Abstract
Subcritical water treatment with pressure-releasing distillation was proposed as a method to obtain essential oil from Japanese mint (Mentha arvensis L. var. piperascens Malinv. cv.). The extraction and fractionation were carried out between 100 °C and 220 °C, and extraction times of 5 to 60 min were investigated. Quantitative analysis of volatile component was conducted by gas chromatography. The yields of both l-menthol and l-menthone converged in the initial fractions as the extraction temperature was increased. The yields of l-menthol, l-limonene, l-menthone, and piperitone increased as the temperature was increased from 100 °C to 180 °C, but decreased as it was raised further to 220 °C. The yields of iso-menthone, 3-octanol, and l-menthyl acetate increased as temperature was increased up to 220 °C. The extraction time of 60 min showed a negative effect on the extraction, and the highest total yield (30.5 mg/g-dry leaves) was obtained at 180 °C with an extraction time of only 5 min.
Introduction
Essential oils are used worldwide in aromatherapy, as the fragrances have various therapeutic and healing effects (Ali et al., 2015). Essential oils are also used as food additives and in food preservation for their antimicrobial and antioxidant activities (Tongnuanchan and Benjakul, 2014). A general definition of essential oils has been given in the seventh edition of the European Pharmacopoeia (Aziz et al., 2018) as: “Odorant product, generally of a complex composition, obtained from a botanically defined plant raw material, either by driving by steam of water, either by dry distillation or by a suitable mechanical method without heating. An essential oil is usually separated from the aqueous phase by a physical method that does not lead to significant change in its chemical composition.” However, the description of extraction methods for obtaining essential oils is somewhat limited in the European Pharmacopoeia, as some extraction techniques that are not encompassed by this definition have been developed, modified, and applied in practice. Generally, the extraction methods can be classified as conventional/classical methods and innovative techniques. The former includes hydrodistillation, entrainment by water steam, vapor-hydrodistillation, steam distillation, hydrodiffusion, cold pressing, and extraction with organic solvents. The latter class includes subcritical water extraction, supercritical carbon dioxide extraction, ultrasound-assisted extraction, microwave-assisted extraction, and instant controlled pressure drop (El Asbahani et al., 2015). Among these extraction methods, subcritical water extraction has been reported to be a powerful alternative based on its merits of short extraction times, low cost, and environmental friendliness (Herrero et al., 2006).
Subcritical water refers to water that has been heated to beyond the boiling temperature of 100 °C but below the critical point of 374 °C, and the liquid state is maintained by its high vapor pressure. Subcritical water has remarkable characteristics of high ionic products and a low dielectric constant (ε). Regarding the ionic products, the self-ionization of pure water is promoted as the temperature increased, and the molar concentrations of hydrogen and hydroxide ions thus increase. For example, the ionization constant of water (pKw) is 14 at 25 °C, but decreases to 11 as the temperature is raised to 250 °C. That is to say that a thousand-fold increase in ionic products occurs as the temperature is increased from 25 °C to 250 °C, and both acid- and base-catalysed hydrolysis are thus enhanced (Marshall and Franck, 1981; Patrick et al., 2001). The dielectric constant is a value related to polarizability. At 25 °C, water has higher dielectric constant (ε = 80) than those of organic solvents such as methanol (ε = 33) and ethanol (ε = 24) (Wagner and Pruß, 2002). However, as the temperature of water is increased from 25 °C to 250 °C, its dielectric constant decreases from 80 to 27, and the polarity becomes closer to that of methanol and ethanol (Alghoul et al., 2018). Since these characteristics of high ionic content and a low dielectric constant benefit the extraction process, subcritical water is regarded as an excellent extractant for recovering functional substances from plants, algae, microalgae, and food and agricultural by-products (Herrero et al., 2006; Chiou et al., 2011; Tangkhavanich et al., 2012; Chiou et al., 2013).
