Agarwood, a highly precious fragrant non-timber forest product, has been used for centuries in fragrances, incense, medicines, aromatherapy, and religion [1,2,3,4]. Due to its unique aroma, agarwood is used for incense and aromatherapy in the Middle East, and burning for fragrance in Japan. Agarwood has also been widely used as a medicine for tranquilizing and reducing excitement in East and South Asia for nearly two centuries. For example, the Compendium of Materia Medica, a pharmaceutical masterpiece written by Shizhen Li during the Ming Dynasty, recorded agarwood being used for treating forgetfulness and fright . However, to our knowledge, only four studies have reported the sedative-hypnotic function of agarwood. Okugawa et al.  reported that agarwood benzene extract reduced spontaneous motility and prolonged hexobarbital-induced sleeping time. Further phytochemical and pharmacological screening showed that jinkoh-eremol and agarospirol had a neuroleptic effect . Agarwood essential oil (AEO), generally considered to have anticancer, anti-inflammation, and anti-oxidant activities [8,9,10], also demonstrated a sedative effect on mice through inhalation. Takemoto et al.  reported that AEO could sedate mice by vapor inhalation. Miyoshi et al.  revealed that benzylacetone, released by heated agarwood, had sedative activity in mice. However, obtaining an exact dose-effect relationship is difficult with drug administration via inhalation. Furthermore, the mechanism of agarwood’s sedative-hypnotic function has not yet been reported. The few investigations on the sedative-hypnotic function of agarwood are largely dissymmetric with the various applications in large quantities. One of the main reasons for the lack of studies might be that the material is becoming hard to obtain from wild sources, and the price is too high.
Sleep disorders, like insomnia, are a growing mental health problem, affecting the quality of life and frequently causing significant functional impairment . Benzodiazepines and benzodiazepine receptor agonists are the most widely used clinical controller of sleep problems, which were introduced into clinical practice in the 1960s [14,15]. Benzodiazepines and benzodiazepine receptor agonists act by binding to the benzodiazepine-receptor binding site of the type A γ-aminobutyric acid (GABAA) receptor, increasing the activity of the inhibitory neurotransmitter GABA, and enhancing inhibitor outputs to all major cell groups in the brainstem and the hypothalamus that promotes arousal. However, long-term exposure to benzodiazepine usually results in many undesirable side-effects, such as dependence and tolerance [15,16,17]. Therefore, substances that could contribute to inducing and improving sleep quality with fewer side-effects would be beneficial.
With the use of artificial methods, such as the whole-tree agarwood-inducing technique for producing agarwood , the agarwood yields are increasing and the price is becoming more reasonable. As a result, agarwood no longer needs to be obtained from wild natural resources, enabling its wider application. To further investigate the sedative-hypnotic function of agarwood, we performed systematic investigations. The sedative-hypnotic effect of AEO was evaluated through animal behavioral tests, and the potential mechanism on GABAergic system was explored.
2.1. AEO Component Analysis
Sixty-eight compounds (Table 1 and Figure 1), representing 98.244% of the AEO, were identified by comparing their MS data with database data. Thirty-four sesquiterpenes, which included gualol (14.089%), dehydrofukinone (4.096%), aristolene (4.063%), 6-isopropyl-4,8-α- dimethyl-1,2,3,7,8,8-hexahydronaphthalene (3.481%), 2,3,3,3,4,5-hexahydro-7-isopropyl-3-methyl-1H-cyclopenta[1,3]cyclopropa-[1,2]benzene-3,6(7H)-dione (3.175%), germacrene B (3.121%), sandal (2.996%), 1,1,7-trimethyl-4-methylenedecahydro-1H-cyclopropa[e]azulene (2.874%) and hinesol (2.745%). were the main (51.132%) components of AEO. Thirteen compounds were aromatics (24.114%), including 7-methyltridecane (11.228%), 2,3,4,5-tetramethyltricyclo[3.2.1.02,7]oct-3-ene (5.716%), pyrethrone (2.316%) and perhydropyrene (1.619%). Other known compounds in the essential oil accounted for 19.823%.
