Mass Spectrometry Analysis And Antidepressant Activity Of Limonoids From Xylocarpus Granatum

Natural products play a dominant role in the discovery of leads for the development of drugs for the treatment of human diseases.Indeed,pharmacologically active compounds from plants and microbes represent an important pipeline for new investigational drugs.Xylocarpus granatum has a variety of biological activities and has been extensively documented in the recent years,but the studies on its antidepressant activity were not reported in literature.Mass spectrometry technology is rapid and sensitive,and has been widely used in natural product research.Mass spectrometry fragmentation pathways efficiently facilitate elucidation of structures,dereplication of known compounds,and exploring novel compounds for further studies.However,the mass spectrometry research of limonoid structures in X.granatum was rarely documented.Therefore,this study present the identification and quantification of limonoids from X.granatum by mass spectrometry,and a preliminary study of the potential antidepressant-like effects of limonoids from X.granatum.This study lays a foundation for discovering novel compound from X.granatum as well as the authentication and quality control of X.granatum.This study provides experimental basis for discovering lead compounds from X.granatum limonoids with antidepressant activity.Part one Identification of limonoids from Xylocarpus granatum by mass spectrometryObjective: To explore the fragmentation pathways of limonoids from Xylocarpus granatum through the analysis of their quadrupole time-of-flight tandem mass spectrometry(Q-TOF-MS/MS)spectra.The fragmentation process and fragmentation patterns were summarized.To identify limonoids from Xylocarpus granatum by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry(UHPLC-Q-TOF-MS/MS).Methods:1 UHPLC-Q-TOF-MS/MS analytical conditions: The LC separation was conducted on a Kinetex column(XB-C18,100 mm×2.1 mm,2.6 ?m)at 40 °C with a 2 ?L injection volume.The mobile phase consisted of acetonitrile(A)and 1 mM ammonium acetate containing 0.1%(v/v)formic acid(B)at a flow rate of 400 ?L/min.The following gradient program was used for quantitation: 0.0-5.0 min,10% A;5.0-10.0 min,10-30% A;10.0-20.0 min,30-35% A;20.0-35.0 min,35-50%% A;35.0-45.0 min,50-95% A;45.0-50.0 min,95% A.The ESI ion spray voltage was 5500 V under positive ionization mode with a turbo spray temperature of 600 °C.The curtain gas,nebulizer gas(Gas 1)and heater gas(Gas 2)were set at 35,50 and 55 psi,respectively.Information dependent acquisition using a TOF-MS survey scan 100-1000 Da(200 ms)and up to 8 dependent TOF-MS/MS scans 50-1000 Da(100 ms)using Collision Energy(CE)of 35 eV with Collision Energy Spread(CES)of ± 15 eV.Empirical formula calculation and fragment ion prediction using PeakView? software.2 Strategy of identification: Unambiguously identification of known compounds was performed by comparing the retention time,exact mass and MS/MS with those of reference compounds.Tentative identification of known compounds and unknown compounds was performed based on fragmentation patterns,exact mass,the retention time and phytochemistry reports.Results:1 The fragmentation patterns of 17 limonoids from Xylocarpus granatum were explored and summarized using Q-TOF-MS/MS.The fragmentation patterns mainly include the loss of acyl/acyloxy side chains,loss of H2 O and loss of CO.2 UHPLC-Q-TOF-MS/MS was used for the identification of limonoids from Xylocarpus granatum.At least 300 compounds were detected in the extract from Xylocarpus granatum fruit.17 known compounds were identified unambiguously compared with those of reference compounds.116 compounds were tentatively identified.7 compounds were characterized as potential new ones.Conclusion:1 The fragmentations patterns of 17 limonoids from Xylocarpus granatum were studied systematically for the first time.2 Limonoids were identified by UHPLC-Q-TOF-MS/MS from Xylocarpus granatum.These limonoids from Xylocarpus granatum fruit were putatively identified by comparing their mass spectral fragmentation patterns with those of their authentic compounds and phytochemistry reports.UHPLC-Q-TOF-MS/MS method is simple,rapidly,high sensitivity and specificity for the identification of limonoids from Xylocarpus granatum.The identification of these chemicals provided essential data for further phytochemical studies,metabolites identification,and the quality control of Xylocarpus granatum.Part two Simultaneous determination of limonoids from Xylocarpus granatum by mass spectrometryObjective: To develope and validate a chemometric-assisted liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for the simultaneous determination of 17 limonoids to reveal the chemical composition and distribution of limonoids in the different parts of Xylocarpus granatum fruit.Methods:1 LC-MS/MS analytical conditions: The LC separation of 17 analytes was conducted on a Phenomenex Kinetex column(XB-C18,100 mm×2.1 mm,2.6 ?m)at 40 °C with a 5 ?L injection volume.The mobile phase consisted of acetonitrile(A)and 0.1%(v/v)formic acid(B)at a flow rate of 300 ?L/min.The following gradient program was used for quantitation: 0.0-1.0 min,45-55% A;1.0-4.0 min,55-60% A;4.0-5.0 min,60-95% A;5.0-10.0 min,95% A;10.0-11.0 min,95-45% A.The ESI ion spray voltage was 5500 V under positive ionization mode with a turbo spray temperature of 600 °C.The curtain gas,nebulizer gas(Gas 1)and heater gas(Gas 2)were set at 30,50 and 55 psi,respectively.Nitrogen was used in all cases.The scheduled MRM algorithm was