Chemotypic Characterization and Biological Activity of Rosmarinus officinalis

Rosemary (Rosmarinus officinalis L.) is a popular herb in cooking, traditional healing, and aromatherapy. The essential oils of R. officinalis were obtained from plants growing in Victoria (Australia), Alabama (USA), Western Cape (South Africa), Kenya, Nepal, and Yemen. Chemical compositions of the rosemary oils were analyzed by gas chromatography-mass spectrometry as well as chiral gas chromatography. The oils were dominated by (+)-α-pinene (13.5%–37.7%), 1,8-cineole (16.1%–29.3%), (+)-verbenone (0.8%–16.9%), (−)-borneol (2.1%–6.9%), (−)-camphor (0.7%–7.0%), and racemic limonene (1.6%–4.4%). Hierarchical cluster analysis, based on the compositions of these essential oils in addition to 72 compositions reported in the literature, revealed at least five different chemotypes of rosemary oil. Antifungal, cytotoxicity, xanthine oxidase inhibitory, and tyrosinase inhibitory activity screenings were carried out, but showed only marginal activities.


Introduction
Rosmarinus officinalis L., "rosemary" (Lamiaceae) is an evergreen shrub with aromatic needle-like leaves. It is native to the Mediterranean and Asia, but is now cultivated in temperate locations around the world as a decorative garden plant and culinary herb. Rosemary leaves have been used to flavor foods such as lamb, pork, chicken, fish, and stuffings, and to prepare herbal oils, butters, and vinegars.
Rosemary essential oil is used in aromatherapy as a nerve stimulant for purposes including memory loss and lethargy [22]. Rosemary oil has shown psychostimulant activity. In a mouse model, a dose of 100 µg/kg caused a significant increase in locomotor activity [23]. In humans, massaged [24] or inhaled [25] rosemary oil has caused significant increased blood pressure, heart rate, and respiratory rate; oral administration of rosemary oil significantly increased blood pressure in hypotensive patients [26]. Inhaled rosemary oil has been shown to increase memory and concentration abilities [27]. In addition to psychostimulatory effects, rosemary essential oil has shown anticholinesterase [28], acaricidal [29,30], antibacterial [31,32], antifungal [33,34], and antinociceptive [35] activities.
The biological activities of R. officinalis essential oils doubtless depend on the chemical compositions, and at least 13 different rosemary oil chemotypes have been previously identified, based on the relative percentages of α-pinene, 1,8-cineole, camphor, borneol, verbenone, and bornyl acetate [31,[36][37][38][39][40][41]. In this work, we have characterized the essential oils of R. officinalis collected from Alabama (USA), Western Cape (South Africa), Victoria (Australia), Kenya, Nepal, and Yemen, and screened these essential oils for antifungal activity. In addition, a hierarchical cluster analysis has been carried out based on the compositions of an additional 72 rosemary essential oils reported in the literature.

Plant Material
Leaves of R. officinalis from flowering plants in Huntsville The obtained oil was subsequently dried over anhydrous Na 2 SO 4 and kept at 4 • C until analysis. After filtration, the yield of the oil was 1.1% w/w. R. officinalis, in the flowering stage, from Jumla, Nepal (29 • 16 28.99" N, 82 • 11 1.79" E, elevation 2500 m asl), was identified by Prasun Satyal, and collected on 2 July 2016. The fresh plant material (100 g) was hydrodistilled for 4 h using a Clevenger-type apparatus and collected to give 500 mg (0.5% yield) colorless essential oil after drying with Na 2 SO 4 .
R. officinalis from Ribeeck Kasteel, Western Cape, South Africa (33 • 23 7.21" S, 18 • 53 54.65" E, elevation 300 m asl), was identified by Prabodh Satyal, and collected on 20 August 2016. The fresh plant material (500 g) was subjected to steam distillation for 4 h using a Clevenger-type apparatus and collected to give 4 g (0.8% yield) colorless essential oil after drying with Na 2 SO 4 .
Flowering R. officinalis from Lancefield, Victoria, Australia (37 • 16 S, 144 • 43 E, elevation 495 m asl), was identified by Chris Burder, and collected on 20 December 2015. The fresh plant material (1 kg) was subjected to steam distillation for 3 h using a Clevenger-type apparatus and collected to give 9 g (0.9% yield) colorless essential oil after drying with Na 2 SO 4 .
R. officinalis from Thika, Kenya (1 • 1 S, 37 • 5 E, elevation 1500 m asl), was identified by Aaron Sorensen, and collected on 8 July 2016. The fresh plant material (1 kg) was subjected to steam distillation for 3.5 h using a Clevenger-type apparatus and collected to give 10 g (1.0% yield) pale yellow essential oil after drying with Na 2 SO 4 .