2.2. Effect of AEO on Locomotor Activity in Mice
Treatment with AEO significantly decreased total distance traveled in a dose-dependent manner after single administration (Figure 2A). A single administration of AEO significantly and dose-dependently reduced distance moved, time moved and average velocity of mice (Figure 2B–D). The positive control diazepam, a commonly used clinical sedative drug, also displayed a sedative effect on total distance, distance moved, time moved and average velocity, which all significantly decreased. However, over time, the sedative effect of diazepam decreased after multiple (7 days and 14 days) injections, which is consistent with previous reports [15,19]. Interestingly, after multiple administrations of AEO, even after 7 or 14 days, sedative effects were maintained without obvious desensitization compared to a single dose.
2.3. Effect of AEO on Pentobarbital-Induced Sleeping in Mice
Compared to the control group (Figure 3), a single administration of AEO (15, 30, and 60 mg/kg) and diazepam (2 mg/kg) significantly increased the rate of sleeping induced by the subthreshold pentobarbital sodium. The effect of AEO showed dose-dependent enhancement. Additionally, the hypnotic function in mice was largely maintained after multiple administrations of AEO, both on day 7 and day 14, without significant differences compared to a single dose, whereas the function of diazepam (2 mg/kg) obviously decreased (Figure 3).
For the hypnotic dose of the pentobarbital-induced sleeping assay, as shown in Figure 4, the results revealed that AEO decreased the latency of sleeping time and prolong the duration of sleeping time in mice in a dose-dependent manner. Treatment with AEO (60 mg/kg) significantly reduced the latency of sleeping time, regardless of length of treatment (1, 7, or 14 days), whereas diazepam (2 mg/kg) only had that effect with single administration (Figure 4A). Similarly, AEO dose-dependently increased the duration of sleeping time with single and multiple administrations without obvious desensitization, whereas the potency of diazepam (2 mg/kg) decreased as administration time increased and significant tolerance appeared between day 1 and day 14 (Figure 4B).
2.4. Effect of AEO on the Brain Neurotransmitters Levels
Neurotransmitters play a critical role in intercellular neural signal transmission, and many sedative-hypnotic agents usually exert their pharmacological effects by altering the concentration of neurotransmitters. So, we assessed the neurotransmitters in the cerebral cortex of the mice by using an ultrafast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) system. The results, as shown in Figure 5, demonstrated that AEO did not have obvious effects on the concentration of glutamic acid (Glu) and GABA. Diazepam significantly elevated the concentration of GABA and did not have an apparent influence on the Glu level (Figure 5).
2.5. Effects of GABAA Receptor Antagonist on Locomotor Activity and Pentobarbital-Induced Sleeping
Even though AEO did not have obvious effect on the concentration of GABA, we supposed that AEO might act on the GABA receptor. So we used bicuculline and flumazenil to investigate the influence of AEO on locomotor activity and pentobarbital-induced sleeping. As the results in Figure 6 and Figure 7 show, both bicuculline (2 mg/kg and 4 mg/kg) and flumazenil (4 mg/kg and 8 mg/kg) antagonized the action of AEO. Only treatment with bicuculline had an indistinct influence on locomotor activity and pentobarbital-induced sleeping. Interestingly, when AEO (60 mg/kg) was administrated with bicuculline, the mice locomotor activities, including total distance, distance moved, time moved, and average velocity significantly increased, pentobarbital-induced latency of sleeping time increased, and duration of sleeping time decreased compared to the administration of AEO only (Figure 6).
Similarly, with a single injection of flumazenil, no apparent influence was observed on mice locomotor activity or sleeping induced by pentobarbital sodium. When treated with AEO (60 mg/kg) along with flumazenil, the sedative and sleep-promoting effect of AEO decreased along with the total distance, distance moved, time moved and average velocity increased, and duration of sleeping time decreased (Figure 7).