A batch-wise method is usually applied for subcritical water extraction since it is easier to carry out and less costly than continuous methods. In our previous study (Nomura et al., 2019), subcritical water was employed in a batch-wise process to extract compounds from Japanese mint leaves between 180 °C and 260 °C, and various substances including aromatics, sugars, proteins, and phenolics were obtained. However, the batch-wise method is not particularly specialized for obtaining essential oil. Based on the Antoine equation (Thomson, 1946), the vapor pressure of water is 0.367, 1.083, and 2.383 MPa at 140 °C, 180 °C, and 220 °C, respectively. Since essential oil is a volatile mixture, it is considered that the essential oil could be extracted more efficiently by superheated steam in a pressure-releasing process. In this study, we proposed a modification of the batch-wise method that was specialized for the recovery of essential oil. As a model plant for extraction of essential oil, Japanese mint (Mentha arvensis L. var. piperascens Malinv. cv.) were used. Essential oil of Japanese mint has been commercially produced by traditional steam extraction in Japan. Generally, the essential oil yield of Japanese mint is approximately 1.5% (w/w, dried above-ground part) and the content of l-menthol is 70–80% (w/w) (Nagasawa, 1960). The steam extraction is the major process for obtaining essential oil, however, the effect of temperature on the composition and yield of essential oil is rarely investigated. To investigate the temperature effect and to improve the extraction efficiencies of mint essential oil, pressure-releasing distillation was performed in conjunction with batch-wise subcritical water treatment, and fractionation was carried out for subsequent evaluations.
Materials and Methods
Japanese mint The material used in this study was Japanese mint (Mentha arvensis L. var. piperascens Malinv. cv) with the cultivar name ‘hokuto’. The crop was grown in Nikoro-cho, Kitami, Hokkaido for about 3.5 months (from mid-March to late August). The mint plants were harvested in early September, and dried under the sun in a field for about 3 weeks. After removing the stems, the dried leaves were stored at −80 °C until use.
Reagents Phenol was purchased from Sigma Aldrich, Japan (Tokyo, Japan). Chloroform was procured from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). l-Menthol, l-menthone, piperitone, l-menthyl acetate, 3-octanol, and l-limonene were purchased from TCI (Tokyo, Japan). All reagents used were reagent grade.
Mint essential oils Essential oils obtained by traditional steam distillation in large and small scales were used for a comparison of the chemical compositions of the oils. For the large-scale extraction, a Tanaka-style mint distillation kettle with a cylindrical tank (1.4 m height × 1.35 m diameter in the upper part, 1.5 m diameter in the lower part) was used to extract the essential oil. Whole dried mint plant (approximately 1 ton) was used as the starting material. Steam was generated by a steam boiler and injected to the bottom of the tank. The operation was carried out under ordinary pressure for approximately 2 hrs. For the small-scale extraction, an Herb oil maker (standard type for lab., Tokyo Seisakushiyo, Tokyo) with a cylindrical tank (12.3 cm i.d. × 27 cm height) was used to extract essential oil from approximately 100 g of the dried mint leaves. The steam was generated by heating distilled water (500 mL) with an induction cooking which was placed under the cylindrical tank. The operation was carried out under ordinary pressure for approximately 1 hr.
Subcritical-water treatment with pressure-releasing distillation The apparatus for the subcritical-water treatment with pressure-releasing distillation is illustrated in Fig. 1. A pressure-resistant SUS-316 stainless steel vessel (TVS-N2 type, Taiatsu Techno, Osaka) with an internal volume of 117 mL (3.0 cm i.d. × 16.6 cm height) was used for the extraction. Maximum operating temperature and pressure of the vessel are 260 °C and 20 MPa, respectively. A mantle heater (TXN-700B, As One, Osaka) was used for heating the vessel.
As shown in Fig. 1, 5 g of dried leaves and 100 mL of distilled water were placed in the vessel (mark c) and the vessel was screwed tightly shut. The vessel was coated and heated by a mantle heater (mark d) connected to a thermal controller (mark a) and a temperature sensor (mark b). The temperature was increased up to the specified value (100, 140, 180, or 220 °C) at a heating rate of 5.1–5.5 °C /min (Jeyashoke et al., 2010), and the specified temperature was held for 5 min for extraction (Chiou et al., 2012). After the 5-min extraction, the valve (mark e) was gently unfastened, and the high-temperature steam flowed through the stainless steel loop (mark f, 3.18 mm o.d. × 1.6 m with a thickness of 0.71 mm) and the glass loop (mark g, 3.2 mm i.d. × 1.5 m; GC-8A series, Shimadzu GLC Ltd., Tokyo). The glass loop was kept at 30 °C in the water bath (mark h), the temperature of which was regulated by a temperature controller (mark i) linked to a cooling loop (mark j). The steam then entered the glass loop (mark g), wherein it was condensed into a liquid state and became visible. The liquid was collected in 10 mL portions per tube (mark k), with 5 tubes collected for each extraction. In addition to the 5-min extractions at 100, 140, 180, and 220 °C, 30-min and 60-min extractions were also carried out at 140 °C.
