Note
Locomotor-Reducing, Sedative, and Antidepressant-Like Effects of Confectionery Flavours Coumarin and Vanillin
2024 Volume 47 Issue 10 Pages 1768-1773
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2024 Volume 47 Issue 10 Pages 1768-1773
Coumarin and vanillin are compounds with comforting scents and are often used for flavouring confectionery. The locomotor-reducing, sedative, and antidepressant-like effects of coumarin and vanillin vapours administered via inhalation were investigated. Coumarin and vanillin showed all these effects. In particular, antidepressant-like effects were observed over a wide range of doses and were stronger than the positive control, fluoxetine (10 mg/kg). These results suggest that coumarin and vanillin may be suitable as antidepressant-like agents without strict dose control.
Coumarin is a characteristic scent compound of salted Oshima cherry leaves (Cerasus speciosa (Koidz.) H. Ohba; Rosaceae). A phenylpropanoid glycoside, 2-O-glucosylcoumaric acid is converted to coumarin via 2-coumaric acid during salting.1) Salted Oshima cherry leaves are very fragrant and are a flavouring ingredient of the Japanese confection sakuramochi. Vanillin is also a favourite scent compound frequently used in confections and cakes that is obtained by repeated fermentation of vanilla beans (Vanilla planifolia Andrews; Orchidaceae).2) These compounds have comforting scents and are widely used as flavouring agents in confectionery manufacture. Even though the vanillin concentration in air is low, it is detected by olfactory systems. Some fragrance compounds have been reported to show sedative and antidepressant-like effects via inhalation in mice, and a representative fragrance compound showing those effects is linalool.3,4) However, it is generally difficult to find plants and items containing these types of fragrance compounds. In contrast, foods and confectionery are easy to obtain, and if the scents of foods and confectionery have sedative and antidepressant-like functions, the functionalities of these fragrance compounds can be used easily without precise knowledge of the flavouring agents.
In humans, memories of scents are linked with experiences,5) and confectionery has comforting scents that are usually not linked with unpleasant memories. These associations are an important feature of flavouring agents for possible use as functional scents. Here, the locomotor-reducing, sedative, and antidepressant-like effects of coumarin6) and vanillin7) were investigated in mice.
Vanillin (>98%) and triethyl citrate (>98%) were purchased from Tokyo Chemical Industry (Tokyo, Japan), and coumarin (>95%) was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). All fragrance compounds used in this study were the highest grade available. Pentobarbital was purchased from Nacalai Tesque (Kyoto, Japan). The positive controls and pentobarbital were dissolved in saline (Otsuka Pharmaceutical, Tokyo, Japan) using the solubilisers propylene glycol (>99%), ethanol (99.5%), and methanol (99.5%) purchased from Nacalai Tesque (Kyoto, Japan), and hydrochloric acid purchased from Junsei Chemical (Tokyo, Japan). Authentic compounds for the GC/MS retention indices were decane, dodecane, tetradecane, octadecane, tetracosane (FUJIFILM Wako Pure Chemical Corporation), eicosane, docosane, hexacosane, (Tokyo Chemical Industry), and hexadecane (Kishida Chemical, Osaka, Japan).
