GC-MS Analysis of Commiphora molmol OleoResin Extracts which Inhibit the growth of Bacterial Triggers of Selected Autoimmune Diseases

Introduction: Myrrh has been used traditionally for the inhibition of microbial growth and for the treatment of rheumatic diseases. Despite this, myrrh extracts are yet to be tested for the ability to inhibit the growth of the bacterial triggers of autoimmune inflammatory diseases. Methods: Solvent extracts prepared from commercially obtained myrrh resin were analysed for the ability to inhibit the growth of bacterial species associated with initiating rheumatoid arthritis (P. mirabilis), ankylosing spondylitis (K. pneumoniae) and multiple sclerosis (A. baylyi, P. aeruginosa) by disc diffusion assay, and quantified by MIC determination. Toxicity was determined by Artemia franciscana bioassay. The most potent inhibitory extract was investigated using non-targeted GC-MS head space analysis (with screening against a compound database) for the identification and characterisation of individual components in the crude plant extracts. Results: Methanolic myrrh extract inhibited the growth of all bacterial species tested. The growth inhibition of this extract was particularly notable against P. mirabilis and K. pneumoniae, with MIC values substantially < 1000 μg/mL for both reference and clinical bacterial strains. Indeed, the MIC values of the methanolic extract against P. mirabilis reference and clinical strains were 572 and 463 μg/mL respectively. The methanolic extract also inhibited the growth of A. baylyi (MIC approximately 3000 μg/mL) and P. aeruginosa (MIC approximately 1800 μg/mL). However, the MICs against these bacteria was indicative of only moderate inhibitory activity. The aqueous, ethyl acetate, chloroform and hexane extracts also inhibited the growth of all bacterial species, albeit with moderate (MIC values 1000-5000 μg/mL) to low efficacy (MIC values >5000 μg/mL) against all bacterial species. All myrrh extracts were non-toxicin the Artemia franciscana bioassay, with LC50 values substantially above 1000 μg/mL. Non-biased GC-MS headspace analysis of the methanolic extracti dentified a high diversity of monoterpenoids and sesquiterpenoid. Conclusion: The lack of toxicity and the inhibitory activity of the methanolic myrrh extract against microbial triggers of rheumatoid arthritis, ankylosing spondylitis and multiple sclerosis indicates its potential in the treatment and prevention of these diseases.