Gas chromatography (GC) analysis A membrane filter (0.45 µm, cellulose acetate; Advantec, Toyo Roshi, Tokyo) was used to filter the extracted liquid. To concentrate the aroma components, 1.0 mL of chloroform was added to 10 mL of the filtered extract liquid and the mixture was mixed thoroughly. A centrifuge (MX-307, TOMY, Tokyo) was used to separate the water and chloroform layers at 10,000 × g for 5 min. The chloroform layer was then mixed with an equal volume of a 2% (w/w) phenol-acetone solution. One micro-millilitre of the mixed sample was injected into a GC (Shimadzu, Kyoto) equipped with a TC-WAX capillary column (30 m × 0.25 mm i.d.; GL Science, Tokyo). Nitrogen gas was used as both the carrier gas and the make-up gas. The temperatures of the injector and FID detector were both set to 250 °C. The oven temperature was held at 50 °C for 3 min, increased to 110 °C at 15 °C /min, to 150 °C at 3 °C /min, to 200 °C at 15 °C /min, and held at 200 °C for 5 min. Including an interval of 10 min, each run took about 40 min. For quantitative analysis, the relative response factors (RRFs) between reference standards (aromatic components) and phenol (as an internal standard) were evaluated in triplicate.
Results and Discussion
The fractionation at different extraction temperatures Since l-menthol and l-menthone are the most two abundant components in the essential oil of Japanese mint, these two components were used as the representative compounds to study the effect of fractionation. The results of the fractionation at different extraction temperatures between 100 °C and 220 °C are shown in Fig. 2A. At 100 °C, both l-menthol and l-menthone were extracted in similar yields in the first three tubes (0–30 mL), and the yields decreased thereafter (31–50 mL). At 140 °C, the yields of both l-menthol and l-menthone were twice as high in the second tube (11–20 mL) than in the first tube (0–10 mL).
At 180 °C, the yields of l-menthone were 1.92, 1.76, 0.41, 0.18, and 0.08 mg/g-dry leaves in tubes 1–5, respectively. The first two tubes (0–20 mL) accounted for about 85% of the overall yield of the five tubes (0–50 mL). The same tendency was observed for l-menthone extracted at 220 °C, although the yields were lower in each of the five tubes compared to the corresponding yields at 180 °C. It was proposed that some of the substances could have degraded under the subcritical water conditions, especially at high temperatures over 200 °C (Chiou et al., 2011; Kuhlmann et al., 1994).
The yields of l-menthol extracted at 180 °C in tubes 1–5 were 9.15, 9.49, 2.49, 1.22, and 0.48 mg/g-dry leaves, respectively, with the first two tubes accounting for about 82% of the combined yield of the five tubes. The yields of l-menthol at 220 °C were lower yields than those at 180 °C, following a tendency similar to that for l-menthone.
For l-menthone, the first two tubes accounted for about 54%, 69%, 85%, and 87% of the total yields of the five tubes when extracted at 100, 140, 180, and 220 °C, respectively. The same tendency was also observed for l-menthol. This indicates that the yields of aroma components converged in the initial fractions as the extraction temperature was increased. These results suggest that extraction efficiency could be improved by collecting the fractions containing greater amounts of aroma components.
To evaluate total amount of aromatic components, seven aromatic components, including l-menthol, l-menthone, iso-menthone, piperitone, l-menthyl acetate, 3-octanol, and l-limonene, were quantitated. The combined yields of the 7 aroma components from the 5 tubes (0–50 mL) obtained at the different extraction temperatures are shown in Fig. 2B. The yield of l-menthol increased as the temperature was increased from 100 °C to 180 °C, and decreased as it was increased further to 220 °C. The highest yield of l-menthol, 22.8 mg/g-dry leaves, was obtained at the extraction temperature of 180 °C. The yields of l-limonene, l-menthone, and piperitone exhibited similar trends to that of l-menthol. Since the yields of all 4 of these compounds decreased when temperature was increased to 220 °C, it was suggested that they were unstable under the subcritical water conditions at 220 °C. The yields of iso-menthone, 3-octanol, and l-menthyl acetate increased as the temperature was increased from 100 °C to 220 °C. In comparison to the behaviors of the previous 4 components (l-menthol, l-limonene, l-menthone, and piperitone), the yields of 3 components (iso-menthone, 3-octanol, and l-menthyl acetate) increased when temperature was increased to 220 °C. This suggests that the latter 3 components have higher thermal resistances than those of the former 4 compounds. According to these results, it was suggested that the composition of the essential oil could be modified by raising the extraction temperature under subcritical water conditions.