Qualitative Analyses Using Solid-Phase Microextraction Gas Chromatography Combined with Mass Spectrometry (SPME-GC/MS)Coumarin in sakuramochi (Matsuemon, Tokyo, Japan) and vanillin in a pudding (Serendip, Takasaki, Japan) were analysed using headspace and direct SPME-GC/MS, respectively. In the headspace method, sakuramochi pieces (10 g) were put in a glass vial, which was capped to capture the scent vapour in the headspace. Next, 100 µm polydimethylsiloxane SPME fibre (Supelco Co., Bellefonte, PA, U.S.A.) was inserted into the headspace of the vial for 20 s to extract the scent vapour. In the direct method, the SPME fibre was inserted into the pudding for 10 s. Then, extracted volatiles were analysed using gas chromatography-quadrupole mass spectrometry (JMS-Q1500GC, JEOL, Tokyo, Japan) equipped with an Inertcap WAX column (GL Sciences, Tokyo, Japan; 60 m × 0.25 mm, 0.25 µm film thickness). Operating conditions were as follows: injector temperature, 240 °C; carrier gas, helium; column flow, 1 mL/min; and injection volume, 1 µL (splitless). The oven program was holding at 100 °C for 4 min, increasing 3 °C/min to 200 °C, and holding at 200 °C for 19 min.8) The components extracted by the SPME fibres were identified by comparing their mass spectra with those in a data library (NIST11, National Institute of Standards and Technology).
AnimalsThe animal studies were designed according to the recommendation of the Committee of Animal Experiments at Daiichi University of Pharmacy, Fukuoka, Japan (Authorisation Number: 16004-24). Three- and four-week-old male ddY mice were obtained from Japan SLC (Hamamatsu, Japan). The mice were housed in colony cages (four mice per cage) at an ambient temperature of 25 ± 2 °C under a 12 h light–dark cycle. They were fed standard pellet chow and water ad libitum. All behavioural observations were conducted from 9:00 to 18:00.
Open Field TestCoumarin and vanillin were dissolved and diluted using triethyl citrate (400 µL), and dropped onto four filter paper disks attached to the four corners of the glass cage (61.2 L). The cage was filled with solution vapour by natural diffusion for 60 min. A mouse was then placed in the centre of the cage and monitored with a video camera for 60 min. The total spontaneous locomotor activity was the area under the curve, which was calculated from the time (min) on the x-axis and the number of times per 5 min the mouse crossed the line drawn on the bottom of the cage at 10 cm intervals on the y-axis.9) The effective compounds showed two-phase effects, and the effects at lower doses were considered true activity because the mice displayed excited behaviours, such as jumping and rearing, at higher doses.10) As the positive control, benzylacetone (4.5 × 10−4 mg/cage; Tokyo Chemical Industry) was administered via inhalation.
Pentobarbital-Induced Sleep TestThe pentobarbital-induced sleep test was conducted following Takemoto et al.11) Pentobarbital induces loss of righting reflex (LRR). Mice that exhibited LRR for over 1 min were regarded as asleep. Pentobarbital (25 mg/kg) dissolved in the vehicle (saline/propylene glycol/ethanol = 87/77/36) was injected intraperitoneally into mice. After administration, the mice were placed in the cage filled with the solution vapour, as in the open field test, and were monitored with a video camera for 60 min. The LRR latency was recorded from pentobarbital injection to LRR onset. LRR duration was recorded from LRR onset to recovery of the righting reflex. The control group inhaled triethyl citrate vapour, and the vehicle group were orally administered saline 30 min prior to administration of pentobarbital under the same conditions as the control group. As the positive control, chlorpromazine hydrochloride (5 mg/kg) dissolved in saline was administered orally 30 min prior to administration of pentobarbital.
Tail Suspension TestThe tail suspension test is generally used as a behavioural model for testing the effect of antidepressant agents. The tail suspension test was conducted according to Tankam and Ito, who reported the antidepressant-like effect of fragrance compounds via inhalation.3) Mice were placed in a cage filled with the solution vapour to inhale the fragrant vapour for 30 min. The mice were suspended from the edge of a table (65 cm high) by adhesive tape placed about 1 cm from the tip of the tail. The mice were considered immobile when they stopped making struggling movements and hung passively. Immobility time was recorded over a period of 6 min. As the positive control, antidepressant agent fluoxetine was dissolved in saline containing one drop of methanol and hydrochloric acid and was administered intraperitoneally at a dose of 10 mg/kg to mice 30 min before starting the tail suspension test.3)
Statistical AnalysisResults are expressed as the mean ± standard error of the mean (S.E.M.). Statistical analyses were performed with Dunnett’s test and Student’s t-test using GraphPad Instat (GraphPad Software, San Diego, CA, U.S.A.).10) A probability level of p < 0.05 was considered statistically significant.
