared, using 250 mL conical asks, and then cultivated at 37 °C for 24 h.The obtained bacterial suspensions were diluted to 10 5 CFU mL −1 .For each bacterial strain, several aliquots (10 mL) of the suspension were added to two different 96-well plates; each well contained 90 mL of Nutrient Broth in addition to different concentrations of BO and BO-ICs.The concentrations of BO-ICs ranged from 0.25 to 4.09 mg mL −1 .BO concentrations ranged from 0.34 to 5.4 mg mL −1 .It is worth noting that the BO was added to the plates as an aqueous microemulsion, and their corresponding BO-ICs were added as an aqueous suspension following their ltration using syringe lters (0.20 mm).For negative control wells, only BO and BO-ICs were added to the culture media.For positive control wells, microbial suspensions were added to the culture media without BO or BO-ICs.The microplates were then incubated for 18 h at 37 °C aer which the optical density was measured at 570 and 600 nm, utilizing a microtiter-plate reader (FLUOstar Omega, BMG Labtech, Ortenberg, Germany).8][49] Under the same conditions, a positive control of an antibiotic, ciprooxacin, was examined within a range of concentrations of 10 to 0.001 mg mL −1 against the same strains of bacteria.Measurements were carried out as three replicates.

Statistical analysis
The mean ± standard deviation was calculated for each measurement.Three duplicates of each formulation were prepared.A p value of less than (or equal to) 0.05 revealed statistically signicant differences.Also, one-way analysis of variances (ANOVA) was chosen to identify statistical differences.

Characterization of BO-ICs
The PDI and Z-average of HPbCD were 1.0 and 1.647 mm, respectively, reecting a signicantly polydisperse and heterogeneous system.The PDI index reects the uniformity of particle sizes in a given dispersion.A system is considered extremely polydisperse with a PDI value higher than 0.7. 51The BO-IC suspension, on the other hand, demonstrated improved stability with considerably smaller and less scattered particles.The PDI and Z-average values determined for the BO-ICs were 0.1045 ± 0.0006 and 448.6 ± 0.6244 nm, respectively.The strong tendency of HPbCD and ICs to aggregate in water, as a result of the cyclodextrins inclination to self-assemble in water, can explain the above results. 44It is worth noting that the complexes formed by cyclodextrins and their hydrophilic aggregates have the ability to dissolve lipophilic substances via developing micelle-like structures and complexation. 52][54] The morphology and size of HPbCD and BO-ICs were investigated using the FE-SEM images, presented in Fig. 1a and b, respectively.Along with some undamaged ovoid-shaped crystals, HPbCD exhibited varying-sized rectangular particles.Conversely, considerable morphological alterations in the crystal sizes and shapes of the BO-ICs were shown, and, more importantly, the BO-ICs showed a signicant decrease in the crystal sizes compared to free HPbCD.Zetasizer analysis further evidenced the change in the particle sizes.In addition, many agglomerations were exhibited by the BO-ICs which can be explained by the increased tendency of the larger particles to gather and attract the smaller ones.The formation of the aggregates of the smaller particles and the changes observed in their morphologies suggest a complexation establishment, in which an amorphous product merged with an additional compound in the complex was developed.Similar ndings were previously reported. 44,53oreover, the encapsulation of BO and the structural and morphological characteristics of both HPbCD and BO-ICs were investigated using the UHR-TEM images obtained (Fig. 2).HPbCD particles (Fig. 2a) were presented with round shaped vesicles with diameters ranging between 82.7 and 334.2 nm and frequently developing larger micellar structures (self-assembled structures).On the other hand, BO-ICs (Fig. 2b) showed presumably spherical vesicles with a diameter ranging from 38.5 to 59.9 nm and a thin layer membrane surrounding the BO.Also, BO particles showed clear evidence of agglomeration in which larger particles attract smaller ones.These agglomerations were shown with diameters ranging from 136.9 to 336.8 nm.These ndings refer to the positive establishment of BO-ICs and the successful encapsulation of BO into HPbCD.Nevertheless, the micellar structures revealed by both samples might be attributed to their preparations in distilled water, where cyclodextrins tend to self-assemble in water forming multiple agglomerates.Similar ndings were reported supporting current results. 44,46,55,56

