【摘要】：在發(fā)現(xiàn)先導(dǎo)化合物用于開發(fā)具有治療人類疾病的藥物過程中,天然產(chǎn)物具有主導(dǎo)地位。事實(shí)上,植物和微生物的藥理活性成分是新藥研發(fā)的重要途徑。木果楝(Xylocarpus granatum Koenig)具有多種活性,并且近年被廣泛證明。但是對(duì)其抗抑郁活性的研究未見報(bào)道�？焖�、靈敏的質(zhì)譜技術(shù)在天然產(chǎn)物中的應(yīng)用越來越廣泛,且質(zhì)譜裂解規(guī)律有助于快速識(shí)別并解析化合物的結(jié)構(gòu),而對(duì)已知物去重復(fù),進(jìn)而探索未知化合物進(jìn)行深入研究。目前,關(guān)于木果楝中含有獨(dú)特結(jié)構(gòu)的檸檬苦素的質(zhì)譜研究還鮮有報(bào)道。因此,本研究運(yùn)用質(zhì)譜方法對(duì)木果楝檸檬苦素類成分進(jìn)行鑒定和定量的快速識(shí)別,并對(duì)木果楝檸檬苦素的抗抑郁活性進(jìn)行了初步實(shí)驗(yàn)研究,本研究為從木果楝中快速發(fā)現(xiàn)新的化合物及鑒定和質(zhì)量控制木果楝奠定基礎(chǔ),為從木果楝檸檬苦素中發(fā)現(xiàn)具有抗抑郁活性先導(dǎo)化合物提供實(shí)驗(yàn)依據(jù)。第一部分木果楝檸檬苦素的質(zhì)譜鑒定目的:通過分析木果楝檸檬苦素的二級(jí)質(zhì)譜(Q-TOF-MS/MS),探索其質(zhì)譜裂解途徑,總結(jié)裂解規(guī)律。運(yùn)用超高效液相色譜串聯(lián)四級(jí)桿飛行時(shí)間質(zhì)譜(UHPLC-Q-TOF-MS/MS)對(duì)木果楝檸檬苦素類化合物進(jìn)行鑒定。方法:1 UHPLC-Q-TOF-MS/MS分析條件:Kinetex-C18色譜柱(100 mm×2.1mm,2.6μm),柱溫:40℃。流動(dòng)相:A為乙腈,B為水-0.1%甲酸-1 m M乙酸銨溶液。梯度洗脫,洗脫程序如下:10%A(0~5 min);10-30%A(5~10min);30-35%A(10~20 min);35-50%A(20~35 min);50-95%A(35~45min);95%A(45~50 min)。流速:0.4 m L/min。進(jìn)樣量:2μL。Duo Spray電噴霧離子(ESI)源;離子源溫度(turbo spray temperature):600℃。氣簾氣(CUR):35 psi;霧化氣(Gas 1):50 psi;加熱氣(Gas 2):50 psi;在正離子模式下,噴霧電壓(ion spray voltage)為5500 V,解簇電壓(DP)為80 V。信息相關(guān)采集模式,包括TOF-MS探測(cè)掃描100-1000 Da(200ms)和最多8個(gè)依賴的TOF-MS/MS掃描50-1000 Da(100 ms),碰撞能量為35 e V,碰撞擴(kuò)展能量為±15 e V。2鑒定策略:經(jīng)驗(yàn)分子式的計(jì)算和碎片離子的預(yù)測(cè)使用Peak View?軟件完成。通過與對(duì)照品比較保留時(shí)間、精確質(zhì)量和MS/MS質(zhì)譜,明確鑒定已知化合物�；谫|(zhì)譜裂解規(guī)律、精確質(zhì)量、保留時(shí)間和植物化學(xué)文獻(xiàn),初步鑒定已知化合物和推測(cè)鑒定未知化合物。結(jié)果:1應(yīng)用Q-TOF-MS/MS分析并總結(jié)了17個(gè)木果楝檸檬苦素的質(zhì)譜裂解規(guī)律。這些裂解規(guī)律主要包括,丟失�；蝓Ｑ趸鶄�(cè)鏈、丟失水分子及CO分子。2應(yīng)用UHPLC-Q-TOF-MS/MS鑒定了木果楝檸檬苦素類化合物。從木果楝果實(shí)提取物中檢測(cè)到了至少300個(gè)化合物。通過與對(duì)照品對(duì)比,明確鑒定了17個(gè)已知化合物。初步鑒定了116個(gè)化合物。鑒定了7個(gè)潛在的新化合物。結(jié)論:1首次系統(tǒng)的研究了17個(gè)木果楝檸檬苦素的質(zhì)譜裂解規(guī)律。2 UHPLC-Q-TOF-MS/MS法鑒定了木果楝檸檬苦素類化合物�；趯�(duì)照品的質(zhì)譜裂解規(guī)律、植物化學(xué)文獻(xiàn),推測(cè)鑒定了木果楝果實(shí)中的檸檬苦素類化合物。UHPLC-Q-TOF-MS/MS法鑒定木果楝檸檬苦素簡(jiǎn)單、快速、靈敏度高、專屬性好。鑒定這些化合物為進(jìn)一步研究木果楝中檸檬苦素類化合物的植物化學(xué)、代謝物鑒定和質(zhì)量控制提供了重要依據(jù)。第二部分木果楝檸檬苦素的質(zhì)譜定量測(cè)定目的:開發(fā)和驗(yàn)證一種化學(xué)計(jì)量學(xué)輔助的LC-MS/MS方法用于測(cè)定17種檸檬苦素,揭示木果楝果實(shí)不同部位檸檬苦素的化學(xué)組成與分布。方法:1 LC-MS/MS分析條件:17個(gè)分析物的LC分離在40℃的Phenomenex Kinetex柱(XB-C18,100 mm×2.1 mm,2.6μm)中進(jìn)行,使用5μL進(jìn)樣體積。流動(dòng)相由乙腈(A)和0.1%(v/v)甲酸(B)組成,流速為300μL/min。以下梯度程序用于定量: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。ESI噴霧電壓為5500 V,正離子模式監(jiān)測(cè),噴霧溫度為600℃。氣簾氣、霧化氣體(Gas 1)和加熱器氣體(Gas 2)分別設(shè)置在30、50和55 psi。在所有情況下使用氮?dú)�。預(yù)設(shè)的MRM算法用于在90 s的MRM檢測(cè)窗口和0.7 s的目標(biāo)掃描時(shí)間,在預(yù)期的保留時(shí)間監(jiān)測(cè)34個(gè)離子對(duì)(每個(gè)化合物2個(gè)離子對(duì))。在增強(qiáng)產(chǎn)物離子(enhanced product ion,EPI)模式中,信息相關(guān)采集(information dependent acquisition,IDA)掃描觸發(fā)相關(guān)的MS/MS質(zhì)譜掃描以便更好地確認(rèn)被測(cè)物。將完整的分子指紋信息保存在EPI譜中,顯著降低假陽性和陰性結(jié)果的風(fēng)險(xiǎn)。使用35 e V的標(biāo)準(zhǔn)化碰撞能(collision energy,CE)以15 e V的擴(kuò)展碰撞能量在4000 Da/s的掃描速度下產(chǎn)生EPI質(zhì)譜。