INTRODUCTION
Myrrh is the common name for the aromatic resin produced by several small thorny trees of the genus Commiphora (family Burseraceae).The main myrrh producing species is Commiphora molmol (synonym Commiphora myrrha; Yemen, Somalia, Ethiopia), although the resins of Commiphora gileadensis (Eastern Mediterranean, Arabian Peninsula), Commiphora wightii (northern India) and several other species are often also referred to as myrrh. 1,2The myrrh resin is harvested by making incisions in the tree to tap the resinous exudate.The gum subsequently hardens to produce a pale yellow resin called myrrh.Myrrh has been traded on the Arabian Peninsula and in Northern Africa since ancient times.It has long been valued for its health promo ting properties and therapeutic effects.There are frequent references to the use of myrrh in religious texts, accounting for its extensive burning as incense in Christian, Jewish and several Orthodox religious ceremonies. 3ndeed, myrrh is perhaps best known in Western cultures through the biblical story of the 'three wise men' presenting gifts of gold, frankin cense and myrrh to the newborn baby Jesus. 4Later in the Christian bible, Jesus was also offered myrrh in wine as an analgesic prior to the cru cifixion.Nowadays, the primary use of myrrh is in perfumery and for aromatherapy, although it is also used as a traditional medicine.A wide variety of therapeutic uses have also been attributed to myrrh, including its use in the treatment of several inflammatory and skin diseases, as an antiseptic, an antipyretic, a mouth wash, to aid wound healing and as a stimulant. 5,6Many of these conditions are rela ted to bac terial infections and several studies have reported the growth inhibitory properties of myrrh against panels of pathogens.One study screened Commiphora molmol extracts for the ability to inhibit the growth of a panel of bacterial and fungal pathogens. 7That study tested myrrh for growth inhibitory activity against 7 bacterial (Staphylococcus aureus, Vibrio tubiashii, Sterptococcus spp., Cellulosimicrobium cellulans, Micrococcus luteus, Legionella pneumophila, Bacillus cereus) and 2 fungal species (Fusarium oxysporum, Aspergillus flavus).The C. molmol extract inhibited the growth of all microbes screened, although it was particularly potent against A. flavus, V. tubiashii, Sterptococcus spp., L. pneumophila and B. cereus.However, the value of that study was limited as MIC's were not determined, making it difficult to compare this activity to that of other antibacterial chemotherapies.Another study examined the growth inhibitory potential of myrrh essential oil against S. aureus and reported that the oil not only inhibited bacterial growth, but also inhibited biofilm formation. 8Furthermore, several myrrh compounds have been isolated and shown to have good antibacterial efficacies.Furanodiene6one and methoxyfuranoguaia9ene8one have been highlighted as being particularly potent growth inhibitors, with MIC values against three bacterial and one fungal species ranging from 0.2-2.8µg/mL. 9Simi Pharmacognosy Journal, Vol 8, Issue 3, May-Jun, 2016 larly, several terpenoids (including the sesquiterpenoids βelemene and Tcadinol) isolated from C. molmol oleoresin inhibited the growth of a panel of pathogenic bacteria, with MIC ranging from 4256 µg/mL. 10 number of studies have also verified the antiinflammatory, anti pyretic, antihistamine 11 and antiarthritic pharmacological claims. 12 recent study reported the myrrh inhibited the production of nitric oxide, prostaglandin E2, tumour necrosis factorα (TNFα), but did not affect interleukin (IL)1β and IL6. 13 The same study reported that myrrh also inhibited cjun NH 2 terminal kinase (JNK), but did not affect extra cellular signal related kinase (ERK), p 38 and nuclear factorκB (NFκB).Furthermore, myrrh administration decreased leukocyte infiltration, providing evidence for a pleuripotent antiinflammatory mechanism.Despite the traditional uses of myrrh in the treatment of inflammatory disease, as well as the wellestablished growth inhibitory activity of myrrh against other bacterial pathogens, myrrh has not been rigorously evaluated for the ability to block the microbial triggers of autoimmune inflammatory diseases.This may result from a poor understanding of many of these disorders.However, recent serotyping studies have identi fied bacterial triggers of some of these conditions and the bacterial antigens responsible for the induction of an immune response, allowing for studies to examine the ability to inhibit the trigger mechanisms of these diseases.The major microbial trigger of rheumatoid arthritis has been identified as Proteus mirabilis, 1416 a normal part of the human gastro intestinal flora.Similarly, Klebsiella pneumoniae has been shown to initiate ankylosing spondylitis 1618 and Acinetobacter baylyi and Pseudomonas aeruginosa have been linked with the onset of multiple sclerosis. 2022he development of antibiotic agents targeted at the specific bacterial triggers of autoimmune inflammatory disorders would enable afflicted individuals to target these microbes and thus prevent the onset of the disease and reduce the severity of the symptoms once the disease has progressed.The current study examined the ability for myrrh extracts to block the initiating events of several autoimmune inflammatory diseases by blocking their microbial triggers.

Myrrh source and extraction
Myrrh was originally sourced from verified Commiphora molmol Engler trees in Somalia by Noodles Emporium, Australia and supplied as a dry resin.Voucher samples have been stored in the School of Natural Sciences, Griffith University.Prior to use, the resin was freshly ground to a coarse powder and 1 g quantities were weighed into separate tubes.Individual 50 mL volumes of methanol, water, ethyl acetate, chloroform or hexane were added to the ground resin and extracted by maceration for 24 h at 4 o C with gentle shaking.The extracts were subsequently filtered through filter paper (Whatman No. 54) under vacuum, followed by drying by rotary evaporation in an Eppendorf concentrator 5301.The resultant dry extract was weighed and redissolved in 10 mL deionised water (containing 0.5% DMSO).All solvents were obtained from Ajax Australia and were AR grade.