2.6. Effects of AEO on mRNA Expression of GABAA Receptors Subunits and Subtypes
Based on the above results, quantitative real time polymerase chain reaction (RT-PCR) was used to investigate the mRNA level of GABAA receptor subunits and subtypes in the cerebral cortex of the mice in two rounds. In the first round, we tested the expression of GABAA receptor subunits α, β, and γ with subunit-nonspecific primers. As shown in Figure 8, AEO significantly increased the mRNA expression of the α subunits on both day 7 and day 14, whereas diazepam had no obvious effect on subunit α. Additionally, subunits γ were increased by AEO and diazepam on day 14, but not on day 7. No difference between AEO and diazepam on subunit β regulation was found compared with the control on day 7 or 14. Based on the results, subunit-specific primers were used in the second round to determine the difference in the subtypes of α-subunits, including α1 α2, α3, α4, and α5. The results showed that AEO considerably increased α subtype 1 to 5 both on day 7 and 14, except for α3 on day 14. Conversely, diazepam mostly increased α3 subunit on day 7, α4 subtype on day 14, and decreased α1 and α5 on day 14.
2.7. Effects of AEO on Cl− Influx in SH-SY5Y Cells
To demonstrate the effect of AEO on GABAA receptor function, intracellular Cl− concentration was tested using a Cl− fluorescence probe N-(Ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) in SH-SY5Y cell. As shown in Figure 9, both AEO and pentobarbital exhibited significant promoting activities on the Cl− influx compared to the control. Treatment with AEO (0.05, 0.1, 0.2, and 0.4 mg/mL) dose-dependently increased the Cl− influx, and AEO (0.1 and 0.2 mg/mL) had comparable potentiation with pentobarbital (0.1 mg/mL), whereas higher concentrations of AEO (0.4 mg/mL) were more efficient than pentobarbital (0.1 mg/mL) on Cl− influx.
This study demonstrated that AEO has a sedative-hypnotic effect through multiple animal behavior tests over different time periods range, which may contribute to the understanding of scientific nature of the traditional agarwood application. Furthermore, this study revealed that the mechanism of AEO on sedative-hypnotic function may potentially be related to the GABAergic system regulation (Figure 10).
3.1. AEO Had Sedative-Hypnotic Effect in Mice
AEO has been reported to inhibit spontaneous motor activity in mice after vapor inhalation . Our previous results also demonstrated that AEO had a sedative-hypnotic effect on mice through inhalation . The advantage of inhalation is avoiding the first pass hepatic metabolism and the drug rapidly transforms into the blood for faster efficacy. However, the disadvantage is that the drug administration dosage is hard to control, leading to an undefined dosage-effect relationship. In this study, we used intraperitoneal injection which avoids the first pass hepatic metabolismsimilar to inhalation, and takes advantage of the most common application of agarwood as incense.
As per the previous reports, the sedative-hypnotic effect of agarwood was assessed after a single dose administration of agarwood extracts or AEO [6,11]. We evaluated the function of AEO on locomotor activity and pentobarbital-induced sleeping in mice with a single dose intraperitoneal injection. The results indicated that AEO could significantly reduce locomotor activity in a dose-dependent manner (Figure 2), in accordance with inhalation results in previous reports [11,20]. Generally, a decrease in the locomotor activity of mice is indicative of a sedative action of pharmacological drugs  and this behavioral alternation is considered as a reflection of decreased excitability in the central nervous system . The inhibitive effect on locomotor activity showed that AEO has sedative and excitability suppressive functions. Simultaneously, AEO demonstrated a synergic effect with pentobarbital, with an increased rate of sleeping (Figure 3), an increased duration of sleeping time and a decreased latency of sleeping time (Figure 4), which revealed the hypnotic-like effect of AEO. Prolonged administration of sedative-hypnotic drugs, such as diazepam, usually creates tolerance. We assessed the effect of AEO by multiple dose applications after 7 and 14 days. Interestingly, the results showed that AEO sustained the sedative-hypnotic action after multiple treatments without apparently increasing locomotor activity (Figure 2), decreasing the rate of sleeping (Figure 3), or significantly reducing the duration of sleeping time (Figure 4B). Conversely, the effect of diazepam decreased (Figure 3 and Figure 4) or even disappeared completely (Figure 2) after prolonged injection, is accordance with a previous report . Overall, the results verified the sedative-hypnotic function of AEO in a dose-dependent manner without apparent tolerance development after prolonged administration.
