【摘要】：中藥多成分藥代動(dòng)力學(xué)(簡稱多成分藥代)是中醫(yī)藥理論和作用機(jī)制闡明的重要研究內(nèi)容。但是由于中藥成分的復(fù)雜和多樣性,以及中藥成分與生物體系相互作用的復(fù)雜性和不確定性,中藥多成分藥代動(dòng)力學(xué)的研究一直面臨著巨大挑戰(zhàn):(1)中藥化學(xué)成分復(fù)雜、體內(nèi)代謝多樣化,使得體內(nèi)成分的結(jié)構(gòu)鑒定困難;(2)藥代動(dòng)力學(xué)研究的目標(biāo)化合物不明確、在中藥及生物樣本濃度低、內(nèi)源性基質(zhì)和組分之間干擾大、對(duì)照品缺乏,導(dǎo)致中藥多成分檢測(cè)和定量方法的建立困難;(3)藥效指標(biāo)不統(tǒng)一、有效成分不明確,導(dǎo)致中藥藥代動(dòng)力學(xué)研究結(jié)果的綜合評(píng)價(jià)和應(yīng)用不充分。因此需要運(yùn)用先進(jìn)的分析技術(shù),能夠快速、準(zhǔn)確、全面的定性分析中藥體內(nèi)多成分,靈敏、快速、準(zhǔn)確、寬動(dòng)態(tài)范圍的定量分析中藥體內(nèi)多成分,建立符合中醫(yī)治病整體觀的更深層次和更廣視角的新策略,為符合中醫(yī)理論的中藥多成分藥代動(dòng)力學(xué)研究提供技術(shù)支持和科學(xué)數(shù)據(jù)。本論文針對(duì)中藥藥代動(dòng)力學(xué)研究存在的一些瓶頸問題,分別選擇了巫山淫羊藿、小續(xù)命湯有效成分組和二仙湯為研究對(duì)象,探索性地開展了中藥多成分藥代動(dòng)力學(xué)研究所需分析新技術(shù)和新策略的研究工作。論文第一部分針對(duì)中藥多成分藥代研究缺乏體內(nèi)成分及其代謝物對(duì)照品的瓶頸問題,探索性地基于體內(nèi)主要成分代謝途徑的全面詮釋,建立多成分藥代指標(biāo)成分的研究策略。該策略首先采用高效液相色譜-高分辨質(zhì)譜/質(zhì)譜聯(lián)用方法(HPLC-HRMSn)和質(zhì)譜樹狀圖相似度過濾(MTSF)技術(shù)對(duì)淫羊藿5個(gè)主要異戊烯基黃酮成分在大鼠體內(nèi)的代謝產(chǎn)物進(jìn)行了分析和鑒定,基于體內(nèi)代謝物的鑒定結(jié)果推斷5種主要異戊烯基黃酮的代謝途徑,將血漿中主要暴露的14個(gè)原型成分和葡萄糖醛酸代謝物選為潛在的藥代指標(biāo)成分,作為測(cè)定對(duì)象,采用高效液相色譜串聯(lián)質(zhì)譜分析技術(shù)(HPLC-MS/MS),初步研究了 5種主要異戊烯基黃酮灌胃給予大鼠后,這14個(gè)體內(nèi)成分在血漿中的主要藥動(dòng)學(xué)參數(shù)特征;由于葡萄糖醛酸結(jié)合型代謝物沒有對(duì)照品,因此采用酶水解的方法將血漿樣本中的這14個(gè)體內(nèi)成分轉(zhuǎn)化為有對(duì)照品、且存在代謝轉(zhuǎn)化關(guān)系的10個(gè)潛在藥代指標(biāo)成分作為藥代動(dòng)力學(xué)的研究對(duì)象,建立了 HPLC-MS/MS分析方法:在色譜分離方面,通過優(yōu)化兩種流動(dòng)相的比例和混合時(shí)間,在二元液相泵上實(shí)現(xiàn)了四元流動(dòng)相的分離效果,消除了朝藿定A-C/淫羊藿苷的cross-talk和淫羊藿素的基質(zhì)效應(yīng);在質(zhì)譜檢測(cè)方面,采用分段多反應(yīng)監(jiān)測(cè)模式,縮短了質(zhì)譜的duty cycle,從而增加了方法的靈敏度。所建立的方法經(jīng)過了方法驗(yàn)證,能夠滿足生物樣品分析,并被應(yīng)用于研究5種主要異戊烯基黃酮和巫山淫羊藿灌胃給予大鼠后,10個(gè)潛在的藥代指標(biāo)成分在血漿中的主要藥動(dòng)學(xué)參數(shù)特征;采用Kendall's tau-b等級(jí)相關(guān)分析法對(duì)酶水解前后血漿中測(cè)得的指標(biāo)成分的濃度隨給藥時(shí)間的變化進(jìn)行相關(guān)性分析。實(shí)驗(yàn)結(jié)果表明淫羊藿素的葡萄糖醛酸結(jié)合型代謝物是巫山淫羊藿灌胃給藥后,異戊烯基黃酮在大鼠體內(nèi)的主要存在形式。另外,酶水解血漿中的總淫羊藿素水平能夠同時(shí)反映異戊烯基黃酮在體內(nèi)的暴露量和動(dòng)態(tài)變化。因此,具有高暴露量和較好相關(guān)性(r0.5)的淫羊藿素可以作為巫山淫羊藿異戊烯基黃酮的藥代指標(biāo)成分。除此之外,我們還利用淫羊藿素的藥代特征建立數(shù)學(xué)模型,用于預(yù)測(cè)大鼠體內(nèi)巫山淫羊藿的暴露量。結(jié)果顯示預(yù)測(cè)值與實(shí)驗(yàn)值有較好的一致性,進(jìn)一步證實(shí)酶水解血漿中的淫羊藿素可以作為巫山淫羊藿中異戊烯基黃酮的藥代指標(biāo)成分。論文第二部分研究了中藥復(fù)方小續(xù)命湯抗腦缺血的有效成分組(AF-XXMD)的多成分藥代。采用HPLC-HRMSn方法和MTSF技術(shù)分析和鑒定AF-XXMD中68種化學(xué)成分,對(duì)大鼠血漿和腦內(nèi)高暴露水平的化學(xué)成分,采用反向分子對(duì)接的方法,預(yù)測(cè)了藥理活性,選擇大鼠血漿和腦組織21個(gè)高暴露且預(yù)測(cè)有活性的化合物作為藥代指標(biāo)成分,建立高分離度快速液相色譜串聯(lián)質(zhì)譜法(RRLC-MS/MS)。前處理方法通過添加抗壞血酸保護(hù)血漿中的黃芩苷和黃芩素不被氧化,通過脂質(zhì)預(yù)處理柱除去磷脂,以消除血漿和腦組織中甘草酸的基質(zhì)效應(yīng);色譜分離采用RRLC法,可實(shí)現(xiàn)3對(duì)同分異構(gòu)體的基線分離;質(zhì)譜檢測(cè)采用分時(shí)間段的正負(fù)離子切換模式,并通過添加微量的甲酸銨(0.08mmol l-1)提高各化合物的質(zhì)譜響應(yīng)。