2024 Volume 30 Issue 6 Pages 689-695
β-caryophyllene (BCP) is a volatile bicyclic sesquiterpenoid found in essential oils from spices and edible foods such as black pepper, basil, and cloves. Orally administered BCP exhibits several bioactivities, including anti-inflammatory and antioxidant effects. In addition to its physiological function as a functional food factor, BCP is volatile. BCP inhalation has been reported to exhibit several bioactivities, suggesting that inhaled BCP is as bioactive as orally administered BCP. Recent studies have shown that both orally administered and inhaled BCP can be transferred to the serum and organs. No studies have compared the distributions of orally administered and inhaled BCP. Therefore, this study compared the distribution patterns for orally administered and inhaled BCP in mice. Our findings showed that the distribution patterns differed between oral administration and inhalation. Consequently, the effects of BCP on biological activity may differ between oral and inhalation routes.
β-caryophyllene (BCP), a natural bicyclic sesquiterpene, is an essential oil component in several spices, including black pepper, basil, thyme, and cloves (Sharma et al., 2016). Several studies have suggested that orally administered BCP exhibits a variety of bioactivities, including anti-inflammatory (Picciolo et al., 2020), antioxidant (Calleja et al., 2013), anticancer (Dahham et al., 2015), and anxiolytic-like (Galdino et al., 2012) effects. These bioactivities may be involved in the agonist activities of BCP for some receptors, including the cannabinoid type 2 receptor and peroxisome proliferator-activated receptors α and γ (Youssef et al. 2019; Wu et al., 2014; Bento et al., 2011; Cheng et al., 2014). Since no serious safety concerns regarding BCP have been reported (Api et al., 2022), BCP is considered to be a promising functional food factor.
In addition to the physiological functions of functional food factors described above, volatility is a characteristic of BCP. In humans, BCP inhalation has been reported to decrease pulse wave velocity in smokers (Yamada et al., 2022), improve the Yale Food Addiction Scale Score, which measures overeating (Alizadeh et al., 2022), and increase testosterone concentrations in the saliva of women (Tarumi and Shinohara, 2020). In mice, BCP inhalation alters liver metabolites (Takemoto et al., 2021) and alleviates nicotine-induced aortic degeneration (Kishi et al., 2022). These reports suggest that inhaled BCP is as bioactive as orally administered BCP.
Recent studies have detected BCP in the serum and organs, including the aortic wall, brain, lung, liver, kidney, white adipose tissue, and brown adipose tissue, of mice that inhaled volatile BCP (Takemoto et al., 2021; Kishi et al., 2022). In humans, BCP is detected in the serum after inhalation of BCP (Yamada et al., 2022). BCP has also been detected in the sera of rats and humans following oral administration (Liu et al., 2013; Mödinger et al., 2022). These studies suggest that the incorporation of intact BCP into organs can affect the biological activities of animals, including humans, by oral administration and inhalation. No reports have compared the distribution for orally administered and inhaled BCP. Therefore, evaluating the distribution patterns of orally administered and inhaled BCP in the body may provide clues for understanding the differences in bioactivity between the two administration types. This study compared the distribution patterns for orally administered and inhaled BCP in mice.
Materials The BCP-rich fraction of clove leaf oil (Plant Lipids, Kerala, India) was obtained by combining 150 g of clove leaf oil with 300 g of potassium hydroxide solution (20 % KOH, 80 % (v/v) methanol) and incubating the mixture at 50 °C overnight for 30 min. The separated upper layer (oil portion) was combined with 100 g of 5 % citric acid in water and shaken thoroughly. The oil layer was then separated by centrifugation (8 500 × g, 5 min) to obtain 3.78 g of the BCP crude fraction. Further treatment under a vacuum (110 °C, 150 Pa) yielded 3.32 g of the β-caryophyllene fraction.
Animals All animal experiments were approved by the Institutional Animal Care and Use Committee and conducted in accordance with Kinki University Experimental Regulations (Approval No. KAAG-2022-009). In addition, 4-week-old male ddy mice (Shimizu Experimental Co., Ltd., Kyoto, Japan) were maintained under a 12-hour light/dark cycle at 25 ±1 °C with ad libitum access to commercially available feed (MF; Oriental Yeast Co., Ltd., Tokyo, Japan) and water.