SPME-GC/MS revealed the presence of coumarin in the headspace volatiles of sakuramochi (Fig. 1a), and a low amount of vanillin was found in a pudding by direct SPME-GC/MS (Fig. 1b).
Inhalation of coumarin significantly reduced the locomotor activity of mice at doses of 4.5 × 10−4–4.5 × 10−3 mg/cage (Fig. 2a). Inhalation of vanillin decreased the locomotor activity at a dose of 4.5 × 10−3 mg/cage (Fig. 2b).
Data are expressed as the mean ± S.E.M. for five mice. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. * p < 0.05 and ** p < 0.01 vs. the control group. AUC, area under the curve.
LRR latency was significantly shortened after administration of coumarin at a dose of 4.5 × 10−3 mg/cage (Fig. 3a). However, LRR duration was not prolonged at any dose in the coumarin groups (Fig. 3b). LRR latency was also significantly shortened after administration of vanillin at a dose of 4.5 × 10−3 mg/cage, and LRR duration was not prolonged in the vanillin group (Figs. 3a, b). In the coumarin groups, shorter LRR latency and longer LRR duration were observed at the higher dose than at the lower dose.
Data are expressed as the mean ± S.E.M. for five mice. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. * p < 0.05 and ** p < 0.01 vs. the control group.
Coumarin showed a significant antidepressant-like effect at doses of 4.5 × 10−7–4.5 mg/cage via inhalation (Fig. 4a). Vanillin also displayed a significant effect at doses of 4.5 × 10−8–4.5 × 10−4 mg/cage via inhalation (Fig. 4b). The antidepressant-like effect of coumarin and vanillin were stronger than those of fluoxetine (10 mg/kg), coumarin at doses of 4.5 × 10−6–4.5 × 10−2 mg/cage, and of vanillin at a dose of 4.5 × 10−6 mg/cage (Figs. 4a, b).
Data are expressed as the mean ± S.E.M. for five mice. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. * p < 0.05 and ** p < 0.01 vs. the control group. Positive control: fluoxetine (10 mg/kg) i.p.
Vanillin has a low olfactory threshold12); therefore, vanillin was detected by direct SPME-GC/MS instead of headspace SPME-GC/MS. 5-Hydroxymethylfurfural, the most detected aroma compound in the pudding, was the target of the study. However, this compound does not originate from vanilla beans and is instead generated from sugars by heating during the pudding preparation.13,14) More 5-hydroxymethylfurfural than vanillin was observed in the pudding, which may be due to the direct SPME-GC/MS method. The lipophilicity of 5-hydroxymethylfurfural is low and it is easily dissolved in water.15) The pudding was 75% water16); therefore, 5-hydroxymethylfurfural was contained in the water in the pudding. Inserting the solid-phase microextraction (SPME) fibres directly into the pudding allowed 5-hydroxymethylfurfural to be adsorbed on the fibre, different from in air. Vanillin has higher lipophilicity17) and is bound by lactalbumin or ovalbumin18); thus, a low amount of vanillin would have been detected. The boiling point and hydrophilicity of 5-hydroxymethylfurfural are higher than those of vanillin,14,16) and its olfactory threshold is 100-fold higher than vanillin.12) Consequently, there would be less 5-hydroxymethylfurfural vapour in the headspace than that expected from the result of direct SPME-GC/MS, although there could be more 5-hydroxymethylfurfural than vanillin. However, vanillin bound by proteins dissociates as the temperature increases,19) releasing vanillin into the air. Vanillin can be detected at low concentration in air,12) and the pudding would not have a fatty 5-hydroxymethylfurfural-like scent but a sweet vanillin-like scent.20)
Locomotor-Reducing Effect of Coumarin and VanillinCoumarin showed a similar locomotor-reducing effect to benzylacetone used as a positive control at the same doses.21) This is the first report of the effect of coumarin administered via inhalation. However, no locomotor-reducing effect was observed after administration of coumarin analogues, such as 4-hydroxycoumarin, via intraperitoneal injection and scopoletin orally.22,23) Tetrahydrozerumbone9) and valerena-4,7(11)-diene24) showed a locomotor-reducing effect via intraperitoneal injection, and the doses were 1000-fold lower than those of 4-hydroxycoumarin and scopoletine. Therefore, the doses of 4-hydroxycoumarin and scopoletin were much higher than the doses at which those compounds show a locomotor-reducing effect. The locomotor-reducing effect could be