對(duì)所建方法進(jìn)行了方法驗(yàn)證,表明該方法符合生物樣品分析方法的技術(shù)要求,滿足大鼠腦組織和血漿中的藥代動(dòng)力學(xué)研究需要;采用正常大鼠與雙側(cè)頸總動(dòng)脈結(jié)扎導(dǎo)致的慢性腦缺血模型大鼠,灌胃給予AF-XXMD,比較研究了 21個(gè)指標(biāo)成分的血漿和靶器官腦的藥動(dòng)學(xué)特征和差異。研究結(jié)果顯示在慢性腦缺血病理模型下AF-XXMD的藥代特征發(fā)生明顯的改變,包括血腦透過率、腦部動(dòng)態(tài)變化、腦部暴露量和腦組織不同部位分布。慢性腦缺血大鼠灌胃給予AF-XXMD后最主要的化合物是黃酮和色原酮類化合物,而腦部最主要的化合物為色原酮和生物堿類化合物。AF-XXMD對(duì)慢性腦缺血的治療效果與色原酮、黃酮和生物堿類化合物作用于多個(gè)靶點(diǎn)和多條代謝通路有關(guān)。論文第三部分以中藥復(fù)方二仙湯為研究對(duì)象,整合中藥復(fù)方中化合物的識(shí)別與鑒定、代謝組學(xué)、生物信息學(xué)的技術(shù)與方法,從中醫(yī)藥治病整體觀角度,探索中藥體內(nèi)藥效物質(zhì)基礎(chǔ),包括來源于中藥的外源性成分、被中藥擾動(dòng)的機(jī)體內(nèi)源性成分,以及這些成分與藥效的關(guān)系和其作用機(jī)制的研究思路和策略。首先,我們建立了快速識(shí)別與鑒定中藥復(fù)方中未知化合物的新策略。這個(gè)新策略能夠快速識(shí)別候選化合物的結(jié)構(gòu)信息,并為結(jié)構(gòu)鑒定提供有效的線索。這個(gè)策略主要包括以下四步:1、建立了 HPLC-HRMSn的中藥成分的分析方法,獲得高分辨質(zhì)量數(shù)和多級(jí)質(zhì)譜數(shù)據(jù),利用MTSF技術(shù),識(shí)別潛在化合物并獲得其母核信息;2、基于保留時(shí)間和高分辨質(zhì)量數(shù),利用判別分析方法,對(duì)潛在化合物按類別進(jìn)行分類;3、將MTSF技術(shù)和判別分析方法獲得的潛在化合物的結(jié)構(gòu)母核信息進(jìn)行匹配,母核信息一致的潛在化合物作為候選化合物進(jìn)行結(jié)構(gòu)鑒定;4、基于同位素分布數(shù)據(jù)對(duì)鑒定的候選化合物結(jié)構(gòu)進(jìn)行確證。這個(gè)新策略能夠在MTSF技術(shù)的基礎(chǔ)上進(jìn)一步排除約41%的不相關(guān)離子信息,同時(shí)還能夠提供553個(gè)候選化合物的準(zhǔn)確結(jié)構(gòu)類型信息,最終鑒定出71個(gè)化合物。以鑒定的71個(gè)化合物為模板,基于MTSF技術(shù),我們從二仙湯灌胃給藥后的大鼠血漿、尿、糞、膽汁中鑒定出13個(gè)原型成分和150個(gè)代謝物。其次,我們建立了高通量不同官能團(tuán)類型甾體激素的UHPLC-MS/MS定量分析方法,實(shí)現(xiàn)了一份生物樣本一次分析中,同時(shí)進(jìn)行羰基和酚羥基甾體激素的衍生化分析。所建立的方法僅需要500 μL血清樣本,可在單次運(yùn)行時(shí)間28 min內(nèi)定量分析20個(gè)甾體激素類化合物。該方法首先采用液液萃取技術(shù)從生物樣本中提取甾體激素,緊接著進(jìn)行離線的吉拉德P衍生化,然后采用自動(dòng)化的進(jìn)樣程序自動(dòng)吸取兩次樣本:第一次將吉拉德P衍生化的羰基類甾體激素加載到色譜柱上;第二次使甾體激素在進(jìn)樣針中進(jìn)行丹磺酰氯的在線衍生化反應(yīng),反應(yīng)結(jié)束后再將丹磺酰氯衍生化的酚羥基類甾體激素加載到色譜柱上。與此同時(shí),質(zhì)譜切換閥與進(jìn)樣程序密切配合,以導(dǎo)入衍生化的甾體激素和去除多余的衍生化試劑,而后質(zhì)譜對(duì)導(dǎo)入的兩種類型的甾體激素衍生化產(chǎn)物按順序分別進(jìn)行分析。方法驗(yàn)證結(jié)果表明:線性范圍為2～400 pg/mL,相關(guān)系數(shù)r值均高于0.988,最低定量限范圍為2～4 pg/mL,各化合物精密度的相對(duì)標(biāo)準(zhǔn)偏差(RSD)值不大于20%,準(zhǔn)確度在80～120%之間,完全能夠滿足生物樣本中甾體激素的分析要求。再次,基于脂質(zhì)組學(xué)開展了二仙湯對(duì)不同絕經(jīng)階段雙側(cè)去卵巢大鼠的調(diào)節(jié)作用研究,設(shè)置了假手術(shù)組、雙側(cè)去卵巢模型組、模型給予二仙湯組和模型給予尼爾雌醇組。將體重、血脂指標(biāo)(膽固醇、高密度脂蛋白、低密度脂蛋白和甘油三酯)和肝臟油紅O染色切片檢查作為藥效學(xué)指標(biāo),評(píng)價(jià)二仙湯給藥后不同時(shí)間點(diǎn)的藥效學(xué)。在藥效學(xué)研究的同時(shí),基于建立的激素、脂質(zhì)和鞘脂組學(xué)分析平臺(tái),結(jié)合統(tǒng)計(jì)學(xué)方法,開展脂質(zhì)代謝組學(xué)研究;研究去卵巢大鼠血漿和肝組織紊亂的脂質(zhì)代謝特征,以及二仙湯干預(yù)后不同絕經(jīng)階段去卵巢大鼠血漿和肝組織紊亂的脂質(zhì)代謝特征,基于脂質(zhì)合成與代謝通路,系統(tǒng)詮釋了二仙湯調(diào)節(jié)脂代謝紊亂的作用機(jī)制,并研究不同絕經(jīng)階段病理變化和二仙湯治療的特點(diǎn)。