Abstract
Chemical compositions of seven essential oils from Taiwan have been analyzed by gas chromatography−mass spectroscopy (GC−MS). A step gradient was utilized to separate the complicated components from essential oils in high throughput and excellent resolution. The eluates have been identified by matching the mass fragment patents to the National Institute of Standards and Technology (NIST) 08 database.
Quantitatively analysis showed the major components of lemon verbena are geranial (26.9%) and neral (23.1%); those of sweet marjoram are -terpinene (18.5%), thymol methyl ether (15.5%) and terpinen-4-ol (12.0%); those of clove basil are eugenol (73.6%) and -(Z)-ocimene (15.4%); those of patchouli are carvacrol (47.5%) and p-cymene (15.2%); those of rosemary are -pinene (54.8%) and 1,8-cineole (22.2%);
those of tea tree are terpinen-4-ol (33.0%) and 1,8-cineole (27.7%); and those of rose geranium are citronellol (28.9%) and 6,9-guaiadiene (20.1%). These components are somewhat different from the same essential oils reported that are obtained from other origins. Lemon verbena has the same major components everywhere. Tea tree, rose geranium, and clove basil have at least one major component throughout different origins. The major components and their amounts for sweet marjoram, patchouli, and rosemary are very different from various places. This result demonstrates that the essential oils have large diversity for their composition with different origins.
The antibacterial activity of the essential oils against Escherichia coli was evaluated by optical density method (turbidimetry). Patchouli is a very effective inhibitor that completely inhibits the growth of E. coli at 0.05%. Clove basil and sweet marjoram are good inhibitors and their upper limits of minimum inhibitory concentration are 0.1%.
Introduction
Essential oils have a long history of being utilized in daily life on both spiritual and practical. In recent decades they have been widely used in aromatherapy as the alternative medicine to smooth mind and emotion, and, consequently, improve health.1 Each essential oil has its unique and complex mixture of components, and, therefore, delivers numerous health benefits.2 Components of essential oils may be direct related to their psychological and physical therapeutic benefits and, therefore, it is important to determine each essential oil’s chemical composition. Documentation of the contents of the essential oils not only makes the investigation of the influence between essential oils and claims possible, but also builds a common ground for discussion.
In recent years, essential oil has been connected with the electrophysiological research.3 Brain waves are electromagnetic waves that neurons emit continuously, and are classified by frequency into four categories, , , , and waves. Each category has its unique forms, and the interpretation is an ongoing research. Brain waves have been investigated in many fields including brain physiology, pharmacology, neurology, and psychiatry. Essential oils are believed to influence the brain waves through the sense of smell and to further affect cognition and/or mood.4-6 Recently
psychopharmacological researchers, Moss and Oliver have found the relationship between the cognitive behavior and rosemary essential oil. The participants were asked to do simple math under the ambient aroma of rosemary essential oil. They found that higher concentration of 1,8-cineole (the major component for rosemary) absorbed in the serum lead to better performance on serial number subtraction in both speed and accuracy.7 Exploration in this research area is expected to escalate.
Plant products were the principal sources of pharmaceutical agents used in
traditional medicine. Some medicinal plants are rich in antimicrobial reagents.8,9 Several essential oils derived from varieties of medicinal plants are known to possess insecticidal, antifungal, antibacterial and anti-inflammatory activities.10-13 On the other hand, some flowering plants (angiosperms) were used as food preservatives in ancient Egyptian times. The main active components in those plants are believed to be the volatile C10 and C15 terpenes that are formed by isoprene units. Terpenes are found in many plants and often have strong smell to repel pests. Harmless antioxidants have been searched from various sources including wild herbs, spices, fruits, nuts, and leafy vegetables. Nowadays many essential oils are also known as strong natural antioxidants.14,15
Many components of the essential oils have distinct flavors and are often used in commercial perfume and fragrance. Some of these components are easy to synthesize chemically. One can image that spike few synthetic compounds into semi-finished product is much easier than extracting natural essential oils. Due to the large difference on the production cost, synthetic oils may be used to counterfeit natural essential oils. Qualitative and quantitative analyses must be incorporated to ensure the quality of commercial essential oils.
The contents of essential oils are mostly hydrophobic liquids which are volatile organic compounds. Gas chromatography (GC) is an ideal analysis tool for the volatile liquids. The foremost application of GC is to identify the purity of a specific sample, or to separate the various components from a mixture. Equipped with highly sensitive flame ionization detector (FID), GC analysis is routine and reliable. FID is best for detecting hydrocarbons and other volatile compounds which has a linear response between a wide range of sample concentrations. However, it lacks the ability to indentify the separated components which can be fulfilled by mass spectrometer (MS). Mass spectrometer, combined with commercial available databases, is ideal for
analyzing unknowns. After the electron ionization, analyte will break into
characteristic and reproducible fragments. Commercial available database facilitates the identification of the unknowns. A combination of GC and mass spectrometer gives the best approach for routine analysis of essential oils. In this chapter, GC−MS has been exploited for both qualitative and quantitative analysis. Due to the complexity of the essential oils, developing a standard method is essential for separation and
analysis. Few standard compounds were chosen for getting the absolute concentration.