Qualitative phytochemical studies
Phytochemical analysis of the myrrh extracts for the presence of saponins, phenolic compounds, flavonoids, phytosteroids, triterpenoids, cardiac glycosides, anthraquinones, tannins and alkaloids was conducted by previously described assays. 2325

Antibacterial screening Test microorganisms
All media was supplied by Oxoid Ltd.Australia.Reference strains of Klebsiella pneumoniae (ATCC31488), Proteus mirabilis (ATCC21721), Acinitobacter baylyi (ATCC33304) and Pseudomonas aeruginosa (ATCC39324) were purchased from American Tissue Culture Collection, USA.All other clinical microbial strains were obtained from the School of Natural Sciences teaching laboratory, Griffith University.All stock cultures were subcultured and maintained in nutrient broth at 4 o C.

Evaluation of antimicrobial activity
Antimicrobial activity of all plant extracts was determined using a modified disc diffusion assay. 2628Briefly, 100 µL of the test bacteria were grown in 10 mL of fresh nutrient broth media until they reached a count of approximately 10 8 cells/mL.An amount of 100 µL of bacterial suspen sion was spread onto nutrient agar plates.The extracts were tested for antibacterial activity using 5 mm sterilised filter paper discs.Discs were impregnated with 10 µL of the test sample, allowed to dry and placed onto inoculated plates.The plates were allowed to stand at 4 o C for 2 h before incubation with the test microbial agents.Inoculated plates were incubated at 30 o C for 24 h, then the diameters of the inhibition zones were measured to the closest whole millimetre.Each antimicrobial assay was performed in at least triplicate.Mean values (± SEM) are reported in this study.Standard discs of ampicillin (10 µg) were obtained from Oxoid Ltd., Australia and served as positive controls for antibacterial activity.Filter discs impregnated with 10 µL of distilled water were used as a negative control.

Minimum inhibitory concentration (MIC) determination
The minimum inhibitory concentration (MIC) of the extracts were determined as previously described. 29,30Briefly, the extracts were diluted in deionised water and tested across a range of concentrations.Discs were impregnated with 10 µL of the test dilutions, allowed to dry and placed onto inoculated plates.The assay was performed as outlined above and graphs of the zone of inhibition versus concentration were plotted for each extract.Linear regression was used to calculate the MIC values.

Toxicity screening Reference toxin for toxicity screening
Potassium dichromate (K 2 Cr 2 O 7 ) (AR grade, ChemSupply, Australia) was prepared as a 1.6 mg/mL solution in distilled water and was serially diluted in artificial seawater for use in the Artemia franciscana nauplii bioassay.

Artemia franciscana nauplii toxicity screening
Toxicity was tested using a modified Artemia franciscana nauplii lethality assay. 3133Briefly, 400 µL of seawater containing approximately 46 (mean 46.3, n=120, SD 11.3) A. franciscana nauplii were added to wells of a 48 well plate and immediately used for bioassay.A volume of 400 µL of diluted plant extracts or the reference toxin were transferred to the wells and incubated at 25 ± 1 o C under artificial light (1000 Lux).A negative control (400 µL seawater) was run in triplicate for each plate.All treat ments were performed in at least triplicate.The wells were checked at regular intervals and the number of dead counted.The nauplii were con sidered dead if no movement of the appendages was observed within 10 sec.After 24 h, all nauplii were sacrificed and counted to determine the total % mortality per well.The LC 50 with 95% confidence limits for each treatment was calculated using probit analysis.

Non-targeted GC-MS head space analysis
Separation and quantification were performed using a Shimadzu GC2010 plus (USA) linked to a Shimadzu MS TQ8040 (USA) mass selective detector system as previously described. 22The system was equipped with a Shimadzu autosampler AOC5000 plus (USA) fitted with a solid phase microextraction fibre (SPME) handling system utilis ing Supelco (USA) divinyl benzene/carbowax/polydimethylsiloxane (DVB/CAR/PDMS).Chromatographic separation was accomplished using a 5% phenyl, 95% dimethylpolysiloxane (30 m×0.25 mm id×0.25 um) capillary column (Restek USA).Helium (99.999%) was employed as a carrier gas at a flow rate of 0.79 mL/min.The injector temperature was set at 230°C.Sampling utilised a SPME cycle which consisted of an agitation phase at 500 rpm for a period of 5 sec.The fibre was exposed to the sample for 10 min to allow for absorption and then desorbed in the injection port for 1 min at 250°C.The initial column temperature was held at 30°C for 2 min, increased to 140°C for 5 m, then increased to 270°C over a period of 3 mins and held at that temperature for the duration of the analysis.The GCMS interface was maintained at 200°C with no signal acquired for a min after injection in splitless mode.The mass spectrometer was operated in the electron ionisation mode at 70 eV.The analytes were then recorded in total ion count (TIC) mode.The TIC was acquired after a min and for duration of 45 mins utilising a mass range of 45450 m/z.