Animal experiments After 5 days of acclimatization, the mice were divided into the Control group (no inhalation + 2 % CMC orally), the BCP oral group (0.04 mg/g BCP essential oil orally), and the BCP inhalation group (60 min BCP essential oil inhalation). In the BCP oral administration group, BCP essential oil emulsified with 2 % CMC was orally administered to mice. Oral administration was performed by gavage. After 30 min of oral administration, the animals were sacrificed using 50 mg/kg sodium pentobarbital. In the BCP inhalation group, BCP essential oil was soaked in 10 mL of absorbent cotton and placed in a net. The BCP-soaked absorbent cotton was then suspended, and the 5 000-mL flask was closed with a silicone stopper for 10 min and filled with volatile BCP. Subsequently, mice were individually placed in a 5 000-mL flask. After 60 min of inhalation, mice were sacrificed using 50 mg/kg sodium pentobarbital. Given that the findings of our previous studies showed that the time to maximum concentration differed between oral administration and inhalation, in this study, we collected serum and organs at different time points for oral administration and inhalation. We established the experimental conditions based on a pilot study examining oral administration (data not shown) and a previous paper (Takemoto et al., 2021).
Sampling Blood was collected from the inferior vena cava under anesthesia. Serum was obtained by centrifugation at 4 °C for 10 min at 800 x g. After perfusion, the lungs, olfactory bulb, brain, heart, liver, kidneys, epididymal adipose tissue, and interscapular brown adipose tissue were removed and frozen on plates cooled with liquid nitrogen without chemical fixation. These samples were stored at −80 °C until use.
Experimental conditions for evaluating BCP concentrations in the serum and organ samples BCP concentrations in the serum and organ samples were analyzed by gas chromatography-mass spectrometry (Agilent 7890B-5977B MSD; Agilent Technologies, Santa Clara, CA, USA), a thermal desorption unit (TDU2), programmable thermal vaporization inlet (CIS4), MSD ChemStation vs. F. 01.03. 2357 (Agilent), Mass Hunter v. B.07. 2357 (Mass Hunter v.B.07. 2357, Mass Hunter v.B.07. 2357, Mass Hunter v.B.07. 2357), and a multi-purpose sampler with a dynamic headspace option (Gerstel GmbH & Co. KG, Mμlheim an der Ruhr, Germany) (Xavier-Junior et al., 2017). Mass Hunter v. B.07.05.2479 (Agilent Technologies) was used for the data analysis. Acetone was used for BCP extraction from serum and organs.
Tenax TA cartridges were released from the Tenax TA trap using a thermal desorption unit (TDU2); Markes International, Ltd. An InertCap WAX column (length: 60 m, df: 0.25 μm, I.D.: 0.25 mm, GL Science Corporation, Tokyo, Japan) was used for component separation. The oven temperature was programmed as follows: initial temperature 40 °C; ramp rates 3 °C/min (40-145 °C), and 10 °C/min (145-240 °C); final temperature 240 °C, 5 min. The inlet pressure was controlled using an electronic pressure control system to achieve a constant column flow rate of 1 mL/min. Mass spectrometry analysis was performed in ionization mode at a voltage of 70 eV.
Statistical analysis All values were expressed as mean ± standard error of the mean (S.E.M.). Multiple comparisons between groups were determined using the Tukey-Kramer test, and comparisons between the two groups were determined using Student’s t-test; p < 0.05 was considered to be statistically significant.
BCP concentrations in serum and organs of orally administered and inhaled BCP in mice The BCP concentrations obtained for each group are shown in Figure 1. BCP was detected in all samples (serum, lung, olfactory bulb, brain, heart, liver, kidney, epididymal adipose tissue, and interscapular brown adipose tissue) from both groups. In the orally administered group, the concentrations in the olfactory bulb and liver were significantly higher than those in the serum, lung, brain, and epididymal adipose tissue (Fig. 1a). The concentrations in the interscapular brown adipose tissue were significantly higher than those in the serum, lung, olfactory bulb, brain, heart, liver, kidney, and epididymal adipose tissue (Fig. 1a). In the inhalation group, the concentrations in the olfactory bulb were higher than those in the serum, lung, brain, heart, liver, kidney, and epididymal adipose tissue (Fig. 1b). The concentrations in the interscapular brown adipose tissue were significantly higher than those in the serum, lung, olfactory bulb, brain, heart, liver, kidney, and epididymal adipose tissue (Fig. 1b). These data suggest that the distribution patterns differed between the two groups.
Comparison of the transfer ratio of BCP between oral administration and inhalation To clarify the distribution patterns of orally administered and inhaled BCP in the body, the values of BCP detected in each organ were divided by the serum concentration and compared (Fig. 2). The values in the olfactory bulb, brain, epididymal adipose tissue, and interscapular brown adipose tissue were significantly higher in the inhaled BCP group than in the oral administration group. No significant differences were observed between groups in the lung, heart, liver, and kidney. These results suggest different distribution mechanisms for oral administration and inhalation.