Biocatalysis represents an effective and sometimes preferable alternative to the standard synthesis of fine and/or optically active chemicals [1,2,3,4,5,6,7]. Overall, reactions catalysed by biological systems frequently exhibit high selectivity (chemo-, regio-, and stereo-selectivity) and can be considered environmentally acceptable because they typically occur under mild conditions. Both isolated enzymes and whole-cells can be used of as biocatalysts, but whole-cell biocatalysts are often preferable to the former because they are more convenient and stable sources of enzymes, with no need for costly enzyme purification and coenzyme addition. Moreover, because of the enzymes are kept within the natural environment of living cells, usually less enzyme inactivation occurs.
Flavours play a very important role in the quality perception of food and beverages, whereas fragrances represent an important part of soap and perfume industry [8,9,10]. Consumers have a strong preference for natural food additives over chemically synthesized ones. Both United States (US)  and European (EU)  laws have already labelled as “natural flavour” all those obtained from living cells, including Generally Regarded As Safe (GRAS) microorganisms . Thus, products obtained by microorganisms and enzymes can be considered natural as long as natural raw materials are used. As a result, the “natural” label, allocated by EU and US food legislation, represents a strong marketing advantage [9,10].
Monoterpenes are one of the largest classes of flavouring compounds (over 400 different naturally occurring structures), and represent a valuable resource for the flavour and fragrance industry. The consumer requests for natural flavours and fragrances have encouraged a growing part of scientific community to study and develop novel biocatalysts for producing this class of molecules. Thus, the microbial and enzymatic biotransformation of some monoterpenoids, in particular a few ketones and aldehydes (e.g., carvone, menthol, citronellol, myrtenal and geraniol) into highly valuable flavouring derivatives is becoming of increasing interest because of their economic potential for the perfume, soap, food, and beverage industry [13,14,15,16,17,18,19,20,21,22]. Carvone is produced by over 70 different plants. It is found basically in two distinct stereoisomeric forms, which differ between them for their flavouring attributes: (i) (4R)-(−)-carvone, which is the principal constituent in spearmint (Mentha spicata) oil, and (ii) S-(+)-enantiomer, which is present in oils extracted from caraway (Carum carvi) seeds and from dill (Anethum graveolens) seeds. Biocatalytic transformation of carvone has recently been the focus of several studies, reporting that some enzymes may catalyze the reduction of C=C and C=O double bonds competitively, affording a mixture of saturated ketones, saturated alcohol and, more rarely, the allylic alcohol [14,20,23,24,25,26,27,28,29,30,31,32]. From an industrial point of view, carvone and related compounds are important flavours and fragrances of industrial interest . In particular, due their high volatility, dihydrocarvones are potent inhibitors of bacteria and filamentous fungi, as well as prospective insect repellents , and have been used as chiral starting compounds in the synthesis of natural products (e.g., striatenic acid, pechueloic acid) [35,36,37], antimalarial drugs  and valuable chiral synthons [39,40]. Dihydrocarveols are valuable fragrance ingredients currently used in decorative cosmetics, fine fragrances, shampoos, soaps and other toiletries as well as in household products such as cleaners and detergents .
Although the use of yeast whole-cells as biocatalysts is a well-established practice, and a few yeast-catalysed processes have been even successfully scaled up from laboratory to the industrial level [5,42,43,44,45], if compared with other microbial domains (e.g., bacteria, filamentous fungi), the number of studies reporting the use of yeast whole-cells to catalyse the biotransformation of monoterpenes represents only a little percentage of the literature published so far . To most people, yeasts are exemplified by the common baker’s yeast (taxonomically defined as belonging to the ascomycetous species Saccharomyces cerevisiae). This is in spite of the fact that this species represents only a small fragment of the huge taxonomic and metabolic diversity occurring in the yeast world. In fact, in recent decades, biotech-oriented research had paid its attention to the so-called non-conventional yeasts (NCYs), which demonstrated sometimes a superior biocatalytic aptitude than S. cerevisiae [43,44,47].
As a part of a program aiming at the selection of yeast strains as novel sources of natural flavouring molecules, the production of flavours and fragrances via bioreduction of (4R)-(−)-carvone and (1R)-(−)-myrtenal by whole-cells of non-conventional yeasts (NCYs), belonging to the genera Candida, Cryptococcus, Debaryomyces, Hanseniaspora, Kazachstania, Kluyveromyces, Lindnera, Nakaseomyces, Vanderwaltozyma, and Wickerhamomyces was studied.
