Bioscience Evidence 2024, Vol.14, No.4, 184-194 http://bioscipublisher.com/index.php/be 190 Target prediction results suggest that 14 BVOCs may interact with 19 protein targets, including CNR2, PPARα, and VDR, which are closely related to neurotransmission and circadian rhythm regulation. CNR2 is closely linked to the regulation of the central nervous system (Ishiguro et al., 2010), while PPARα is involved in the regulation of sleep and the biological clock (Mezhnina et al., 2022). These findings provide important clues for further research into the mechanisms by which Cypress cone shells may affect anxiety and insomnia, though further experimental validation is needed to confirm the accuracy of these predictions. Compared to previous studies, this research systematically identified the volatile organic components in Cypress cone shells and predicted their potential targets. Previous research has hypothesized that these specific small aromatic molecules may be inhaled through the respiratory tract or absorbed through the skin via direct contact with a pillow, thereby influencing the function of the central nervous system (Li et al., 2023; Dong, 2024). In contrast, this study not only identified the key BVOCs but also predicted their potential protein targets, providing new perspectives and evidence for the pharmacological mechanism of Cypress cone shells. Despite revealing the potential mechanisms by which Cypress cone shells may alleviate anxiety and insomnia through GC-MS and target prediction, this study has certain limitations. The GC-MS analysis was conducted under in vitro conditions, and the absorption, metabolism, and bioavailability of BVOCs in actual applications may be influenced by various factors. Although target prediction offers valuable insights, these results have not yet been validated through in vivo experiments, and the actual biological effects require further investigation. Future research should focus on validating the biological activity of BVOCs through in vivo studies and exploring their clinical application potential in different populations. 4 Materials and Methods 4.1 Experimental materials The Cypress (Platycladus orientalis (L.) Franco) cones were collected from the Dabie Mountains in Anhui Province during the autumn when the cones were mature. Random sampling was conducted in typical Cypress forests, with sample trees selected every 50 meters, resulting in a total of 50 trees being sampled. The maturity of the cones was determined based on the color and hardness of the shells, with all selected trees being at least five years old. Each tree produced no fewer than 50 mature cones to ensure the representativeness of the sample. The cones were air-dried naturally for 7 days, with the ambient temperature controlled at (25±2) ℃ and relative humidity at (40±5)%, ensuring the stability of the volatile components. After air-drying and natural seed shedding, the dry Cypress shells were collected. 4.2 BVOCs extraction and detection The collected Cypress shells were sampled using the quartering method, with 2.419 7 g of the sample placed in a 20 mL headspace vial to allow the volatile components to naturally disperse. After equilibrating the sample for 2 hours, 1 mL of the headspace was sampled and analyzed using GC-MS (Agilent, GC QQQ8890-7000) to determine the BVOCs composition. The gas chromatography conditions were as follows: the silica capillary column was Agilent HP-5MS (250 μm × 0.25 μm, 30 m); the carrier gas was high-purity helium (purity not less than 99.999%) with a constant flow rate of 1.0 mL/min. The injection port temperature was set at 250 ℃, with a splitless injection. The temperature program started at 40 ℃ for 1 minute, then increased at 4 ℃/min to 230 ℃, and finally ramped at 100 ℃/min to 260 ℃, held for 11.7 minutes. Mass spectrometry conditions were as follows: the ion source was an electron impact (EI) source, with the ion source temperature at 230 ℃, quadrupole temperature at 150 ℃, and interface temperature at 280 ℃. The electron energy was set at 70 eV, and the scan mode was full scan (SCAN) with a mass range of m/z 50-500. The solvent delay was set to 8 minutes. 4.3 Quantification of BVOCs components The total ion chromatogram was matched against the NIST 2.0 standard library for similarity, retention index, and
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