Comparison of BCP concentration in the organs To compare the differences in the concentration of BCP in the organs between oral administration and inhalation, the relative values for each organ are shown in Figure 3. Here, the concentration of BCP in the heart was set to 1. The patterns for BCP concentration differed among the oral administration and inhalation routes. In the oral administration group, the concentration in the olfactory bulb was significantly higher than those in the lung, brain, heart, and epididymal adipose tissue (Fig. 3a). The concentration in the liver was significantly higher than that in the lung, brain, and epididymal adipose tissue (Fig. 3a). The concentration in the interscapular brown adipose tissue was significantly higher than that in the lung, brain, heart, kidney, and epididymal adipose tissue (Fig. 3a). In the inhalation group, the concentrations in the olfactory bulb and the interscapular brown adipose tissue were significantly higher than those in the lung, brain, heart, liver, kidney, and epididymal adipose tissue (Fig. 3b). When the relative concentrations for each organ were compared between the oral administration and inhalation groups, the values obtained for the lung, olfactory bulb, brain, epididymal adipose tissue, and interscapular brown adipose tissue were significantly higher in the inhalation group (Fig. 3c). On the other hand, the value obtained for the liver was significantly higher in the oral administration group than in the inhalation group (Fig. 3c).
In this study, BCP was detected in all samples. The distribution patterns for BCP were observed to differ between oral administration and inhalation. Because BCP doses cannot be perfectly matched between oral administration and inhalation, we used two calculated values to compare the distribution patterns for BCP.
A comparison of values corrected for serum concentration showed significantly higher values in the olfactory bulb, brain, epididymal adipose tissue, and interscapular brown adipose tissue in the inhalation group than in the oral administration group. If the distribution route for each organ was the same between the two groups, then no significant difference would have been observed in the values between the two groups. Consequently, these results suggest different distribution mechanisms for oral administration and inhalation. One possible pathway involved in the transfer to the olfactory bulb and brain is through the nasal cavity. Previous studies have reported a significantly higher bioavailability for nasally administered drug distributions than for intravenous injections (Anand Kumar et al., 1982). It has also been reported that intranasally administered drugs pass through the blood-brain barrier, reach the brain via the systemic circulation pathway, pass through the olfactory region of the nasal cavity, and are transported directly to the olfactory bulb and other brain tissues (Erdő et al., 2018). It has been speculated that the nasal transfer route may be a potential pathway for inhaled BCP to reach the olfactory bulb and brain. However, no studies have demonstrated how volatile components reach the brain via the nasal cavity, and further research is required.
The different distributions of BCP in the epididymal adipose tissue and interscapular brown adipose tissue may be related to the differences in the presence of BCP in the blood between oral administration and inhalation. Orally administered BCP is incorporated into the body through intestinal absorption. In this system, BCP circulates in the same form as other dietary lipophilic constituents. In contrast, inhaled BCP is likely to be present in the blood in a form that differs from the dietary fat-soluble component transport system because BCP is incorporated into the body via the lungs. It is speculated that inhaled BCP, which is not incorporated into the fat-soluble component transport system, volatilizes easily in the blood and is more likely to be taken up by lipophilic organs such as white and brown adipose tissues. The higher amount of BCP in the interscapular brown adipose tissue than in the epididymal adipose tissue may be attributed to the density of the microvessels. Brown adipose tissue contains more microvessels than white adipose tissue (Frontini and Cinti, 2010). Consequently, higher contact areas with the microvessels may result in different concentrations of BCP. Measurement of the form of BCP in the blood is needed to validate this hypothesis.
A comparison of the BCP components in each organ revealed a higher percentage of inhaled BCP in epididymal adipose tissue and interscapular brown adipose tissue. This could be related to the fat solubility of BCP and its presence in the blood, as discussed above. The higher percentages in the liver for the oral administration group may be related to the excretion of BCP from the body. Because orally administered BCP is presumably incorporated into the transport system of dietary fat-soluble components, it may be easier to excrete by the xenobiotic metabolic system than inhaled BCP, which may not be incorporated into this system. Higher concentrations of BCP in the olfactory bulb were observed in the oral administration group; however, the data obtained in this study do not allow us to discuss the mechanism underlying BCP distribution. Future studies are needed to clarify whether the distribution is unique to BCP inhalation or whether the same distribution pattern is observed with other aromatic compounds. In conclusion, the findings of this study showed that different BCP administration pathways differentially affect the distribution of BCP in the body. This finding suggests that the effects of BCP on biological activities might differ between the oral and inhalation pathways. A limitation of this study is the lack of time-dependent change data for BCP. More quantitative data, such as the area under the curve values, would facilitate more accurate comparisons of BCP incorporated into the body via different pathways.
Authors’ contributions M.H. analyzed the data and wrote the manuscript. N.M. performed animal experiments, analyzed the data and edited the manuscript. T.Y. and T.S. edited the manuscript. Y.Y. and S.M. performed the GC-MS analysis. T.M. edited the manuscript. N.Z. designed the studies, wrote and edited the manuscript, and secured funding.
Acknowledgements This work was supported by Grant-in-Aid for Exploratory Research (21K19095).
Conflict of interest There are no conflicts of interest to declare.