Statistical analysis
Data are expressed as the mean ± SEM of at least three independent experiments.

Liquid extraction yields and qualitative phytochemical screening
Extraction of 1 g of myrrh with selected solvents yielded dried extracts ranging from 162 mg (hexane extract) to 728 mg (aqueous extract) (Table 1).Ethyl acetate extracted a relatively high yield (642 mg) similar to that of the aqueous extract.In contrast, methanol extracted a similarly low yield (181 mg) to the hexane extract.Chloroform extracted an intermediate amount (307 mg).The dried extracts were resuspended in 10 mL of deionised water (containing 1% DMSO), resulting in the extract concentrations shown in Table 1.Qualitative phytochemical studies showed that all solvents extracted similar classes of phytochemicals, with polyphenolic compounds (parti cularly water insoluble polyphenolic compounds), flavonoids, saponins and triterpenoids generally present in the highest levels.All extracts were generally devoid of all other classes of phytochemicals.Interest ingly, despite having much lower extraction yields, the methanolic extract contained a similar phytochemical profile and similar relative levels compared to the aqueous extract.

Antimicrobial activity
To determine the growth inhibitory activity of the myrrh extracts against the bacterial triggers of the autoimmune inflammatory diseases, aliquots (10 µL) of each extract were tested in the disc diffusion assay.Reference and clinical strains of P. mirabilis were inhibited by all myrrh extracts (Figure 1).The methanolic extract was the most potent P. mirabilis growth inhibitor (as assessed by the sizes of the zones of inhibition), with 9.5 ± 0.5 and 9.0 ± 0 mm for the reference and clinical strains respectively.The ethyl acetate and chloroform extracts were also moderate inhibitors of P. mirabilis growth, with zones of inhibition >8 mm for each extract against both strains.Whilst the aqueous and hexane extracts also inhibited P. mirabilis growth, their inhibition was weaker, with inhibition zones generally ≤7.5 mm in diameter (compared to >10.5 mm for the ampicillin control).The growth of K. pneumoniae (Figure 2) was generally more susceptible to inhibition by the myrrh extracts than was P. mirabilis (as judged by the zones of inhibition).Both polar and low polarity extracts were gener ally good K. pneumoniae growth inhibitors, however the higher polarity aqueous and methanolic extracts were generally more potent.Indeed, both the methanolic and aqueous extracts showed zones of inhibition ≥8.3 mm against both the reference and clinical strains of the bacterium.This compares well with the growth inhibition of the positive control (ampicillin, 10 µg), which had inhibition zones of 77.3 mm against both strains.The lower polarity chloroform extract was also a good inhibi tor of K. pneumoniae growth (zones of inhibition 7.3 mm).Whilst also inhibiting K. pneumoniae growth, the ethyl acetate and hexane extracts were substantially less potent, with smaller zones of inhibition (6.87.5 mm).Both reference and clinical strains of A. baylyi were also inhibited by all of the myrrh extracts (Figure 3).Both strains appeared to be approxi mately equally susceptible to each extract.The methanolic extract was the most potent growth inhibitor, with zones of inhibition of 7.7 ± 0.3 and 8.0 ± 0.5 mm for the reference and clinical bacterial strains respec tively.The ethyl acetate extract was also a good growth inhibitor (zones of inhibition of 7.5 ± 0.4 mm and 7.3 ± 0.3 mm).In addition, the aqueous, chloroform and hexane extracts all inhibited A. baylyi growth, with zones of inhibition of 6.67.0 mm.However, it is noteworthy that these zones of inhibition are indicative of only weak growth inhibition, and are substantially less than the inhibition by the ampicillin control (8.3 mm for both strains).Both reference and clinical strains of P. aeruginosa were also inhibited by all of the myrrh extracts, albeit with relatively small zones of inhibition (Figure 4).The methanolic extract was the best P. aeruginosa growth inhibitor with zones of inhibition of 7.5 ± 0.5 mm and 7.3 ± 0.3 mm for the reference and clinical