The objective of this study was to propose metrics that could be used in the quality control of Melaleuca armillaris oil, extracted from plants cultivated in Brazil, to differentiate it from Melaleuca alternifolia oil. The oil was obtained by hydro-distillation using steam stripping. The physicochemical characteristics of the M. armillaris essential oil were evaluated, obtaining the following results: density 0.9146 g/mL, pH4.36, specific rotation 2.38, refractive index 1.45948, viscosity of 3 centipoises, 35901 particles ≥ 10 µm and 1743 particles between 10 and 25 µm/5 mL, moisture content 0.84% and decomposition temperature of 115.19 ºC. Osmolarity was not detected. The components of the M. armillaris oil were identified using the following techniques: gas chromatography (GC), high performance liquid chromatography (HPLC) with a mass detector, Raman and near infrared (NIR) spectroscopy. A total of 24 compounds were identified by GC, the main one being 1,8 cineole, and 145 substances were identified by HPLC. In the identification by Raman the transmittance of the oil was found to be 785 nm, and using the NIR technique, the greatest absorbance was at 2350 nm. In this way the M. armillaris oil was characterized by physicochemical analyses and its components identified.
Keywords: Melaleuca; Essential oil; Physicochemical analysis; Characterization
Australia was the pioneering country to work with the essential oil (EO) of the Melaleuca plant [1]. The different species of this genus are cultivated in various parts of the world, including in Brazil [2], where the area cultivated is still small due to a series of factors such as the absence of technical and handling information concerning the species, amongst others [3].
The first literature reports concerning the extraction of EO from the genus Melaleuca occurred in Brazil at the st beginning of the 21 century [3, 4]. Other important reports (2007) [5] and Pereira et al. (2010) [6], who researched the effect of the oil in controlling a fungus that was attacking strawberry cultures in the south of Minas Gerais State, Brazil. Silva et al. (2010) [7] carried out research with the species M. hypericifolia and M. thymifolia at the Federal University of Viçosa, and a couple of years later, Pereira & Teixeira (2012) [8], also working in the south of Minas Gerais State (MG), working with the oil of Melaleuca sp., cultivated as an ornamental plant in the town of Pouso Alegre (MG), aimed to verify the efficacy of the oil in the control of the bacteria Staphylococcus sp. in vitro. Subsequent research aimed to verify the efficiency of the oil Gerais State, in controlling hospital pathogens [9, 10] as well as other properties such as anti-histaminic activity [11]. The chromatographic identification of the EO was also carried out by Falci et al., 2015, which demonstrated a chemodiversity of the oil content, such as Eucaliptol, Limonene, isomers of Terpineol, and others [9].
Interest in the use of Melaleuca EO is increasing in Brazil, and thus the quality of these oils is important to maintain the control of microorganisms in the areas of health, agriculture and in industries. The studies in these areas have been focused on the antimicrobial actions of the oil in the control of diverse types of fungi, bacteria, and virus [9, 12, 13]. In Brazil, the Melaleuca EO commercialized is produced on small agricultural farms, or imported from various other countries, and for this reason, divergencies in the quality and efficacy of the oil in the combat of microorganisms has been constantly observed [14]. Essential oils can present quality problems due to environmental conditions that intervene in the composition and percentage of the principle active constituents [15]. In addition, the product may be adulterated by the addition of synthetic compounds or of less valuable EO or even the complete falsification of the oil by mixing with synthetic substances dissolved in an inert vehicle [15]. Also, the sale of M. armillaris oil as M. alternifolia oil is common in Brazil, due to errors in species identification. Thus, this study aimed to propose some metrics that could be used in the quality control of Melaleuca armillaris EO, extracted from plants cultivated in Brazil, to differentiate it from Melaleuca alternifolia oil.
The M. armillaris used in this study was cultivated in a green area at the Botany Laboratory of UNIVAS, Pouso AlegreMG, Brazil (register nº 003, Herbário UNIVAS). The oil was extracted from leaves of the M. armillaris by hydrodistillation, a technique standardized for this type of essential oil [9]. A total of 5 kg of dry M. armillaris leaves were used to obtain 150mL of EO. The following tests were used to characterize the EO and identify its components:
® The density of the EO was evaluated using a 30 PX densimeter (Mettler Toledo, Brazil), which uses the oscillating body method. The equipment was calibrated at the reference temperature and duly cleaned and dried.