FTIR analysis
The encapsulation and complexation formation of BO-ICs were assessed by obtaining the FTIR spectra of HPbCD, BO, and BO-ICs (Fig. 3).
The FTIR spectrum of BO showed absorption bands at 3408.2 cm −1 (O-H stretching), 2920.7 cm −1 (methylene group stretching vibration), 2873.For the BO-IC FTIR spectrum, apart from the shiing and narrowing of the O-H bands as well as some other band shis, all the BO absorption bands in the BO-IC FTIR spectrum were covered by the overlapping exerted by more intense bands of HPbCD.The encapsulation of some BO's compounds inside the HPbCD cavity and the possible interactions outside the HPbCD cavity may explain the prominent alteration presented in the O-H bands. 52The emergence of some inter-molecular hydrogen bonds between HPbCD and some BO components might indicate the above interactions. 40Furthermore, the broader band of the -CH 2 chemical group, shown at 2920 cm −1 , may indicate the successful entry of BO's lipophilic components into the HPbCD cavity. 40Consequently, these results show that BO was effectively encapsulated into HPbCD, making stable ICs. 31,39,40It is noteworthy that aer six months, a re-examination of the corresponding FTIR spectra of BO and BO-ICs has been conducted to assess the stability of their primary functional groups and chemical constituents.Remarkably, identical spectra were acquired, indicating the stability of the chemical composition of BO and BO-ICs.

BO-ICs encapsulation efficiency (% EE) and drug loading capacity (% DL)
The calculated % EE and % DL of BO-ICs were 96.79 ± 1.17% and 10.87 ± 0.12%, respectively.The lengthy complexation allowed during the preparation procedure, whilst maintaining the ICs solutions well protected and rmly sealed throughout the preparation and drying processes, could explain the high encapsulation efficiency of BO-ICs.It was previously observed that the complexation time and drying processes signicantly affect the proportion of the EOs encapsulated into HPbCD. 54Furthermore, several compounds detected in the BO, using the GC-MS analysis, have shown remarkable encapsulation affinities towards the

H NMR spectra analyses
1 H NMR was utilized to conrm the encapsulation and investigate the precise positioning of the BO components inside the hydrophobic cavity of HPbCD.Once BO (the guest molecule) approaches the inner side of the HPbCD's hydrophobic cavity, the proton atoms located at position 3 and position 5 (H3 and H5) at the HPbCD's inner cavity are chemically shied. 57he 1 H NMR spectra of BO, HPbCD, and BO-ICs are shown in Fig. 4. BO's spectrum revealed recognizable proton peaks at 7.33, 7.07, and 6.85 ppm, conrming the presence of aromatic rings inside the molecular structure of some components present in the BO.The presence of double bonds was also indicated by the noticeable proton peaks appearing at 6.35 and 6.12 ppm.Protons shown at 3.76 and 3.37 ppm indicate that the molecular composition of BO has a C-O-C structure.Another characteristic proton peak of a methylene or a methyl structure linked to a double bond was observed at 1.86 ppm.These ndings were in agreement with the analytical results obtained by the GC-MS analysis and FTIR.
Moreover, the BO-IC 1 H NMR spectrum showed the proton peaks of both HPbCD and BO (Fig. 4).Additionally, Fig. 4B and  C and Table 1 reveal the changes in the chemical shis of the protons associated with the D-glucopyranose units of the HPbCD molecules.H1 and H2 were slightly shied upeld (H1 is located in the middle structure of the hydrophobic cavity of HPbCD, whereas H2 is attached to the outside).Also, H6 was slightly shied downeld (H6 is attached to the outermost side of the hydrophobic cavity).In contrast, H4 (located outside the cavity) showed a stronger shi downeld.Finally, H3 and H5

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Nanoscale Advances could also show stronger shis upeld (H5 is located inside the depth of the HPbCD cavity, whereas H3 is closer to the wider edge).Based on these ndings, the higher upeld shis observed with H3 and H5 refer to the greater electron cloud densities developed around these protons.The greater electron cloud densities could have been induced by the stronger shielding exerted by the double bonds and the aromatic structures present in the BO, suggesting the effective development of the BO-ICs.On the other hand, 2D-HNMR NOESY (Nuclear Overhauser Effect Spectroscopy) was conducted on BO-ICs to investigate the inclusion mechanisms showing the entry of BO into the hydrophobic cavity of HPbCD.This method can detect possible cross correlations inuenced by the spatial proximity that could develop among the BO components (guest) and HPbCD (host).The establishment of the BO-ICs, as shown by NMR spectroscopy, can be claried most effectively by the Nuclear Overhauser Effect (NOE), which becomes more prominent by the spin polarization transfer, occurring between nearby atoms, moving from one population (HPbCD) to another (BO).Therefore, an interaction impacted by a couple of protons presented near each other, within 0.4 nm, in the space could be detected in a NOESY spectrum represented by the NOE cross correlation (crossed peaks). 58,59Hence, the obtained 2D HNMR NOESY