Gas Chromatography-Mass Spectrometry (GC-MS)
The essential oils of R. officinalis were analyzed by GC-MS using a Shimadzu GCMS-QP2010 Ultra operated in the electron impact (EI) mode (electron energy = 70 eV), scan range = 40-400 atomic mass units, scan rate = 3.0 scans/s, and GC-MS solution software. The GC column was a ZB-5 fused silica capillary column with a (5% phenyl)-polymethylsiloxane stationary phase and a film thickness of 0.25 µm. The carrier gas was helium with a column head pressure of 552 kPa and flow rate of 1.37 mL/min. Injector temperature was 250 • C and the ion source temperature was 200 • C. The GC oven temperature program was programmed for 50 • C initial temperature, temperature increased at a rate of 2 • C/min to 260 • C. A 5% w/v solution of the sample in CH 2 Cl 2 was prepared and 0.1 µL was injected with a splitting mode (30:1). Identification of the 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 [43], and stored in our in-house MS library.

Chiral Gas Chromatography-Mass Spectrometry
Chiral analysis of the essential oils was performed on a Shimadzu GCMS-QP2010S operated in the EI mode (electron energy = 70 eV), scan range = 40-400 amu, scan rate = 3.0 scans/s. GC was equipped with a Restek B-Dex 325 capillary column (30 mL × 0.25 mm ID × 0.25 µm film). Oven temperature was started at 50 • C, and then gradually raised to 120 • C at 1.5 • C/min. The oven was then raised to 200 • C at 2 • C/min and held for 5 min. Helium was the carrier gas and the flow rate was maintained at 1.8 mL/min. Samples were diluted 3% w/v with CH 2 Cl 2 and then a 0.1 µL sample was injected in a split mode with a split ratio of 1:45.

Antifungal Screening
Antifungal activity was carried out as previously described [82]. Briefly, minimum inhibitory concentrations were determined using microdilution methods for Candida albicans (American Type Culture Collection, ATCC #18804), Cryptococcus neoformans (serotype D or var. neoformans) (ATCC #24067), and Aspergillus niger (ATCC #16888). Single colonies from potato dextrose agar plates were grown in potato dextrose broth for three days. Cells were diluted to 2 × 10 3 cells/mL using MOPS (3-(N-morpholino)propanesulfonic acid) buffered RPMI (Roswell Park Memorial Institute) medium and aliquoted into sterile 12 × 75 mm tubes (900 µL). Essential oil (100 µL) was added to each tube followed by incubation at 37 • C for three days in a shaking incubator (175 rpm). Minimum inhibitory concentrations (MICs) were calculated from microdilution in 96-well plates, performed in triplicate. Serial dilution was performed by adding 50 µL of RPMI to each well, then an equal volume of essential oil to be tested to the first row. After mixing, 50 µL was removed and added to the next row. The procedure was repeated for each row, discarding the final 50 µL removed. To the mixture in each well, 50 µL of cells were added. The plates were incubated for two days at 37 • C before growth was quantitated from turbidity.

Xanthine Oxidase Inhibition Assay
Using xanthine as substrate, the xanthine oxidase (XO) activity of R. officinalis oil from Yemen was assayed spectrophotometrically according to Apaya and Hernandez [83]. A mixture containing 1 mL of 100 µg/mL of R. officinalis oil or allopurinol, 1.9 mL of 50 mM potassium phosphate buffer, and 1 mL of xanthine substrate (0.6 mM) was preincubated for 10 min at 25 • C, and the reaction was started by the addition of 0.1 mL of XO enzyme (0.1 U/mL in phosphate buffer). The reaction was incubated at 25 • C for 30 min, and the absorbance was measured against phosphate buffer as blank at 295 nm using quartz cuvettes. Allopurinol was used as standard enzyme inhibitors. The percent xanthine oxidase inhibition was calculated according to the following formula: where A 1 is the activity of the enzyme in presence of the oil, B is the absorbance in absence of the enzyme, and A o is the absorbance in absence of the oil or inhibitor.