The pH value of the M. armillaris oil was evaluated using a model 913 pH-meter (Metrohm, USA). The equipment was calibrated with buffers at pH values of 4, 7 and 9 to increase the reliability, and the results obtained for the asymmetric pH value and the slope parameters used to measure the sensitivity and efficiency of the electrode, were satisfactory.
® This test was carried out using an Alemmar ADP220 polarimeter (Bellingham & Stanley, USA) belonging to the Physicochemical Quality Control Laboratory of the União Química Farmacêutica Nacional S/A, Brazil. The reading was made directly with no sample preparation, with the sample at 20 ºC. The equipment was autozeroed using air before starting the test, and the sample then inserted into a glass tube belonging to the equipment and placed in the reading chamber. After a few seconds the value of the rotation angle appeared in the viewfinder, allowing for the measurement of the value of polarized light.
This test was carried out in the Physicochemical Quality Control Laboratory of the União Química Farmacêutica Nacional S/A, Brazil, and the refractometer verified before carrying out the test using water at 25 ºC. The temperature was carefully adjusted and maintained, since the refractive index varies significantly with temperature [16]. After filling the whole orifice and adjusting the temperature in the viewfinder, the refractometer was verified, and the sample read.
A Vapro model 5520 (Wescor, USA) osmometer was used, calibrated with 290mmol/kg, 1000mmol/kg and 100mmol/kg standards to guarantee the precision of the result. A volume of 10μL of M. armillaris EO was aspirated with the aid of an automatic pipette and dispensed in the sample reading chamber of the equipment. After dispersion of the sample on the paper membrane destined for this purpose, the collection key of the equipment was switched on and the reading taken.
The viscosity was determined in the Physicochemical Quality Control Laboratory of the União Química Farmacêutica Nacional S/A, Brazil, using a Brookfield model LVDV viscosimeter (BrasEq, Brazil), which allows for the velocity to be adjusted in rotations per minute using the display. The equipment was adjusted to a velocity of 10 rotations per minute (RPM), an auto-adjustment carried out to certify there was no type of interference, and spindle nº 34 selected for the determination [17].
A model 8000A HIAC counter was used (Beckman Coultre, Brazil), coupled to a sampler which sucked in 5 mL three times, ignoring the first reading, and using the other two for the calculations. This particle counter works based on the light blocking principle, allowing for the determination of the particle sizes and their numbers according to the dimensions. One of the reading channels provided results for particles smaller than 10μm and another for particles larger
The moisture content was determined in the laboratory of the company Metrohm, using a Karl Fischer 915 KF Ti® Touch equipment (Metrohm, Switzerland). Before starting the analysis the equipment was conditioned with Karl Fischer solution to equilibrate it and remove any water. The analysis was carried out in duplicate to minimize possible analytical errors. The first sample weighed 1.3831 grams and the second 1.3743 grams. When titration started a graph of volume by time was provided by the software coupled to the equipment, and after a few minutes an inflection was formed on the curve followed by the constant volume, and hence the results were provided by the program.
® This analysis was carried out using the TGA, DSC equipment (Mettler Toledo, USA) in the laboratory of the company Mettler Toledo. The sample was placed in the small platinum dish and the equipment temperature programmed. Subsequently 80.3182 mg of sample were inserted into the equipment, which was programmed for a temperature range of from 30 ºC to 600 ºC, with a heating rate of 10ºC per minute.
A new method to identify M. armillaris oil by gas chromatography was elaborated in the present study, and compared with that developed by Falci et al., 2015 [9]. The present method presented a chromatographic profile similar to that found in the literature but with reduced time and reduced carry-over risk. The method with which it was compared used a mass selection detector whereas the present study used a flame ionization detector.
The oil components were identified in the laboratory of the company Agilent Technology using a method that did not use the headspace but used the model 7693 sampler ® (Agilent , USA). The solvent dichloromethane (Merck batch: K51006944911) was used to extract the components, the use of 2 mL vials with no cuts on the septum being imperative so that neither the solvent nor the analytes suffered any significant component losses.
Liquid chromatography was carried out in the application laboratory of the company Waters Corporation using the ® ACQUITY H-class UPLC, XEVO equipment (Waters, USA). The HPLC chromatogram obtained was compared with the equipment library of 6000 compounds, thus aiding identification of the substances.
