Studies On The Antidepressant Effect And Mechanism Of Yueju Volatile Oil

Objectives:Yueju pill is composed of medicinal herbs with high volatile oil content,but the part of volatile oil has long been neglected in the research and applica tion of its antidepressant effect.Therefore,this study aims to explore the antid epressant effect and mechanism of Yueju volatile oil（YJVO）,clarify its value in the use of this formula,and provide scientific basis for the rational and eff ective use of Yueju pill.Methods:1.Extracting and analyzing the volatile oil of Yueju and the herbs.The v olatile oil of the Yueju and the herbs was extracted by steam distillation.The chemical components of each volatile oil were analyzed by the gas chromatogr aphy-mass spectrometry（GC-MS）method.The GC-MS detection conditions for YJVO,LSVO（volatile oil of Ligusticum striatum DC.）,ALVO（volatile oil of Atractylodes lancea（Thunb.）DC.）,CRVO（volatile oil of Cyperus rotundus L.）,and GJVO（volatile oil of Gardenia jasminoides J.Ellis）were established.Based on the NIST20.L standard mass spectrometry library,compounds with a matching degree greater than 80%were screened and retained.Determined the components of the volatile oils by referring to other literature.Calculated and compared the components and contents of the volatile oils.2.An acute oral toxicity experiment to determine the safety of YJVO.Th e C57BL/6J mice were used for the acute oral toxicity evaluation of YJVO.A pre-test was conducted before the formal experiment,and the 100%lethal dos e and 0%lethal dose were determined to be 10 m L·kg^-1and 1.25 m L·kg^-1res pectively.Within this range,groups with doses of 10 m L·kg^-1,8 m L·kg^-1,6 m L·kg^-1,4 m L·kg^-1,and 2 m L·kg^-1were set up,In addition,a solvent group of0.1%Tween-80 was set up.After administering a single dose of medication to mice,observe the general state and calculate the mortality rate of mice.The median lethal dose(half lethal dose,LD₅₀)of YJVO was determined using the Bliss method.3.Based on the experiment of the acute oral toxicity,set the dosage reas onably to explore the oral antidepressant effect of YJVO.The antidepressant ef fects and mechanisms of YJVO through oral administration:C57BL/6J mice we re used to establish a CUMS mouse depression model,and the depressive beha vior of animals was evaluated using behavioral tests as the basis for efficacy e valuation.Compared the antidepressant effects of YJVO,Yueju aqueous extract（YJAE）,and Yueju crude drug（YJW）at the same dose.Set up a low dose(0.15 m L·kg^-1),a medium dose(0.3 m L·kg^-1),and a high dose(0.6 m L·kg^-1)g roups to compare the antidepressant effects of YJVO at different doses and det ermine the optimal oral dose for the antidepressant effect.To investigate the m echanism of the antidepressant effect of YJVO through oral administration,ultr asound imaging and laser speckle imaging were used to detect the blood flow in mice;the targeted metabolomics based on ultra-high performance liquid chro matography tandem quadrupole linear-ion-trap mass spectrometry（UHPLC-Qtrap-MS）was used to analyze the concentrations of neurotransmitters in hippocamp al tissue as glutamic acid（Glu）,corticosterone（Cort）,histamine（HA）,histidine（His）,tyrosine（Tyr）,γ-aminobutyric acid（GABA）,kynurenine（Kyn）,dopamin e（DA）,glutamine（Gln）,tryptophan（Trp）,and 5-hydroxytryptamine（5-HT）;th e expression levels of related proteins in hippocampal tissue as Akt,ERK1/2,a nd GLT-1 were detected using immunofluorescence.4.To explore the antidepressant and mechanism of YJVO administered thr ough inhalation.C57BL/6J mice were selected to establish a CUMS mouse dep ression model,and three groups of low dose（1.5%）,medium dose（3%）,and high dose（6%）were set up;The CUMS model mice were treated with inhalat ion of YJVO,and then behavioral tests were used to evaluate the depression b ehavior of mice as a basis for efficacy evaluation.After determining the optim al dose of YJVO,the mechanism of the antidepression effect was studied.The antioxidant activity of YJVO in vitro was detected using the DPPH as well a s ABTS radical scavenging assay;then oxidative stress markers in mouse hippo campal tissue were detected:the 4-hydroxynonenal（4-HNE）content was detect ed by ELISA and the malonaldehyde（MDA）content was evaluated by thiobar bituric acid reactive substance（TBARS）;Reactive oxygen species（ROS）expres sion in mouse hippocampal cells was detected using the DCFH-DA（2,7-Dichlo rodihydrofluorescein Diacetate）probe combined with flow cytometry;the ultrast ructure of mitochondria in the hippocampus was observed using transmission el ectron microscopy（TEM）to explore the effect of inhalation of YJVO on mito chondria in mice.Results:1.The YJVO,LSVO,ALVO,CRVO,and GJVO were extracted by steam distillation,and the yield of each volatile oil was 0.27%,0.65%,1.00%,0.55%,and 