結(jié)果表明,二仙湯能有效調(diào)節(jié)雙側(cè)去卵巢引起的甾體激素和脂質(zhì)的合成與代謝紊亂,其中以給藥后4周發(fā)現(xiàn)的能夠同時(shí)指征雙側(cè)去卵巢模型脂代謝紊亂和二仙湯調(diào)節(jié)作用的潛在生物標(biāo)志物的數(shù)量最多,包括血漿中15種甘油酯、10種磷脂酰甘油酯和4種甾體激素,肝臟中8種甘油酯和10種磷脂酰甘油酯。除此之外,基于不同絕經(jīng)階段發(fā)現(xiàn)的潛在生物標(biāo)志物的數(shù)量、種類、重復(fù)出現(xiàn)的生物標(biāo)志物等研究,發(fā)現(xiàn)二仙湯的調(diào)節(jié)作用與時(shí)間密切相關(guān)。最后,基于反向分子對(duì)接的方法,對(duì)二仙湯進(jìn)入體內(nèi)的主要成分進(jìn)行反向分子對(duì)接,篩選出導(dǎo)致代謝通路變化的潛在藥效物質(zhì),構(gòu)建與藥效相關(guān)的內(nèi)源性代謝物和外源性中藥成分之間的關(guān)系(物質(zhì)基礎(chǔ)),并采用Western-blot和ELISA方法,驗(yàn)證了二仙湯參與調(diào)節(jié)血漿和肝臟中甘油三酯的合成通路,進(jìn)一步揭示了二仙湯調(diào)節(jié)雙側(cè)去卵巢大鼠脂代謝紊亂的物質(zhì)基礎(chǔ)和作用機(jī)制,從而進(jìn)一步揭示二仙湯的作用機(jī)制。
[Abstract]:Multicomponent pharmacokinetics of traditional Chinese medicine (TCM) is an important research content in clarifying the theory and mechanism of TCM. However, due to the complexity and diversity of TCM components and the complexity and uncertainty of interaction between TCM components and biological systems, the study of multicomponent pharmacokinetics of TCM has been confronted with great challenges. (2) The target compounds of pharmacokinetics research are not clear, the concentration of Chinese medicine and biological samples is low, the interference between endogenous matrix and components is large, and the lack of reference substances, which makes it difficult to establish the method of multi-component detection and quantitative determination of Chinese medicine; (3) The pharmacokinetics research of Chinese medicine is difficult. The results of pharmacokinetic studies of traditional Chinese medicine are not fully evaluated and applied because of the inconsistency of effective indexes and the unclearness of effective components.Therefore, it is necessary to use advanced analytical techniques to analyze the multi-components in traditional Chinese medicine quickly, accurately and comprehensively. The new strategy of the holistic view of TCM treatment provides technical support and scientific data for the study of multi-component pharmacokinetics of TCM in line with TCM theory. In the first part of this paper, the bottleneck problem of multi-component pharmacokinetics of traditional Chinese medicine (TCM) is the lack of in vivo components and their metabolites. A multi-component pharmacokinetic strategy was developed. Firstly, the metabolites of five main isoprenyl flavonoids from Herba Epimedii in rats were analyzed and identified by high performance liquid chromatography-high resolution mass spectrometry/mass spectrometry (HPLC-HRMSn) and mass spectrometry dendrogram similarity filtration (MTSF) techniques based on in vivo metabolism. The metabolic pathways of five main isoprenyl flavonoids were deduced. Fourteen