IDA設(shè)置中,選擇響應(yīng)信號(hào)最高的前體離子(質(zhì)量差異范圍為250 m Da),每個(gè)探測(cè)掃描都扣除動(dòng)態(tài)背景,信號(hào)閾值為100cps。應(yīng)用Master View?軟件進(jìn)行EPI鑒定,純度值設(shè)置在大于等于70%為陽性結(jié)果。常規(guī)MRM與預(yù)設(shè)MRM相比,駐留時(shí)間是唯一的不同參數(shù),它用20 ms替換了每對(duì)離子的監(jiān)測(cè)時(shí)間窗口。2對(duì)預(yù)設(shè)MRM LC-MS/MS方法進(jìn)行驗(yàn)證,包括,線性、最低檢測(cè)限、精密度、重復(fù)性、穩(wěn)定性、回收率和基質(zhì)效應(yīng)。響應(yīng)面優(yōu)化方法用于優(yōu)化超聲輔助提取。主成分分析(principal component analysis,PCA)和正交偏最小二乘法判別分析(orthogonal partial least squares discriminant analysis,OPLS-DA)用于分析LC-MS/MS數(shù)據(jù),可提供化學(xué)計(jì)量學(xué)模型用于簡(jiǎn)便的可視化和解釋木果楝果實(shí)不同部位的差異。結(jié)果:1本研究中,開發(fā)和驗(yàn)證了化學(xué)計(jì)量學(xué)輔助的LC-MS/MS法同時(shí)測(cè)定木果楝果實(shí)不同部位的17種檸檬苦素。與常規(guī)MRM相比,預(yù)設(shè)MRM提高了靈敏度和精度。預(yù)設(shè)MRM LC-MS/MS方法得到了良好的驗(yàn)證,包括,線性(R0.99)、最低檢測(cè)限(0.20-10.0 ng/m L)、精密度(日內(nèi)1.2%-7.6%和日間1.1%-2.6%)、重復(fù)性(2.1%-6.9%)、穩(wěn)定性(4.3%-7.2%)、回收率(88.2%-106.2%)和基質(zhì)效應(yīng)(90.1%-103.3%)。2通過RSM獲得的最優(yōu)超聲輔助提取條件比一般單次單因素法更準(zhǔn)確。該方法用于分析35批木果楝果實(shí)不同部位的檸檬苦素。所有17種檸檬苦素顯示明顯的排序,種仁種子果實(shí);13種檸檬苦素顯示另一種排序,種仁種子果實(shí)果皮種皮�；贚C-MS/MS數(shù)據(jù)的PCA和OPLS-DA分析,確定了木果楝果實(shí)不同部分分類的潛在化學(xué)標(biāo)記物。結(jié)論:本研究中,為了揭示木果楝果實(shí)不同部位檸檬苦素的化學(xué)成分組成與分布,開發(fā)和驗(yàn)證了化學(xué)計(jì)量學(xué)輔助的LC-MS/MS法同時(shí)測(cè)定17種檸檬苦素。另外,本研究表明針對(duì)LC-MS/MS進(jìn)行多組分定量的開發(fā)、優(yōu)化和數(shù)據(jù)分析,由RSM,PCA和OPLS-DA組成的化學(xué)計(jì)量學(xué)輔助的綜合策略是有效和可行的。此外,本研究提供了鑒定和質(zhì)量控制木果楝果實(shí)作為民間藥物的化學(xué)成分組成與分布的證據(jù)。第三部分木果楝檸檬苦素的抗抑郁活性初步實(shí)驗(yàn)研究目的:研究木果楝檸檬苦素在急性應(yīng)激誘導(dǎo)的小鼠抑郁模型中潛在的抗抑郁活性和作用機(jī)制。方法:通過行為學(xué)實(shí)驗(yàn)研究木果楝檸檬苦素xylocarpin H、cipadesin A和xyloccensin K的抗抑郁活性,通過神經(jīng)內(nèi)分泌系統(tǒng)研究其潛在的作用機(jī)制。木果楝檸檬苦素xylocarpin H、cipadesin A和xyloccensin K的抗抑郁活性(5,15,50 mg/kg/day for 7days,intragastrically)通過強(qiáng)迫游泳實(shí)驗(yàn)、懸尾實(shí)驗(yàn)和曠場(chǎng)實(shí)驗(yàn)評(píng)價(jià)。通過酶免疫法測(cè)定血清皮質(zhì)酮和促腎上腺皮質(zhì)激素,評(píng)價(jià)xylocarpin H和cipadesin A對(duì)下丘腦-垂體-腎上腺軸的作用。結(jié)果:木果楝檸檬苦素能顯著降低小鼠強(qiáng)迫游泳實(shí)驗(yàn)中的漂浮不動(dòng)時(shí)間(xylocarpin H、cipadesin A和xyloccensin K在15-50 mg/kg),顯著降低小鼠懸尾實(shí)驗(yàn)中懸尾不動(dòng)時(shí)間(xylocarpin H和cipadesin A在15-50mg/kg,xyloccensin K在5-50 mg/kg)。木果楝檸檬苦素顯著增加小鼠曠場(chǎng)實(shí)驗(yàn)中在曠場(chǎng)中央?yún)^(qū)域停留時(shí)間(xylocarpin H在5-50 mg/kg,cipadesin A在15-50 mg/kg,xyloccensin K在50 mg/kg),并且不影響自發(fā)活動(dòng)。另外,重復(fù)給予xylocarpin H和cipadesin A顯著降低了處于強(qiáng)迫游泳實(shí)驗(yàn)小鼠血清中皮質(zhì)酮CORT(xylocarpin H在15-50 mg/kg,cipadesin A在5-50 mg/kg)和促腎上腺皮質(zhì)激素ACTH(xylocarpin H和cipadesin A在15-50 mg/kg)的水平,但對(duì)于未經(jīng)應(yīng)激處理的小鼠無影響。結(jié)論:木果楝檸檬苦素xylocarpin H、cipadesin A和xyloccensin K在急性應(yīng)激誘導(dǎo)的小鼠抑郁模型中具有潛在的抗抑郁活性,潛在的作用機(jī)制可能是通過抑制下丘腦-垂體-腎上腺軸對(duì)應(yīng)激應(yīng)答的活性。這些數(shù)據(jù)支持對(duì)開發(fā)木果楝檸檬苦素作為潛在的抗抑郁和焦慮藥物做進(jìn)一步的研究。