2. Results and Discussion
The biotransformations of the α,β-unsaturated ketone (4R)-(−)-carvone (1) catalyzed by whole-cells of NCYs in aqueous media were investigated. The possible reaction pathway is illustrated in Scheme 1. According to the proposed scheme, the biotransformation resulted in the reduction of the α,β-unsaturated C=C bond of the cyclic ketone, catalyzed by ene-reductases (ERs) associated to the yeast cells, to give two dihydrocarvones 2a,b. The ER-catalysed reduction was thus followed by the subsequent reduction of the carbonyl group of both dihydrocarvone isomers, catalyzed by carbonyl reductases (CRs), which determined the formation of a mixture of four dihydrocarveols 3a–d (Scheme 1).
Scheme 1. Bioconversion pathway of (1R)-(-)-myrtenal by whole-cells of NCYs
Scheme 1. Bioconversion pathway of (1R)-(-)-myrtenal by whole-cells of NCYs
Although the use of purified ERs needs an accompanying regeneration system for the nicotinamide cofactor [NAD(P)H] to close the catalytic cycle and improve the bioreduction efficiency, we assumed that lyophilized cells contain the needed recycling system except for the co-substrate. Accordingly, glucose was added because we found that this compound was the best co-substrate for co-factor recycling system . In fact, from a quantitative point of view, the presence of glucose in the reaction mixture [acting as auxiliary substrate for NAD(P)H regeneration] resulted critical for enhance the % of conversion of precursor, in close agreement with current literature [23,48]. With no glucose addition, whole-cells of NCYs showed only a little ability to reduce (4R)-(−)-carvone. Only three strains gave acceptable results: Hanseniaspora guilliermondii DBVPG 6790 (conversion about 14%), Lindnera amylophila DBVPG 6346 (about 10%) and Vanderwaltozyma polyspora DBVPG 6243 (about 8%). The prevalent catalytic activity of whole-cells of NCYs was the ER-catalysed reduction of (4R)-(−)-carvone into a mixture of (1R,4R)- and (1S,4R)-dihydrocarvone, with a clear-cut preference towards the production of (1R,4R)-diastereomer. Only traces of dihydrocarveols 3a-d, derived from the subsequent CR-catalysed reduction of the carbonyl group of (4R)-(−)-carvone were found. As expected, the addition of glucose to the reaction mixture, visibly increased the aptitude of some strains to reduce (4R)-(−)-carvone 1 (Table 1).
Table 1. Bioconversion of (4R)-(−)-carvone 1 into derivative products after 120 h by whole-cells of NCYs in the presence of glucose.
|Species and strain||Conversion (mol %)||Products (mol %)|
|C. maltosa DBVPG 6021||12.93 ± 3.87||9.76 ± 4.49||0.55 ± 0.04||2.63 ± 0.93||0||0||0|
|Cr. gastricus DBVPG 6057||4.71 ± 0.15||3.73 ± 0.25||0.33 ± 0.03||0.66 ± 0.09||0||0||0|
|C. oregonensis DBVPG 6149||14.81 ± 4.46||6.69 ± 1.22||0.57 ± 0.08||7.40 ± 3.19||0||0.15 ± 0.13||0|
|C. sake DBVPG 6162||0.05 ± 0.05||0.05 ± 0.04||0.01 ± 0.01||0||0||0||0|
|C. freyschussii DBVPG 6208||1.09 ± 0.26||1.00 ± 0.23||0.01 ± 0.01||0.07 ± 0.02||0||0||0|
|W. canadensis DBVPG 6211||2.05 ± 0.24||0.36 ± 0.12||0||1.32 ± 0.15||0.13 ± 0.22||0.24 ± 0.22||0|
|Cr. albidus DBVPG 6237||0.74 ± 1.08||0.43 ± 0.56||0.30 ± 0.53||0||0||0||0|
|Cr. terreus DBVPG 6242||7.38 ± 12.43||7.24 ± 12.21||0.14 ± 0.22||0||0||0||0|
|V. polyspora DBVPG 6243||13.45 ± 17.77||13.35 ± 17.65||0.10 ± 0.13|