prototype components and glucuronic acid metabolites in plasma were selected as potential pharmacokinetic indicators. High performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) was used to analyze five main isoprenyl flavonoids. After intragastric administration, the main pharmacokinetic parameters of the 14 components in the plasma were characterized; since there was no reference substance for glucuronide-binding metabolites, enzymatic hydrolysis was used to convert the 14 components in the plasma samples into reference substances, and 10 potential pharmacokinetic indicators with metabolic transformation were used as the reference substances. A method of HPLC-MS/MS was established for the determination of pharmacokinetics. In the aspect of chromatographic separation, by optimizing the ratio and mixing time of the two mobile phases, the separation effect of Quaternary mobile phase was realized in binary liquid pump, and the matrix effect of cross-talk and icariin of pilocarpine A-C/icariin was eliminated. The proposed method was validated to be suitable for the analysis of biological samples and was applied to the study of 5 main isoprenyl flavonoids and Herba Epimedii wushanensis in rats after intragastric administration of 10 potential pharmacokinetic components in plasma. Kendall's tau-b hierarchical correlation analysis was used to analyze the correlation between the concentration of the target components in plasma before and after enzymatic hydrolysis and the time of administration. In addition, the level of total icariin in the enzymatic hydrolyzed plasma can reflect the exposure and dynamic changes of isoprenyl flavonoids in vivo. Therefore, icariin with high exposure and good correlation (r0.5) can be used as a pharmacokinetic index of isoprenyl flavonoids of icariin wushan. In addition, a mathematical model was established to predict the exposure of Epimedium wushanensis in rats by using the pharmacokinetic characteristics of icariin. The results showed that the predicted values were in good agreement with the experimental values. It was further confirmed that the enzymatic hydrolysis of plasma icariin could be used as a pharmacokinetic index component of isoprenyl flavonoids in Epimedium wushanensis. In the second part, the multicomponent pharmacokinetics of Xiaoxuming Decoction (AF-XXMD) was studied. 