This test was carried out in the laboratory of the company Metrohm using the model Mira M3 Raman equipment (Metrohm, USA). The sample was introduced into the equipment vial and then placed in the reading compartment.
This test was carried out in the laboratory of the company ® Metrohm using the model NIRS XDS infrared spectroscope (Metrohm, USA). A tip specific for liquid samples was introduced onto the probe of the laser, using a ring according to the viscosity of the sample, and the spectral reading of the M. armillaris oil subsequently made.
The value found for the density of the M. armillaris oil (Table 1) was close to that proposed by the monograph for Melaleuca alternifolia, which shows a specification between 0.885 and 0.906 g/mL. Small variations can occur due to the glassware or instrument used, and on an industrial scale the density is very important to determine the filling volume. The result observed was different from that suggested for M. alternifolia oil.
a Identification by GC 24 compounds b Identification by HPLC 145 molecules Identification RAMAN Identified (Absorption at 785nm) Identification by NIR Identified (Absorption at 2350nm) GC: Gas Chromatography HPLC: High performance liquid chromatography NIR: Near infrared a = described in Table 2 b = described in Table 3 The M. armillaris EO is more acid than that of M. alternifolia, which has a pH value of 5.03 [18]. Thus, the correct identification of the EO and measurement of the pH value is extremely important, since this is the main factor influencing the decomposition of drugs. The association of drugs with distinct pH values can lead to instability, increasing the risk of incompatibility [2]. In addition, if the EO is used in a product destined for topical application the control of the cutaneous pH value must be controlled due to the aggressivity of inadequate topical products [19]. The acid pH value found in the present study (Table 1) favors its antimicrobial action for agricultural formulations and for most microorganisms, both for those that deteriorate foods and for human bacteria such as Staphylococcus aureus [9]. The specific rotation test or polarimetry as used in the identification of M. armillaris EO, demonstrated a polarized light deviation of the value described in Table 1. The specific rotation for M. alternifolia is between 5-15º and the result can be influenced by various factors such as temperature and distinct chemical constituents [17]. This is an important parameter that differentiates the oil of M. armillaris from that of M. alternifólia.
The refractive index is employed mainly to characterize fats, fatty oils, waxes, sugars, and organic solvents, and some drugs. It is used to determine the purity of volatile oils. The value obtained for M. armillaris oil (Table 1) was below the values for M. alternifolia oil (1.475 – 1.482), possibly due to chemical differences between them [17]. Thus, this technique is important in the differentiation between the oils produced by the two species.
The osmotic pressure or osmolarity plays a critical role in all biological processes that involve solute diffusion of fluid transfer through membranes [16]. The equipment did not detect a reading for the osmolarity of M. armillaris oil (Table 1), signifying no evaporation of the components of this oil at room temperature.
The value obtained in the viscosity test showed low resistance of the fluid to flow. A low viscosity directly influences the characteristics of creams and ointments, guaranteeing stability and quality to the herbal medicine and aiding in consumer acceptance, since this depends on the appearance, sensation on the first contact with the skin, spreadability and residual oiliness after application [20]. The low viscosity of 3 centipoises of the M. armillaris oil (Table 1) provides these results and facilitates the filling process of the oil using industrial scale machinery.
Membrane filtration showed large amounts of particles ≥ 10μm and between 10 and 25μm (Table1). The large number of particles found in the particulate material test could be an indication of pollutants coming from the environment, collection and other factors that lead to undesirable effects in the formulation [21]. Thus, the filtration of the EO before incorporating it into a formulation is imperative. Considering the literature review carried out, it appears that this was the first time this test was applied to M. armillaris oil in Brazil, and no papers were found concerning this type of information for M. alternifolia oil. The M. armillaris oil showed a low moisture content, favoring its use in formulations. The moisture of essential oils interferes in the quality of the formulation, and on its shelf life and stability. An excess of water in vegetable raw materials makes enzyme action possible and allows for the development of fungi and bacteria [22].
The thermogravimetric analysis demonstrated the initial allowed for its non-refrigerated storage (Table 1), thus avoiding increases in moisture content, hydrolysis and other phenomena. Similar storage properties are recommended for Melaleuca alternifolia [17]. The test also allowed for the determination of the ash content, which evaluates the quantity of inorganic impurities present in the raw material, such as, for example, metal ions. This may have been the first time this type of thermogravimetric analysis was applied to M. armillaris oil in Brazil and in addition, no papers providing this type of information were found for M. alternifolia.