0.07%,respectively.The relative content of the chemical components of each volatile oil is calculated using the peak area normalization method.Th ere were 52 compounds in YJVO have been identified,accounting for 99.27%of the total peak area,mainly terpenoids,and sesquiterpenoids.The highest con tent was（E）-Ligustilide（30.4%）,followed byβ-eudesmol（8.2%）,Atractylodin（7.5%）,Senkyunolide（4.87%）,Cyperotundone（4.76%）.Guaiol（3.23%）,α-Pinen e（3.18%）,Atractylon（3.06%）,4-Carvomenthenol（2.75%）,andα-Phellandrene（2.21%）.The main species were monoterpenoids and sesquiterpenoids,with a s mall amount of aliphatic and aromatic compounds.The relative peak area is m ainly composed of terpenoids and lactones.2.The results of acute oral toxicity evaluation showed that the LD₅,LD_95,and LD₅₀of YJVO were 3.156 m L·kg^-1,8.404 m L·kg^-1,and 5.780 m L·kg^-1(95%confidence interval:4.670-6.845 m L·kg^-1).According to the European Unio n Classification,Labelling and Packaging（CLP）Regulation,YJVO was virtuall y non-toxic.3.Compared the antidepressant effects of YJVO,YJAE,and YJW through oral administration,the results showed that YJVO displayed more significant a nd stable antidepressant effects;the optimal dose of YJVO for antidepressant w as the medium dose(0.3 m L·kg^-1).The carotid artery blood flow and cerebral blood flow were significantly reduced in CUMS model mice but increased afte r YJVO oral administration.Targeted metabolomics results showed that YJVO oral treatment significantly reversed the increase in Glu（P<0.0001）,Cort（P<0.01）,GABA（P<0.01）,His（P<0.05）,Kyn（P<0.05）,and Trp（P<0.001）concentration which induced by CUMS;increased the concentration of 5-H T in the hippocampus of CUMS model mice;and the HA concentration was si gnificantly reduced（P<0.05）in YJVO group.However,there was no signific ant effect on the content of Tyr,Gln,DA,and the ratio of Kyn/Trp.Immunofl uorescence of mouse hippocampal DG region showed that CUMS modeling sig nificantly reduced GLT-1 expression level in mice（P<0.0001）,while YJVO o ral administration reversed this change（P<0.01）;Akt（P<0.0001）and ERK1/2（P<0.001）in hippocampal DG region of CUMS depression-like model m ice were lower than the control group,while the expression of these proteins i n YJVO group was significantly increased（P<0.0001 for Akt;P<0.01 for ERK1/2）.4.By treating CUMS mice with different concentrations of YJVO for inh alation,it was found that inhalation of YJVO significantly alleviated depressive-like behavior in mice.Treating with inhalation of YJVO increased the sucrose preference and reduced the immobility time of model mice in the forced swi m test（FST）and tail suspension test（TST）,with the low dose（1.5%）been th e most significant.Subsequently,the antidepressant mechanism of YJVO nebuli zation inhalation was studied.In vitro antioxidant experiments found that YJVO has a strong in vitro scavenging effect on DPPH(IC₅₀=0.5398 mg·m L^-1)an d ABTS(IC₅₀=0.4681 mg·m L^-1);in mice,research of antioxidant showed tha t the content of 4-HNE（P<0.0001）and MDA（P<0.001）in hippocampal ti ssue was increased in model mice when compared with the control group,and YJVO inhalation treatment significantly reduced the content of 4-HNE（P<0.05）and MDA（P<0.0001）in the hippocampal tissue;TEM observed the mito chondrial ultrastructure in hippocampal cells of mice found that chronic stress caused mitochondrial damage in the hippocampus of mice,specifically manifest ed as obvious mitochondrial outer membrane blurring,reduced or disappeared c ristae,and partial vacuolization.YJVO inhalation administration can partially re verse this change.Conclusions:1.For the first time,it has been confirmed that volatile oil,as an important component of Yueju pill’s antidepressant effect,exhibits good antidepressant effects in both oral and inhalation administration.2.52 compounds were identified from YJVO.There are 47 compounds from its constituent medicinal materials,and 5 compounds are newly generated during the mixed extraction process of the entire formula.It indicating that the use of Yueju compound is not a simple superposition of medicinal herbs,but rather the mutual influence of multiple components.3.Oral administration of YJVO had a high safety profile with an LD₅₀of 5.780m L·kg^-1.According to the European Union CLP toxicity classification standard,YJVO was virtually non-toxic.4.The different mechanisms of action of YJVO through oral and inhalation administration may be related to the significant differences in the gastrointestinal and inhalation pathways involved in these two administration methods..The potential mechanism of oral antidepressant effect of YJVO is Akt/ERK1/2/GLT-1 mediated Glu clearance.The potential antidepressant mechanism of YJVO inhalation route is to reduce the degree of oxidative stress in the hippocampus to alleviate nerve injury.