strains respectively.The chloroform extract was also a moderate P. aeruginosa growth inhibitor (zones of inhibition of 7.0 ± 0.3 mm and 6.8 ± 0.2 mm for the reference and clinical strains respectively).All other myrrh extracts gave substantially lower zones of inhibition.The hexane extract was a particularly weak growth inhibitor, with zones of inhibition ≤6 mm against both strains.However, it is note worthy that both P. aeruginosa strains were also resistant to the antibiotic control (10 µg ampicillin), with only small zones of inhibition also noted (6.0 ± 0 and 5.3 ± 0.3 mm for the reference and clinical strains respectively).The antimicrobial efficacy was further quantified by determining the MIC values for each extract against each microbial species.The metha nolic extract was generally most effective at inhibiting bacterial growth, particularly growth of P. mirabilis and K. pneumoniae (Table 2), with MIC values ≤1000 µg/mL (≤10 µg impregnated in the disc), indicating the potential of these extracts in controlling rheumatoid arthritis and ankylosing spondylitis.The methanolic extract was particularly potent against P. mirabilis with MIC values of 572 and 463 µg/mL (approximately 6 and 5 µg impregnated in the disc) against the reference and clinical P. mirabilis strains respectively.As P. mirabilis has been identified as a trigger of rheumatoid arthritis, methanolic myrrh extracts have potential for the prevention of this disease in genetically susceptible individuals.The methanolic extract also was a good inhibitor of K. pneumoniae growth with MIC values of 850950 µg/mL (approximately 9 µg impreg nated in the disc).Notably, the methanolic extract also showed moderate inhibitory activity against the bacterial triggers of multiple sclerosis (A.baylii, MIC approximately 3000 µg/mL; P. aeruginosa, approximately 1500 µg/mL).The aqueous extract was a moderate inhibitor of K. pneumoniae growth (MIC values of 3721 and 4866 µg/mL for the reference and clinical strains respectively).Similarly, the ethyl acetate extract was a    moderate inhibitor of P. mirabilis (MIC 177502500 µg/mL); chloroform was a moderate inhibitor of P. mirabilis and K. pneumoniae growth (MIC 17505000 µg/mL).Low growth inhibition was noted for all other extract/bacterium combinations, with MIC values generally>5000 µg/mL.

Quantification of toxicity
All extracts were initially screened undiluted in the Artemia nauplii bioassay (Figure 5).For comparison, the reference toxin potassium dichromate (1000 µg/mL) was also tested.The potassium dichromate reference toxin was rapid in its onset of mortality, inducing nauplii death within the first 3 h of exposure and 100% mortality was evident following 45 h (results not shown).With the exception of the hexane extract, all myrrh extracts induced 100% mortality within 24 h.To further quantify the effect of toxin concentration on the induction of mortality, the extracts were serially diluted in artificial seawater to test across a range of concentrations in the Artemia nauplii bioassay.Table 2 shows the LC 50 values of the myrrh extracts towards A. franciscana.All myrrh extracts were determined to be nontoxic, with LC 50 values substantially greater than 1000 µg/mL following 24 h exposure.Extracts with an LC 50 of greater than 1000 µg/mL towards Artemia nauplii have previously been defined as being nontoxic. 34

Non-targeted GC-MS headspace analysis of the myrrh extracts
As the methanolic myrrh extract had the greatest bacterial growth inhibitory efficacy (as determined by MIC; Table 2), it was deemed the most promising extract for further phytochemical analysis.Optimised GCMS parameters were developed and used to examine the phyto chemical composition of this extract.The resultant gas chromatogram is presented in Figure 6.Major peaks were presentin the methanolic extract chromatogram at approximately 11.1, 12.9, 14.4, 17.0, 19.5, and 34.1 min.Numerous smaller peaks were also evident throughout all stages of the chromatograms.In total, 138 unique mass signals were noted for the myrrh methanolic extract (Table 3).Putative empirical formulas and identifications were achieved 36 of these compounds (26%).