[Abstract]:Natural products play a leading role in the discovery of lead compounds for the development of drugs for the treatment of human diseases. In fact, the pharmacological active ingredients of plants and microorganisms are important approaches for the development of new drugs. Rapid and sensitive mass spectrometric techniques have been widely used in natural products. Mass spectrometric pyrolysis is helpful to identify and analyze the structures of compounds quickly, and repeat the known compounds to explore the unknown compounds. Mass spectrometry is rarely reported. Therefore, this study used mass spectrometry to identify and quantify the limonin components of Melia azedarach, and carried out a preliminary experimental study on the antidepressant activity of Melia azedarach limonin. This study laid a foundation for the rapid discovery of new compounds from Melia azedarach and the identification and quality control of Melia azedarach. The first part is the mass spectrometric identification of limonin from Melia azedarach. Objective: By analyzing the secondary mass spectrometry of limonin from Melia azedarach (Q-TOF-MS/MS), to explore its mass spectrometric cleavage pathway and summarize the cleavage rule. Methods: 1 UHPLC-Q-TOF-MS/MS analysis conditions: Kinetex-C18 column (100 m m *2.1 m m, 2.6 micron), column temperature: 40 C. Mobile phase: acetonitrile A, water-0.1% formic acid-1 M ammonium acetate solution. Gradient elution procedure: 10% A (0-5 min); % A (5-10 min); 30-35% A (10-20 min); 35-50% A (20-35 min); 50-95% A (35-45 min); 95% A (45-50 min). Flow rate: 0.4 m L/min. Sample volume: 2 mu L. Duo Spray Electrospray Ion Source (ESI); Turbospray temperature: 600 (?) CUR: 35 psi; Gas 1: 50 psi; Heating gas (50 psi); Spray: 50 psi in positive ion mode The voltage (ion spray voltage) is 5500 V and the unwrapping voltage (DP) is 80 V. Information correlation acquisition mode includes TOF-MS probe scan 100-1000 Da (200 ms) and up to 8 dependent TOF-MS/MS scan 50-1000 Da (100 ms), collision energy 35 e V, collision spread energy (+15 e V.2) identification strategy: empirical molecular formula calculation and debris ion prediction enabled The known compounds were identified by comparing retention time, accurate mass and MS/MS mass spectrometry with the reference materials. Based on the mass spectrometric pyrolysis law, accurate mass, retention time and phytochemical literature, the known compounds were identified and the unknown compounds were speculated. Mass spectrometric fragmentation of Limonin in Melia azedarach fruit. These fragmentation patterns mainly include loss of acyl or acyloxy side chains, loss of water and CO molecules. 2 UHPLC-Q-TOF-MS/MS was used to identify limonin compounds in Melia azedarach fruit. At least 300 compounds were detected from the extracts of Melia azedarach fruit. The results were compared with those of the control. Seventeen known compounds were identified. Seven potential new compounds were identified. CONCLUSION: 1. Mass spectrometric fragmentation of 17 limonins from Melia azedarach was systematically studied for the first time. 2 U-HPLC-Q-TOF-MS/MS methods were used to