A new gas chromatographic method was developed in the present study in which the run time was reduced from 39 to 26 minutes, when compared with Falci et al., 2015 [9]. Another advantage of the new method was the adjustment of the parameterized injector syringe such that it only collected the gas formed by the boiling of dichloromethane (solvent), which could significantly decrease contamination phenomena. Using gas chromatography, 24 chemical compounds were identified in the M. armillaris EO, the main one being 1,8 cineole (Table 2), as already reported in other papers [1, 23-26], the active principal of Melaleuca alternifolia being terpinene-4-ol.
The identification test using HPLC with mass spectrometry allowed for the identification of 145 compounds in the M. armillaris oil (Table 3; Figure 1). Since this equipment recognizes isomers, some of the compounds appear to be repeated. This test was very interesting since it showed a much greater number of compounds than that found by gas chromatography.
In the identification of M. armillaris EO by Raman, the spectrum showed a single identity (wavelength of 785nm), as shown in Table 1 and Figure 2, any spectrum obtained using the same technique and presenting different bands possibly being an indication of adulteration, problems in obtaining the oil, or cross contamination due to inadequate cleaning of the extraction apparatus. Similarly, using NIR spectrophotometry, a single identity was found for the M. armillaris oil (absorption at 2350nm) (Table 1; Figure 3). Due to the characterization of M. armillaris oil using two identification techniques, NIR and Raman, the test using ATR-IR was not carried out. Infrared and Raman spectroscopy are constantly used in the identification, characterization and elucidation of structures of molecules and compounds, as well as in the monitoring and control of chemical reactions. Both techniques measure the levels of vibrational energy associated with chemical bonds in the sample through the interaction of electromagnetic radiation with the vibrational movement of the nuclei, which generates the vibrational spectrum. Such spectra display a greater wealth of details about the analyzed material to the detriment electronic spectra, which are made up of broad, deformed bands, while vibrational spectra are unique spectra of each material and the molecules that make it up [27]. Dimethyl methylene bicyclic heptane Methylene methylethenyl cyclohexane Methyl methylethylidene Tetramethyl benzene Isopropylidene methyl bicyclic hexane Terpineol isomer Terpineol isomer Terpineol isomer Decahydro trimethyl methylene Cyclopropuzulene Aloaromadendrene Octahydro tetramethyl cyclopropazulene Hexahydro Dimethyl methyl ethyl naphthalene Octahydro Dimethyl methyl ethenyl azulene Hexahydro Dimethyl methylethyl naphthalene
2 6.47 0.86 3 6.64 0.88 Myrcene 4 6.79 0.90 5 7.02 0.93 6 7.28 0.96 7 7.51 0.99 Limonene 8 7.56 1.00 Eucalyptol 9 8.12 1.07 Terpinene 10 8.70 1.15 11 10.28 1.36 12 10.45 1.38 13 10.70 1.42 14 17.43 2.31 15 19.81 2.62 16 21.17 2.80 17 22.20 2.94 18 22.47 2.97 19 23.50 3.11 Unknown 20 24.00 3.17 Unknown 21 24.32 3.22 Globulol 22 24.53 3.24 23 24.85 3.29 Eudesmol 24 25.12 3.32 RT = Retention time Relative RT = Retention time in relation to 1,8 cineole %A = percentage of component in the sample