used to monitor 34 ion transitions(2 ion transitions per compound)at the expected retention time with an MRM detection window of 90 s and a target scan time of 0.7 s.For improved confirmation,an information dependent acquisition(IDA)survey scan triggered dependent MS/MS spectra acquisition in enhanced product ion(EPI)mode.Saving the complete molecular fingerprint into the EPI spectra significantly reduced the risk of false positive and negative results.Both the MRM and EPI experiments were performed in a single run.EPI spectra were generated at a scan speed of 4000 Da/s using a standardized collision energy(CE)of 35 eV with a collision energy spread of 15 e V.For the IDA setting,the most intense precursor ions were used with a mass tolerance of 250 mDa,the dynamic background was subtracted for each survey scan,and the intensity threshold was set to 100 cps.EPI identification was performed in MasterView? software with the PURITY value set to 70% or higher for positive identification.The dwell time is the only parameter that is different between conventional MRM and scheduled MRM,and it was used to replace the detection time window with 20 ms for each ion transition.2 The scheduled MRM LC-MS/MS method was validated for linearity,LOD,precision,repeatability,stability,recovery and matrix effects.Ultrasonic-assisted extraction(UAE)was optimized by response surface methodology(RSM).Furthermore,principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA)were used to analyze the LC-MS/MS data to provide chemometric model for easy visualization and interpretation for the classification of different parts of X.granatum fruit.Results:1 In this study,a chemometric-assisted LC-MS/MS method was developed and validated for the simultaneous determination of 17 limonoids in different parts of X.granatum fruit.The scheduled MRM had increased sensitivity and precision compared to conventional MRM.The scheduled MRM LC-MS/MS method was well validated for linearity(R>0.99),LOD(0.2-10.0 ng/mL),precision(intra-day 1.2%-7.6% and inter-day 1.1%-2.6%),repeatability(2.1%-6.9%),stability(4.3%-7.2%),recovery(88.2%-106.2%)and matrix effects(1.6%-8.4%).2 The optimal UAE obtained by RSM was more accurate than that of the general OVAT.The method was used to analyze 35 batches of different parts of X.granatum fruit.In view of the chemical composition and distribution,all 17 limonoids showed a rank order of seed kernels > seed > fruit,and 13 limonoids showed a rank order of seed kernels > seed > fruit > fruit peel > seed coat.Based on the PCA and OPLS-DA analysis of the LC-MS/MS data,potential marker compounds were identified for the classification of different parts of X.granatum fruit.Conclusion: In this study,a chemometric-assisted liquid chromatography-tandem mass spectrometry(LC-MS/MS)method was developed and validated for the simultaneous determination of 17 limonoids to reveal the chemical composition and distribution of limonoids in the different parts of X.granatum fruit.In addition,the study indicated that the chemometric-assisted strategy consisting of RSM,PCA and OPLS-DA for the development,optimization and data analysis of multi-component quantitation using LC-MS/MS is effective and feasible.Moreover,this study provided chemical composition and distribution evidence for the authentication and quality control of X.granatum fruit as a folk medicine.Part Three Antidepressant activity of limonoids from Xylocarpus granatum: a preliminary studyObjective: To investigate the potential antidepressant-like effects and the underlying mechanisms of limonoids from Xylocarpus granatum in acute stress-induced depression mouse models.Methods: Antidepressant-like effects of limonoids from Xylocarpus granatum(xylocarpin H,cipadesin A and xyloccensin K)were investigated through behavioral tests,and potential mechanism was assessed by neuroendocrine system.Antidepressant-like effects of limonoids from Xylocarpus granatum(xylocarpin H,cipadesin A and xyloccensin K)(5,15,50 mg/kg/day for 7days,intragastrically)were estimated through forced-swimming test(FST),tail suspension test(TST)and open field test(OFT).Effects of xylocarpin H and cipadesin A on hypothalamus-pituitaryadrenal(HPA)axis were evaluated by analysis of serum corticosterone(CORT)and adrenocorticotropic hormone(ACTH)using enzyme-linked immunosorbent assay(ELISA).Results: Limonoids from Xylocarpus granatum administration significantly reduced the floating time in the FST(xylocarpin H,cipadesin A and xyloccensin K at 15-50 mg/kg)and immobility time in the TST(xylocarpin H and cipadesin A at 15-50 mg/kg;xyloccensin K at 5-50 mg/kg).Limonoids from Xylocarpus granatum dose-dependently increased the time in the central zone in the OFT(xylocarpin H at 5-50 mg/kg;cipadesin A at 15-50 mg/kg;xyloccensin K at 50 mg/kg),without altering the locomotor activity.Moreover,repeated xylocarpin H and cipadesin A treatment significantly inhibited the increase levels of serum CORT(xylocarpin H at 15-50 mg/kg;cipadesin A at 5-50 mg/kg),ACTH(xylocarpin H and cipadesin A at 15-50 mg/kg)following the forced swimming,but not in the absence of stress.Conclusion: Limonoids from Xylocarpus granatum(xylocarpin H,cipadesin A and xyloccensin K)has antidepressant-like activities in acute stressed mice model of depression,which likely occurs by inhibiting the HPA axis activity response to stress(xylocarpin H and cipadesin A).These data support further exploration for developing limonoids from Xylocarpus granatum(xylocarpin H,cipadesin A and xyloccensin K)as a potential agent to treat depression and anxiety disorders.