DISCUSSION
The current therapeutic regimens for the treatment of autoimmune inflammatory diseases utilise disease modifying antirheumatic drugs (DMARDs) to alleviate the symptoms of these diseases and/or alter the disease progression.Unfortunately, none of these therapies are entirely effective and many have been associated with numerous adverse effects. 35urthermore, many of the current treatments are aimed at treating the symptoms without addressing the underlying causes and pathogenic mechanisms.Therefore, whilst these treatments may alleviate pain, redness, swelling etc., they do not address the tissue damage that occurs as a consequence of the disease etiology.Furthermore, all of these drugs are used as treatments and there are currently no preventative therapy options.A better understanding of the mechanisms for initiation and progression of the autoimmune inflammatory diseases is important for developing new drugs to target specific processes and thus more effec tively treat autoimmune inflammatory diseases.The studies reported here examined the ability of Commiphora molmol oleoresin (myrrh) extracts to block microbial triggers of 3 autoim mune inflammatory disorders (Proteus mirabilis: rheumatoid arthritis; K. pneumonia: ankylosing spondylitis; Acinitobacter baylyi and Pseudomonas aeruginosa: multiple sclerosis).The methanolic extract was identified as being a particularly potent inhibitor of P. mirabilis (MIC values of 572 and 463 µg/mL for the reference and clinical strains respectively).Thus, this extract has potential for the development of rheumatoid arthritis inhibitory therapies.Interestingly, as previous studies have demonstrated that C. molmol oleoresin extracts and essential oils also regulate cytokine production, 13 the methanolic myrrh extract, may well have similar prop      erties and further studies are required to test this.If these extracts are subsequently found to modulate cytokine production, they may prove to be particularly useful for individuals suffering from rheumatoid arthritis, as they would provide both preventative and treatment mechanisms.The methanolic myrrh extract also displayed good K. pneumonia growth inhibitory properties (MIC values of 850950 µg/mL for both the reference and clinical strains), indicating that it may also be useful in the prevention of ankylosing spondylitis.The myrrh extract may also have further effects on other phases of ankylosing spondylitis disease etiology.Indeed, it is possible that the extract may also modulate cytokine production and therefore also block later inflammatory disease events, 13 although this has yet to be tested for our extracts.Similarly, the methanolic myrrh extract also inhibited A. baylyi and P. aeruginosa growth, albeit with higher MIC values indicative of only moderate inhibition.However, the therapeutic properties of myrrh in the treatment of autoimmune diseases may be of greater efficacy as synergistic actions may exist between various therapeutic mechanisms (antibacterial, antiinflammatory, antioxidant, immunestimulatory etc.), providing combined affects on these complex diseases.Of further note, the antibacterial and MIC assays performed in our study utilised agar based methods.Whilst these are effective in many cases, they often under estimate the efficacy of very low polarity mixtures such as essential oils as the low polarity compounds do not diffuse well within the agar gels.Furthermore, volatile compounds in essential oils are often lost due to evaporation, resulting in falsely low efficacies.Whilst our study tested extracts rather than oils, GCMS analysis of the methanolic extract detected a number of low polarity, volatile terpenoids.Thus, perhaps testing by liquid dilution MIC techniques may have yielded lower MIC values, indicative of greater efficacy and further studies are required to test this.GCMS headspace analysis of the methanolic myrrh extract detected a number of interesting compounds, including a wide diversity of terpenoids.Monoterpenoids were particularly prevalent, with 8 mono terpenoids putatively identified.The monoterpenoids cineole (Figure 7a), sabinol (Figure 7b), 3thujen2one (Figure 7c), terpinene4ol (Figure 7d), αterpineol (Figure 7e), myrtenol (Figure 7f), verbenone (Figure 7g), and Lαbornyl acetate(Figure 7h) were identified.Monoterpenes have been reported to exert a wide variety of biological effects including antibacterial, antifungal, antiinflammatory and antitumour activities 36 and therefore may contribute to the growth inhibitory activity against the bacterial triggers of the autoimmune diseases reported here.Indeed, many of the monoterpenoids putatively identified in our study have been previously reported to have potent broad spectrum antibacterial activity. 