2.58 0.04 3.28 cyclohexane 0.22 0.23 9.42 69.70 0.41 0.08 0.02 0.09 0.45 0.12 0.06 0.05 0.10 0.66 0.08 0.05 0.09 0.38 0.12 0.09 13 268.1680 49 1β-Hydroxycolartin 14 Dihydroactinidiolide 180.1154 50 15 Dihydroeugenol 166.0995 51 16 3-Phenyl-2-butanone 148.0893 52 17 (25R)-Spirostan-4-ene-3,12-dione 426.2751 53 18 Nardosinone 250.1572 54 19 Δ1(10)-Aristolen-9-ol 220.1829 55 20 Dihydroeugenol 166.0996 56 21 Thymol isobutyrate 220.1462 57 22 Δ1(10)-Aristolen-9-ol 220.1830 58 23 M- Isopropyltoluene 134.1099 59 24 3-Phenyl-2-butanone 252.1730 60 25 14(R )-Hydroxy-7ß- 336.2304 61 isovaleroyloxyplop-8(10)en-2-0ne 26 Nardosinone 250.1575 62 27 Dihydroeugenol 166.0996 63 28 Orientalol E 254.1887 64 29 Nardosinone 250.1572 65 30 Cavacrol 150.1047 66 31 Nardosinone 250.1575 67 32 Nardosinonediol 252.1729 68 33 Cumene 120.0944 69 34 25-O-Acetyl-cimigenol 530.3582 70 35 Progesterone 314.2253 71 36 Diethylamine hydrochloride 108.0579 72 73 Apocynin 166.0633 110 74 Turmerone 218.1674 111 75 Campest-7,24(28)-dien-3ß-ol 398.3544 112 76 Turmerone 218.1873 113 77 Debilone 234.1624 114 78 1-Methyl-3(1-methyl –ethyl)- 148.1257 115 benzene 79 1-Methyl-3(1-methyl –ethyl)- 148.1256 116 benzene 80 Nardosinonediol 252.1730 117 81 410.3553 118 Δ7-Stigmasterol 82 2-octyphenol 206.1675 119 83 Debilone 234.1624 120 84 Turmerone 218.1677 121 85 m-Isopropyltoluene 134.1099 122 86 Δ(10)-Aristolen-9-ol 220.1830 123 87 Dendronobilin B 284.1611 124 88 Ergot-7,22-diene-3ß,5α,6ß-triol 430.3445 125 89 Allyl Disulfide 146.0228 126 90 Carvacrol 150.1048 127 91 1,3-cyclooctadiene 108.0942 128 92 m-Isopropyltolueno 134.1100 129 93 Epi-α-Bisabolol 222.1988 130 220.1832 Δ1(10)- Aristolen-9-ol Debilone 234.1624 Epi-α- Bisabolol 222.1986 Fureanodiene 261.1518 Debilone 243.1625 220.1828 Δ1(10)- Aristolen-9-ol Debilone 234.1622 Debilone 234.1623 Δ1(10)- Aristolen-9-ol 220.1831 Chenodeoxycholic acid 392.2914 (-) Calamenene 202.1722 Methyllisoeugenol 178.0993 Vellediol 236.1779 (-) Calamenene 202.1724 1 methyl-3-(1methyl-ethyl)-benzene 148.1256 Bufalin 386.2463 Orientalol E 254.1889 Vellediol 236.1779 Spinasterone 410.3555 Calamenene 202.1726 Tetramethoxy-trans-stilbene 300.1347 Piperitone 152.1202 Debilone 234.1625 Flavenochromane 440.1818 Piperitone 152.1204 ß-Myrcene 136.1257 Nardosinone 250.1573 Dehydrololiolide 194.0946 14(R ) Hydroxy-7ß- 218,1677 isovaleroyloxyoplop-8(10)-en-2-one Turmerone 218.1677 Chenodeoxycholic acid 392,2908 2α-Acetoxycostic acid 292.1683 Diisobutyl phatel 278.1527 Sinenofuranol 238.1939 3-Butanone 72.0575 Guaiene 204.1884 Turmerone 218.1676 Carvacrol 150.1049 Guaiene 204.1881 Chenodeoxycholic acid 392.2908 Turmerone 218.1678 Calamenene 202.1726 1-Methyl-3-(1 methyl-ethyl)-benzeno 148.1258 Debilone 234.1627
standardization of its use and more predictable results for formulations that use it. In addition, the identification of the compounds in the oil helped in the creation of new products and drugs, diversifying the application of M. armillaris oil in the health area. Knowledge concerning the biomass of M. armillaris oil allows for the identification of possible interactions with other chemical substances which could lead to risks for the patients.
This study established some parameters for M. armillaris EO. Amongst these, the main parameters that differentiate this EO from that of Melaleuca alternifolia are the principle active constituents (1,8 cineole and terpineol, respectively). The pH value, specific rotation, refractive index and Raman are also important metrics in the differentiation of these oils, since they establish very particular characteristics for the Melaleuca armillaris EO.
The authors thank the companies União Química Farmacêutica Nacional®, Metrohm®, Mettler Toledo®, where the tests in this study were carried out.
Author declares no conflict of interest.
This study is self funding and in collaboration with companies União Química Farmacêutica Nacional®, Metrohm®, Mettler Toledo®.
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