weaker at high doses in an open field test.9–11,21) Coumarin was dissolved in corn oil and administered to mice intraperitoneally, and the locomotor activity of mice was significantly reduced at dose of 1.0 × 10−5 mg/kg (Fig. 5). This suggested that coumarin in the bloodstream could decrease spontaneous locomotor activity, and thus the effect at high doses may have been caused by coumarin in the bloodstream. No appetite-enhancing effect was observed when fragrance compounds were administered intraperitoneally, although the effect was observed when the compounds were administered via inhalation.7,25) The effect may have arisen from the intranasal olfactory receptors15) or transient receptor potential (TRP) channels.26) Thus, 4-hydroxycoumarin and scopoletin did not show a locomotor-reducing effect because intraperitoneal or oral administration would not have stimulated the olfactory receptors or TRP channels. In contrast, coumarin showed the effect at a dose of 4.5 × 10−4 mg/cage via inhalation, which may be due to stimulation of the olfactory receptors or TRP channels. Oral administration of high-dose scopoletin can stimulate olfactory receptors or TRP channels in the nasal cavity; however, Carpa et al.23) found that it did not affect spontaneous locomotor activity. The hydroxy group of scopoletin makes its boiling point of 413.5 °C at 1013 hPa more than 100 °C higher than that of coumarin.27)
Data are expressed as the mean ± S.E.M. for five mice. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. * p < 0.05 vs. the control group. AUC, area under the curve.
Ueno et al. reported that no differences were observed in the locomotor-reducing effect of vanillin between the high-dose group administered 13.3 mg/cage of vanillin, which is close to a saturated solution, and the control group.28) This dose is approximately 3000 times higher than the effective dose in this study of 4.5 × 10−3 mg/cage. However, Ueno et al. ventilated the cage at a rate of 5 L/min, decreasing the concentration in the cage, and thus mice could have been unable to inhale enough vanillin to exhibit the effect. In our methods, the cage was ventilated naturally,9) and we predicted that the vapourised fragrance compounds would stay in the bottom of the cage. Therefore, the locomotor-reducing effect was observed at lower doses in our studies.
Pentobarbital-Induced Sleep TestDose-dependent tendencies on LRR latency shortening and LRR duration elongation were observed when mice were administered coumarin by inhalation. The pentobarbital-induced sleep tests provide information about the stimulation of sedative-related receptors, such as the GABAA receptor, or the inhibition of stimulant receptors, such as adrenergic receptors.29,30) The locomotor-reducing effect is an index of the sedative effect; however, the significant locomotor-reducing effect observed at a dose of 4.5 × 10−4 mg/cage did not match the result of pentobarbital-induced sleep test at the same dose because the LRR latency was not significantly shortened. This suggested that the locomotor-reducing effect at the coumarin dose was not related to the stimulation or inhibition of receptors in the central nervous system observed in the pentobarbital-induced sleep test at a dose of 4.5 × 10−3 mg/cage. This effect may arise from other mechanisms, like the stimulation of olfactory receptors. Inhalation of valerena-4,7(11)-diene11) and kabuchii citrus essential oil31) caused LRR latency shortening and LRR duration elongation at a higher dose than coumarin. The LRR latency shortening and LRR duration elongation of coumarin was dose-dependent; doses higher than 4.5 × 10−3 mg/cage showed significant LRR latency shortening and LRR duration elongation. Vanillin showed significant LRR duration elongation, similar to coumarin. However, it was reported that the LRR duration in in mice intraperitoneally administered 15 mg/kg of vanillin was shorter than that of the control group.32) The dose of 4.5 × 10−3 mg/cage in this study would correspond to a maximum dose of 0.18 mg/kg, if all the vanillin were vapourised. These differences suggested that the pentobarbital-induced sleep test for vanillin should have a U-shaped dose–response curve like the locomotor-reducing effect in this study. Thus, the strongest effect in the pentobarbital-induced sleep test should be at doses higher than 0.18 mg/kg and lower than 15 mg/kg.