Nanoscale Advances Paper
spectrum of BO-ICs (Fig. 5) could reveal a substantial spatial proximity and correlation, between BO protons and HPbCD protons (H3 and H5).This correlation supports the chemical shis revealed in the 1 H NMR spectra and hence denotes the successful encapsulation of BO into the HPbCD cavity while effectively forming their BO-ICs.Similar results were reported previously showing the effective encapsulation of various molecules into HPbCD cavities while revealing similar strong correlations as could be shown by 2D NMR spectroscopy analyses. 60,61It is worth noting that the 2D-HNMR NOESY analysis was conducted ve months aer the initial preparation of BO-ICs, supporting their high stability prole.

Stability investigations
BO's physical characteristics were closely monitored over a period of 18 months following their extraction, including BO's color, viscosity, and aroma, while being kept in a sealed container in the dark at 4 °C.Interestingly, these characteristics have not changed over time, referring to the high stability of the extracted BO.Furthermore, in order to conrm the positive establishment and investigate the thermal stability and behavior of the BO-ICs, the DSC curves of HPbCD, BO, BO-ICs, and a BO-HPbCD physical mixture were obtained.Briey, the disappearance or shiing of the endothermic melting peak of BO (the guest  Paper Nanoscale Advances molecule) on the DSC thermogram of BO-ICs served as evidence of the successful formation of BO-ICs and effective encapsulation of BO into HPbCD. 44,62he DSC thermogram of HPbCD (Fig. 6) revealed two endothermic peaks.The rst peak appeared below 100 °C, indicating the vaporization of water molecules, whereas the second peak was shown at 343.8 °C, depicting the HPbCD melting.Furthermore, BO's DSC thermogram showed a sharp endothermic peak at 113.8 °C associated with the BO boiling point.
The DSC thermogram of the BO-ICs exhibited one endothermic peak below 100 °C, associated with the water molecules vaporization, in addition to two sharp endothermic peaks at 347.1 and 362.7 °C indicating the successive processes of melting of HPbCD followed by boiling of the released BO, respectively.Furthermore, the DSC thermogram obtained for the physical mixture of HPbCD and BO showed the melting peaks of HPbCD and BO at 351.2 and 134.0 °C, respectively.Hence, the disappearance of the endothermic peaks shown at 113.8 °C and at 134.0 °C in the BO and in the physical mixture's thermograms, respectively, from the BO-ICs thermogram refers to the successful encapsulation of the BO into HPbCD and the formation of BO-ICs.Therefore, investigating the BO-ICs thermogram might depict the following incidents associated with the thermal decomposition process.First, the relatively thermostable structure of HPbCD started to melt at around 330 °C, resulting in the release of the thermolabile components of BO and their rapid decomposition.These results verify the successful formation of the BO-ICs and superior thermal stability compared to free BO.Similar observations were previously reported. 44,45,62,63It is important to note that the DSC analysis of the BO-HPbCD physical mixture was carried out four months aer the initial preparation of BO-ICs.This may also support the high stability prole of BO, where the same BO boiling points were observed.
The non-hygroscopic, homogeneous, and crystalline structure of HPbCD coupled with a hydrophobic cavity and a hydrophilic surface can explain the greater stability proles developed upon their use to encapsulate other molecules, forming larger micellar/self-assembled structures known with superior characteristics (as revealed by both the UHR-TEM and FE-SEM analyses herein), encompassing molecules of various properties and polarities. 64,65These features further depict the signicant stability, therapeutic properties, and bioavailability of the encapsulated molecules.In addition to the molecules' encapsulation into the hydrophobic cavities of HPbCD, the possibility of other interactions on the surfaces of the HPbCD particles greatly supports the stable proles of the developed complexes as supported by stability and morphological Nanoscale Advances Paper ndings. 64,65In fact, previous reports showed that some drugs formed covalent linkages with the OH functional groups furnished by the cyclodextrins, increasing their stability, targeting ability, and sustained release of their cargo. 65