identify the limonins from Melia azedarach. The identification of limonoids in the fruit of Melia azedarach by UHPLC-Q-TOF-MS/MS is simple, rapid, sensitive and specific. The identification of these compounds provides an important basis for further study on the phytochemistry, metabolites identification and quality control of the limonoids in Melia azedarach. Objective: To develop and validate a chemometric-assisted LC-MS/MS method for the determination of 17 kinds of Limonin in Melia azedarach fruits, and to reveal the chemical composition and distribution of Limonin in different parts of Melia azedarach fruits. Methods: 1 LC-MS/MS analysis conditions: LC separation of 17 analytes on a Chemometric Kinetex column (XB-MS/MS) The mobile phase consists of acetonitrile (A) and 0.1% (v/v) formic acid (B) at a flow rate of 300 muL/min. The following gradient procedures are used for quantitative quantification: 0.0-1.0 min, 45-55% A; 1.0-4.0 min, 55-60% A; 4.0-5.0 min, 60-50 min, 60-95% A; 5.0-0-10.0 min, 95% A; 10.0-10-10-0-95% A; 10.0-10-0-0-11.0-11.0, 95-45.0, 95-45-45.95-45.0-45% A; 1.0-0-1-0-4-4.0-4-4.0-4-4-4.0 positive ion mode Gas curtain gas, atomized gas (Gas 1) and heater gas (Gas 2) are set at 30, 50 and 55 psi, respectively. Nitrogen is used in all cases. The default MRM algorithm is used to monitor 34 ion pairs (2 ion pairs per compound) in the 90 s MR detection window and 0.7 s target scan time. In the enhanced product ion (EPI) mode, the information dependent acquisition (IDA) scan triggers the associated MS/MS mass spectrometry scans to better identify the object under test. The collision energy (CE) produces EPI mass spectrometry at a scan speed of 4000 Da/s with a 15 e V extended collision energy. In IDA settings, the precursor ions with the highest response signal (mass difference range 250 m Da) are selected, and the dynamic background is deducted for each detection scan. The signal threshold is 100 cps. EPI is identified by Master View? Residence time is the only difference between conventional MRM and preset MRM. It replaces the monitoring time window for each pair of ions with 20 ms. 2 The preset MRM LC-MS/MS method was validated, including linearity, minimum detection limit, precision, repeatability, stability, recovery and matrix effect. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) are used to analyze LC-MS/MS data. Chemometric models can be used to visualize and interpret neem easily. Results: 1 In this study, a chemometric-assisted LC-MS/MS method was developed and validated for simultaneous determination of 17 limonins in different parts of Melia azedarach fruit. Compared with conventional MRM, preset MRM improved sensitivity and accuracy. Preset MRM LC-MS/MS method was well validated, including linear (R0.99), minimum detection. The optimal ultrasonic-assisted extraction conditions obtained by RSM were more accurate than those obtained by single factor analysis in 35 