Antidepressant-Like Effect of Coumarin and VanillinCoumarin and vanillin showed an antidepressant-like effect at low doses. In previous reports, the effect occurred at high doses, similar to 2-phenylethanol (equivalent dose in this study: 23.1 g/cage)33) and Asarum heterotropoides essential oil (equivalent dose: 10.7 g/cage).34) In comparison, coumarin and vanillin showed the effect at doses 1000-fold lower, of 4.5 × 10−7–4.5 mg/cage and 4.5 × 10−8–4.5 × 10−4 mg/cage, revealing strong effects. At those doses, excluding the doses of coumarin of 0.45–4.5 mg/cage, no excited behaviours, like jumping or increasing of rearing, were observed in the open field test; thus, the decrease in immobility time in this study was due to a true antidepressant-like effect. In contrast, the decrease in immobility time in mice administered 0.45–4.5 mg/cage of coumarin was related to excited behaviours because excited behaviours have been observed for several fragrance compounds at doses over 0.45 mg/cage.10) Tankam and Ito reported the antidepressant-like effect of linalool at low doses of 0.45–4.5 × 10−3 mg/cage using the same method as in the present study.3) The difference between the maximum and minimum doses showing an antidepressant-like effect in linalool was 100 times; however, the differences were 10 million times in coumarin and 10000 times in vanillin. These results suggested that scents such as coumarin and vanillin that are easily obtained from confectionery could show an antidepressant-like effect without strict dose control compared with linalool.
We revealed that coumarin and vanillin have locomotor-reducing, sedative, and antidepressant-like effects. The antidepressant-like effect occurred over a wide range of doses that overlapped with those for the locomotor-reducing and sedative effects. Because coumarin showed a sedative effect and no appetite-enhancing effect, it may be useful for treating overeating, insomnia, and nervousness.8) Vanillin showed an antidepressant-like effect that did not overlap with the sedative effect but that did overlap with the appetite-enhancing effect. Therefore, vanillin may be useful for treating loss of appetite.7) The scents of coumarin and vanillin, characteristic of readily available confectionery, could be used for their locomotor-reducing, sedative and antidepressant-like effects. Confectioneries are sometimes consumed alone, although they are also consumed with beverages, such as green or black teas. The scent of tea contains linalool, which also has an antidepressant-like effect.35) Furthermore, the amino acid theanine has an antidepressant-like effect and improves sleep.36,37) The combination of these compounds may result in a stronger sedative effect and improve depressant symptoms. The effects of coumarin and vanillin have not been reported before, and further work is necessary. For example, the doses of aroma compounds were expressed as the amounts of compounds in the cage, that is, as relative doses, because there are no methods or equipment for measuring amounts of vaporised compounds. It is necessary to develop these methods to administer accurate amounts of aroma compounds to confirm the antidepressant-like and sedative effects of coumarin and vanillin. The results of this study may provide useful knowledge for developing new uses of fragrance agents and research on their bioactivity.
The authors declare no conflict of interest.