Tyrosinase Inhibition Assay
In vitro mushroom tyrosinase inhibitory activity of R. officinalis oil from Yemen was determined by a spectrophotometric approach using L-tyrosine as the substrate [84]. In a total volume of 200 µL, the enzyme activity was measured in buffer containing 50 mM phosphate buffer, pH 6.5, 50 U/mL mushroom tyrosinase, and 50 µg/mL L-tyrosine. The reaction (conversion of L-tyrosine to DOPAchrome) was conducted at 37 • C for 30 min, and absorbance was then measured at the wavelength of 490 nm using a microplate reader. Blanks were run for the same concentration of essential oil in the absence of the enzyme. The inhibition assays were carried out in the presence of 100 µg/mL of the essential oil. Kojic acid and arbutin were used as positive control inhibitors in these assays. The absorbance of the same mixture without the essential oil was used as the negative control. The percent inhibition of tyrosinase activity was calculated as follows: where A is the absorbance difference at 490 nm of the negative control (enzyme activity without oil)-the absorbance of the blank (no enzyme, no oil); B is the absorbance of test sample (with enzyme)-the absorbance of test sample blank (no enzyme).

Cytotoxicity Screening
Human colorectal cancer cell lines (SW480 and HCT116) were generously provided by Dr. Rick F. Thorne (University of Newcastle, Australia) and were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal calf serum (Bio Whittaker, Verviers, Belgium). The rosemary oil from Yemen was tested for acute cytotoxic effect; the essential oil was dissolved in dimethylsulfoxide (DMSO) and different dilutions in culture buffer were prepared. The acute cytotoxic effect of the essential oil on colorectal cancer cells was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [85]. Briefly, cells were seeded at 5000 cells per well onto flat-bottomed 96-well culture plates and allowed to grow for 24 h before the desired treatment. Cells were then incubated with 200 µL of essential oil concentrations (0-200 µg/mL) for 72 h. Cells were then labeled with MTT from the Vybrant MTT Cell Proliferation Assay Kit (Molecular Probes, Eugene, OR, USA) according to the manufacturer's instruction and resulting formazan was solubilized with DMSO. Absorbance was read in a microplate reader at 540 nm.

Antifungal Activity
Antifungal activity was tested against three common opportunistic fungal pathogens. Yeast-like C. albicans and mold-like A. niger are Ascomycota while C. neoformans is a Basidiomycota. Overall, no inhibitory activity at or below 2500 ppm for the R. officinalis essential oil chemotypes was overserved against either ascomycete. Inhibitory activity was observed against C. neoformans for the majority of R. officinalis chemotypes tested. Though not exceedingly promising, R. officinalis from Australia did demonstrate an MIC of 625 ppm. Previous investigations of antifungal activity rosemary oil against A. niger [76,96,97] and C. albicans [19,98,99] have also found rosemary oil to be inactive, while against C. neoformans it had been found to be moderately active (MIC = 156 µg/mL) [100]. Thus, rosemary oil cannot be considered a worthwhile antifungal agent.

Conclusions
Rosemary (Rosmarinus officinalis) essential oil has been extensively studied for its chemical composition and its biological activities. α-Pinene and 1,8-cineole generally dominate the essential oil compositions, but camphor, verbenone, camphene, and myrcene may also appear in high concentrations. The enantiomeric distributions of monoterpenoids in rosemary oils seem to remain relatively constant regardless of environmental factors. Based on the relative concentrations of the major components in rosemary oils, we can define at least five separate chemotypes. The different chemotypes are likely to present different biological activities, but rosemary oil, in general, is relatively inactive in terms of antifungal, enzyme inhibitory, or antitumor potential.