Effect of extraction time on yield The combined yield of the 7 components previously discussed reached the highest value of 30.5 mg/g-dry leaves when extracted at 180 °C for 5 min, as shown in Fig. 3. At 140 °C, extractions of 30 min and 60 min were carried out in addition to the 5-min extraction; the results are compared in Fig. 3. The total yield from the 5-min extraction at 140 °C was 23.6 mg/g-dry leaves, and increased to 29.5 mg/g-dry leaves when the extraction time was extended to 30 min. As the extraction time was further extended to 60 min, however, the yield decreased. Since the yield of the extraction conducted at 180 °C for 5 min was similar to that of the extraction carried out at 140 °C for 30 min, the higher temperature and shorter extraction time were deemed to be the optimal conditions, with respect to saving time.
Essential oil composition The compositions of the essential oils obtained via the modified subcritical water treatment and by steam distillation are summarized in Fig. 4. Since Japanese mint has a high content of l-menthol (Shimizu, 1995), all the essential oils had proportions of l-menthol of over 70%. The proportions of l-menthol were 74.1–74.9% in the oils extracted at temperatures of 100 °C to 180 °C, and only 70.3% in that extracted at 220 °C. The proportions of l-menthone were 17.2–17.3% in the oils extracted at 100–140 °C, and decreased to 14.1–14.3% in the oils extracted at 180–220 °C. Conversely, the proportions of iso-menthone were higher in the oils extracted at 180–220 °C than in those extracted at 100–140 °C. The proportion of l-menthyl acetate increased as the temperature was increased from 100–220 °C. The oil obtained by small-scale steam distillation exhibited a similar composition to those of the oils extracted via the modified method at 100–180 °C. However, the oil obtained by large-scale steam distillation exhibited a distinct composition from those of all the other oils. It was suggested that this was a result of the mint sample used in the large-scale steam distillation containing branches and stems, whereas only leaves were used in the other treatments.
The extraction temperature and extraction time were the two key parameters that affected the extraction yield and composition of the mint essential oil. In traditional steam distillation, free aroma ingredients are extracted by steam from the broken oil glands located on the surfaces of dried mint leaves (Iijima, 2014). However, it has traditionally been considered that it is difficult or excessively time-consuming to extract other substances that are fixed or embedded in the cross-linked structures of plant tissue (Saulnier and Thibault, 1999). Since subcritical water was reported to have the ability to hydrolyze lignin in plant matter, it was suggested that these bound substances could be extracted via the hydrolysis of phenolic esters in the presence of significant ion contents under subcritical conditions (Funazukuria et al., 1990; Khuwijitjaru et al., 2012). Moreover, the solubilities of flavor compounds were reported to increase as the temperature was increased from 25 °C to 200 °C, and it was thus proposed that the extraction yield of the essential oil could thus be improved by increasing the extraction temperature (Miller and Hawthorne, 2000). However, subcritical water at high temperatures might also cause certain components of the essential oil to be degraded, and thus selection of the appropriate conditions for subcritical water extraction is necessary.
Conclusion
In this study, we demonstrated the use of subcritical water treatment combined with pressure-releasing distillation to obtain essential oil from Japanese mint. Approximately 3% (w/w) of essential oil was extracted from dried mint leaves at 180 °C in only 5 min, whereas the comparative small-scale steam distillation required more than one hour to achieve a similar extraction yield. The efficiency of the batch-wise extraction was also improved by saving the typical step of removing non-volatile substances such as solid residues and carbohydrates. Since the aroma components were highly concentrated in the initial fractions, subcritical water treatment coupled with pressure-releasing distillation is considered a useful method for recovering essential oil from Japanese mint.
Acknowledgements The authors are grateful to Mr. Tadashi Oiwa of the Kitami Mint Research Institute for providing dried leaves of Japanese mint. This study was planned and supported by the Research Center for Okhotsk Agriculture-, Forestry- and Fisheries-Engineering Collaboration (CAFFÈ).
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