36A wide variety of monoterpenoids including camphor, carvone, cineole, borneol, menthone, pinene, terpinene, as well as their derivatives, inhibit the growth of an extensive panel of pathogenic and food spoilage bacteria. 37nterestingly, several of these monoterpenoids have also been reported to suppress NFκ B signaling (the major regulator of inflammatory diseases). 3841Thus, the terpene components may have a pleuripotent mechanism in blocking the autoimmune inflammatory diseases and relieving its symptoms by acting on both the initiator and downstream inflammatory stages of the disease.Further phytochemical evaluation studies and bioactivity driven isolation of active components are required to further evaluate the mechanism(s) of bacterial growth inhibition.Several sesquiterpenoids including γelemene (Figure 7i), βlongipinene (Figure 7j), 1Hcyclopropa[a]naphthalene, decahydro1,1,3a (Figure 7k), epicurzerenone (Figure 7l), isospathulenol (Figure 7m), γeudesmol (Figure 7n) and αcadinol acetate (Figure 7o) were also detected in the methanolic myrrh extract.Previous studies have reported bacterial growth inhibitory activities for many sesquiterpenoids including cadinol and its derivatives, 42 caryophyllene, 43 copaene, epicubenol and cubenene. 44ndeed, a previous study reported that several sesquiterpenoids detected in our study (including the sesquiterpenoids elemene and Tcadinol) inhibited the growth of a panel of pathogenic bacteria, with MIC ranging from 4256 µg/mL. 10Thus, these compounds are likely to contribute to the growth inhibitory activity determined in our study.Notably, despite triterpenoids being detected as a major component for the methanolic myrrh extract in the qualitative screenings, no triterpe noids (nor diterpenoids or sesterterpenoids) were detected by GCMS headspace analysis.It is perhaps not surprising that triterpenoids were not detected as a mass range cut off of 450 m/z was used in these studies.Therefore, many triterpenoids would have molecular masses that would be near this cut off and may not be detected.However, given the diver sity of other terpenoids detected, it is perhaps surprising that no diand sesterterpenoids were detected in the extract.Due to the low polarity of these compounds, perhaps an analysis of the lower polarity extracts (chloroform, hexane) may have detected these classes of phytochemical.This highlights an important point: An important consideration of any metabolomic technique is that it will not detect all compounds in a com plex mixture, but instead will only detect a portion of them.This is not necessarily a problem when a directed/biased study is undertaken to detect a particular compound or class of compounds and the separation and detection conditions can be optimised for the study.However, when the aim of the study is metabolomic profiling rather than metabolomic fingerprinting, the technique conditions must be chosen and optimised to separate and detect the largest amount of compounds, with the broadest possible physical and chemical characteristics.GCMS analysis is limited to the detection of the lower polar compounds.Therefore, it is likely that mid and high polarity compounds are present in these extracts and these compounds may also contribute to the growth inhibitory activity reported here.HPLCMS is a good choice for the detection of midhighly polar compounds.Thus, further studies are required, focus sing on these techniques, to build a more comprehensive understanding of the complete metabolomic profile of the myrrh resin.Furthermore, mass spectral techniques are generally not capable on their own of differ entiating between structural isomers.Further studies using a wider variety of techniques are required to confirm the identity of the compounds putatively identified here.Whilst these studies have demonstrated the potential of the myrrh extracts to treat autoimmune disease, more work is required.This study has only tested these extracts against some microbial triggers of 3 auto immune diseases (rheumatoid arthritis, ankylosing spondylitis and multiple sclerosis).The microbial triggers for several other autoimmune inflammatory disorders are also known.Borrelia burgdorferi is linked with Lyme disease. 45Similarly, members of the Enterobacteriaceae family are associated with Graves' disease and Kawasaki syndrome.Mycoplasma pneumoniae is associated with several demyelinating diseases. 46Whilst microbial triggers have also been postulated for lupus, the specific causative agents are yet to be identified.It would be interesting to extend our studies to also screen for the ability of the extracts to block these microbial triggers of autoimmune diseases.