Seven local essential oils in Taiwan were analyzed by the method developed. The chemical composition of essential oils (even the same) from plants is dependent on many factors, including the location and climate of the origin, and the season that the plants have been harvested. In this chapter, we have determined the chemical
composition of seven essential oils that are extracted from the plants that were grown and harvested in Ji-an Town, Hualien, Taiwan. Their antibacterial activities, one of the intriguing powers of essential oils, against Escherichia coli (E. coli) were also
investigated. The chemical composition and antimicrobial activity were compared to same species from other origins according to literature reports.
Experimental Section
Materials
Ethyl acetate was ACS grade and was purchased from Mallinckrodt. Geraniol (99%) and 1,8-cineole (99%) were purchased from Acros. -caryophyllene (98.5%), n-paraffin C7, C8, C9, C10 mix and C10, C12, C14, C16 mix were purchased from Aldrich. Sodium chloride was purchased from Merck. Tryptone (casein hydrolysate) enzymatic digest, yeast extract and agar were purchased from USB Corporation. All chemicals were used as received without further purification. Fresh leaves of Lippia
citrodora (Paláu) Kunth (lemon verbena), Origanum majorana L. (sweet marjoram), Ocimum gratissimum L. (clove basil), Pogostemon cablin Benth. (patchouli),
Rosmarinus officinalis L. (rosemary), Melaleuca alternifolia Cheel (tea tree) and Pelargonium graveolens L’Hér. (rose geranium) were collected from the plants grown in Ji-an Town, Hualien, Taiwan. The plant samples were identified and the voucher specimens were deposited at the Herbarium (professor Jenn-Che Wang’s lab) of the Department of Life Science, National Taiwan Normal University. Voucher numbers are as the following: Pogostemon cablin Benth (TNU055241); Pelargonium
graveolens L’Hér (TNU055242); Origanum majorana L. (TNU055243); Ocimum gratissimum L. (TNU055246); Melaleuca alternifolia Cheel (TNU055247); Lippia citrodora (Paláu) Kunth (TNU055248); Rosmarinus officinalis L. (TNU055249). All seven essential oils were extracted from air-dried leaves of the plant by
hydrodistillation for 40 minutes using a Clevenger type apparatus. The oil samples obtained were dried over anhydrous sodium sulfate (Na2SO4) and stored in sealed vials in a cool and dark place before analyses.
Sample preparation
All the samples were volumetrically diluted in ethyl acetate prior to gas
chromatography injection. Absolute concentration calibration curves were established for geraniol, 1,8-cineole and -caryophyllene. Standards were diluted to five
appropriate concentrations, from 25 to 500 ppm. Essential oils were diluted to a thousand times.
Instrument
Gas chromatography (GC) was performed on Agilent Technologies 6850 Series II equipped with a flame ionization detector (FID). The capillary column was
HP-5MS cross bond (5% diphenyl- and 95% dimethyl- polysiloxane, 30 m x 250 m x 0.25 m). The injector and detector temperatures were both set at 250°C. Nitrogen was utilized as carrier gas, and the flow rate was set to constant mode (1 mL/min).
Injection volume was 1 L and the injection mode was splitless. The temperature was raised by a step gradient which start at 60°C for 15 minutes then quickly go up to 80°C in 4 minutes, followed by a slow rise to 135°C in 55 minutes, and finally a rapid rise to 260°C in 8 minutes and hold for 2 minutes. Gas chromatography−mass
spectrometer (GC−MS) analysis was performed by Hewlett-Packard 6890 Gas Chromatograph and Hewlett-Packard 5973 Mass Selective Detector. The capillary column was also HP-5MS cross bond. The injector and detector temperatures were set at 250°C and 230°C, respectively. Helium was utilized as carrier gas, and the flow rate was set to constant mode (1 mL/min). Injection volume was 1 L and the injection mode was split with 50:1 ratio. Two temperature rising programs were utilized, a linear gradient and a step gradient. The former increases the temperature from 60ºC to 260ºC at a rate of 2ºC/min. The latter was same with GC condition.
Ionization voltage was 70 eV by electron impact. The acquisition mass range was set from 30 to 650 amu. The mass spectra were searched and compared through database of National Institute of Standards and Technology (NIST) 08 libraries for
characterization. Relative percentage amounts were calculated on the basis of peak areas for both GC and GC−MS analysis and the results were very similar. To confirm the high percentage components, standards were used to spike into the sample.