batches of neem fruits. All 17 kinds of Limonin showed distinct order, seed and fruit; 13 kinds of Limonin showed another order, seed peel and seed coat. Potential chemical markers for different parts of neem fruit were identified by PCA and OPLS-DA analysis based on LC-MS/MS data. Chemometric-assisted LC-MS/MS method was developed and validated for the simultaneous determination of 17 kinds of Limonin in different parts of Melia azedarach fruit. In addition, this study showed that LC-MS/MS was a multi-component quantitative development, optimization and data analysis, and a chemometric-assisted synthesis consisting of RSM, PCA and OPLS-DA. In addition, this study provides evidence for identifying and controlling the chemical composition and distribution of the fruit of Melia azedarach as a folk drug. Part III Preliminary experimental study on the antidepressant activity of Melia azedarach limonin Objective: To study the effect of Melia azedarach limonin on acute stress-induced depression in mice. Potential antidepressant activities and mechanisms. METHODS: The antidepressant activities of xylocarpin H, cipadesin A and xyloccensin K in Melia azedarach were studied by behavioral experiments, and their potential mechanisms were studied by neuroendocrine system. The antidepressant activities of xylocarpin H, cipadesin A and xyloccensin K in Melia azedarach (5,15,50). The effects of xylocarpin H and cipadesin A on the hypothalamus-pituitary-adrenal axis were evaluated by enzyme immunoassay. Results: Melia azedarach limonin significantly decreased the forced swimming in mice. The floating immobility time (xylocarpin H, cipadesin A and xyloccensin K, 15-50 mg/kg) was significantly decreased in mice tail suspension test (xylocarpin H and cipadesin A, 15-50 mg/kg, xyloccensin K, 5-50 mg/kg). Azadirachtin significantly increased the residence time (xylo) in the central area of the open field in mice tail suspension test. Carpin H was 5-50 mg/kg, cipadesin A was 15-50 mg/kg, xyloccensin K was 50 mg/kg, and did not affect spontaneous activity. In addition, repeated administration of xylocarpin H and cipadesin A significantly reduced serum corticosterone CORT (xylocarpin H was 15-50 mg/kg, cipadesin A was 5-50 mg/kg) and ACTH (adrenocorticotrophic hormone ACTH) levels in forced swimming mice. The levels of xylocarpin H and cipadesin A were 15-50 mg/kg, but had no effect on mice without stress treatment. CONCLUSION: Melia azedarach limonin xylocarpin H, cipadesin A and xyloccensin K have potential antidepressant activity in mice with acute stress-induced depression. The potential mechanism may be through inhibiting hypothalamus-pituitary. - The activity of the adrenal axis in response to stress. These data support further studies on the development of Limonin as a potential antidepressant and anxiety drug.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R285.5

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