CONCLUSION
The results of this study demonstrate the ability of myrrh extracts to block the growth of bacterial species associated with the onset of rheu matoid arthritis, ankylosing spondylitis and multiple sclerosis, and thus their therapeutic potential in genetically susceptible individuals.Further studies aimed at the purification and identification of bioactive components are needed to examine the mechanisms of action of these agents.

Figure 2 :
Figure 2: Antibacterial activity of the myrrh extracts against K. pneumoniae measured as zones of inhibition (mm).The blue bars represent the inhibitory activity against the reference strain (ATCC:213488) and the green bars represent the zones of inhibition against the clinical strain.M = methanolic extract; W = water extract; E = ethyl acetate extract; C = chloroform extract; H = hexane extract; NC = 0.5% DMSO; PC = ampicillin (10 µg) control.Results are expressed as mean zones of inhibition ± SEM.

Figure 1 :
Figure 1: Antibacterial activity of the myrrh extracts against P. mirabilis measured as zones of inhibition (mm).The blue bars represent the inhibitory activity against the reference strain (ATCC:21721) and the green bars represent the zones of inhibition against the clinical strain.M = methanolic extract; W = water extract; E = ethyl acetate extract; C = chloroform extract; H = hexane extract; NC = 0.5% DMSO; PC = ampicillin (10 µg) control.Results are expressed as mean zones of inhibition ± SEM.

Figure 4 :
Figure 4: Antibacterial activity of the myrrh extracts against P. aeruginosa measured as zones of inhibition (mm).The blue bars represent the inhibitory activity against the reference strain (ATCC:39324) and the green bars represent the zones of inhibition against the clinical strain.M = methanolic extract; W = water extract; E = ethyl acetate extract; C = chloroform extract; H = hexane extract; NC = 0.5% DMSO; PC = ampicillin (10 µg) control.Results are expressed as mean zones of inhibition ± SEM.

Figure 3 :
Figure 3: Antibacterial activity of the myrrh extracts against A. baylyi measured as zones of inhibition (mm).The blue bars represent the inhibitory activity against the reference strain (ATCC:33304) and the green bars represent the zones of inhibition against the clinical strain.M = methanolic extract; W = water extract; E = ethyl acetate extract; C = chloroform extract; H = hexane extract; NC = 0.5% DMSO; PC = ampicillin (10 µg) control.Results are expressed as mean zones of inhibition ± SEM.

Figure 5 :
Figure 5: The lethality of the myrrh extracts and the potassium dichromate control (1000 µg/mL) towards Artemia nauplii.M = methanolic extract; W = water extract; E = ethyl acetate extract; C = chloroform extract; H = hexane extract; PC = positive control (1000 µg/ml potassium dichromate); NC = negative (seawater) control.All tests were performed in at least triplicate and the results are expressed as mean ± SEM.

Figure 6 :
Figure 6: Head space gas chromatograms of 0.5 μL injections of methanolic myrrh extract.The extract was dried and resuspended in methanol for analysis

Table 1 : The mass of dried extracted material, the concentration after resuspension in deionised water, qualitative phytochemical screenings and antioxidant capacities of the myrrh extracts Extract Mass of Dried Extract
large response; ++ indicates a moderate response; + indicates a minor response; indicates no response in the assay.M=methanolic myrrh extract; W=aqueous myrrh extract; E=ethyl acetate myrrh extract; C=chloroform myrrh extract; H=hexane myrrh extract.

Table 2 : Minimum bacterial growth inhibitory concentration (µg/mL) of the myrrh extracts and LC 50 values (µg/mL) in the Artemia nauplii bioassay
Numbers indicate the mean MIC and LC 50 values of triplicate determinations.indicatesno inhibition.

Table 3 : GC-MS analysis of the myrrh methanolic extract, elucidation of empirical formulas and putative identification (where pos- sible) of each compound Molecular Mass
The relative abundance expressed in this table is a measure of the area under the peak expressed as a % of the total area under, or % of the total height of all chromatographic peaks.