Antimicrobial activity investigation
The antibacterial activities of HPbCD, BO, BO-ICs, and cipro-oxacin were assessed against S. aureus, P. aeruginosa, and E. coli bacteria, and the obtained MIC values are shown in Table 2.
The abundance of monoterpenes and oxygenated monoterpene compounds in might be attributed to the potent antibacterial properties of BO.7][68] Additionally, the integration and disruption of the lipid bilayers of microbial cells would make them more vulnerable to being targeted by terpenoid compounds and their analogs, owing to their lipophilic nature.These properties enhance the permeability of the microbial cell membranes, impairing the essential cellular transport mechanisms leading to bacterial death. 66iprooxacin is a potent antibiotic which exerts a broad spectrum of activity against both Gram-positive and Gramnegative bacteria. 69ree HPbCD could not show any antibacterial activities (>100 mg mL −1 ) against the microorganisms tested, suggesting the absence of possible synergistic effects imparted by HPbCD particles.Also, free BO failed to reveal any antibacterial activity against P. aeruginosa within the range of concentrations examined.However, BO-ICs showed signicant antibacterial activity with an MIC90 of 3.93 mg mL −1 and an MIC50 of 0.57 mg mL −1 .Also, BO-ICs showed an increase in BO activity against E. coli with an MIC95 of 3.97 mg mL −1 , compared to free BO which failed to reveal an MIC95 value within the range of concentrations tested.Additionally, BO-ICs depicted a more signicant activity of BO against S. aureus with an MIC95 of 3.92 mg mL −1 , compared to free BO which failed to show an MIC95 value within the range of concentrations examined.
These ndings explicitly demonstrate the signicance of encapsulating BO into HPbCD, improving their penetration capability, solubility, stability, and ultimately their antibacterial activity at lower BO concentrations.Since EOs are well-established for exerting their antibacterial activity at the bacterial cell membranes and in the bacterial cytoplasm, the potential increase of the BO aqueous solubility upon their encapsulation into HPbCD may have improved their targeting of the active sites of bacteria. 46,70It has been documented that limonene, one of the main components detected in BO by GC-MS (ESI Fig. 1 and Table 1 †), exerts its antibacterial activity against E. coli by increasing the bacteria membrane permeability, as could be shown by Attenuated Total Reectance Infrared Micro-Spectroscopy. 713][74] Nevertheless, these active components in addition to others found in BO have been reported to face several challenges, like other EOs, where they have poor liquid solubility, limiting their diffusion in biological uids, coupled with the intrinsic nature of the high volatility of EOs, negatively impacting their stability in similar uids. 701][72] In summary, HPbCD, as an acclaimed drug delivery carrier, facilitated the access of BO's components to their active sites in the targeted bacteria while protecting their cargo from degradation/elimination and controlling their release, augmenting their stability, targeting ability, and therapeutic efficacy.

Conclusions
EOs derived from different plants have exhibited favorable antimicrobial, antitumor, and antioxidant properties.BO has demonstrated outstanding therapeutic properties mainly attributed to its abundant content of terpenoids and other bioactive phytochemicals.In this study, BO was extracted from B. sacra resins via hydrodistillation and then encapsulated into HPbCD by freeze-drying.The obtained BO-ICs exhibited high entrapment efficiency, stability, and antimicrobial activities.
The BO-ICs formation and stability were successfully veried using DSC, FTIR, FE-SEM, UHR-TEM, 1 H NMR, and 2D HNMR NOESY analyses.Ultimately, as compared to free BO, signicant improvement in the antibacterial activities of BO-ICs was demonstrated against E. coli, S. aureus, and P. aeruginosa, supporting the advantageous impacts of BO encapsulation into HPbCD to achieve high stability, bioavailability, penetration ability, and therapeutic effects.Therefore, these ndings, further augmented by the established safety proles of BO and HPbCD, support the promising utility of BO-ICs for controlling bacterial growth in food and for other cosmetic and therapeutic uses.

Fig. 1
Fig.1FE-SEM images of HPbCD (a) and BO-ICs (b).The HPbCD field exhibited varying-sized rectangular particles with some intact ovoidshaped crystals, whereas the BO-ICs showed significant decrease and change in the sizes and shapes of the particles with many agglomerations.These alterations, shown by the BO-ICs, depict the successful formation of an amorphous product incorporating another compound in the complex, indicating the effective establishment of the inclusion complexes.

Fig. 2
Fig. 2 UHR-TEM images of HPbCD (a) and BO-ICs (b) show their structural and morphological characteristics and, in addition, BO encapsulation into HPbCD.Both samples showed round vesicles with thin layer membranes surrounding the particles.Also, several aggregates were shown by BO-ICs, and different shapes of micellar structures could be observed in both fields owing to the self-assembly of cyclodextrins.