68 kinds of chemical constituents in AF-XXMD were analyzed and identified by HPLC-HRMSn and MTSF. The chemical constituents of high exposure level in plasma and brain of rats were identified. The pharmacological activities were predicted by reverse molecular docking. A high-resolution rapid liquid chromatography-mass spectrometry (RRLC-MS/MS) was developed for the determination of 21 highly exposed and predictably active compounds in rat plasma and brain tissues. Three pairs of isomers can be separated at baseline by using RRLC method for chromatographic separation; the positive and negative ion switching mode is used for mass spectrometry detection, and the mass spectrometric response of each compound is improved by adding a small amount of ammonium formate (0.08mmol l-1). The method meets the technical requirements of biological sample analysis and meets the needs of pharmacokinetic study in rat brain tissue and plasma. The pharmacokinetic characteristics and differences of plasma and target organ brain of 21 index components were studied by intragastric administration of AF-XXMD in normal rats and rats with chronic cerebral ischemia induced by bilateral common carotid artery ligation. The results showed that the pharmacokinetic characteristics of AF-XXMD in chronic cerebral ischemia model were significantly changed, including blood-brain permeability, brain dynamic changes, brain exposure and distribution in different parts of brain tissue. The therapeutic effect of AF-XXMD on chronic cerebral ischemia is related to the action of chromogenic ketone, flavonoids and alkaloids on multiple targets and multiple metabolic pathways. The technology and method of bioinformatics explore the material basis of pharmacodynamics in traditional Chinese medicine from the perspective of holistic view of Chinese medicine treatment, including the exogenous components from traditional Chinese medicine, the intrinsic components disturbed by traditional Chinese medicine in vivo, the relationship between these components and pharmacodynamics, and the research ideas and Strategies of their mechanism of action. This new strategy can quickly identify the structural information of the candidate compounds and provide effective clues for structural identification. This strategy mainly includes the following four steps: 1. An analytical method of HPLC-HRMSn was established to obtain high resolution mass fraction and multi-stage mass spectrometry data. Using MTSF technology to identify potential compounds and obtain their parent nucleus information; 2. Based on retention time and high resolution mass number, potential compounds are classified by category by discriminant analysis method; 3. Matching the parent nucleus information of potential compounds obtained by MTSF technology and discriminant analysis method, potential compounds with identical parent nucleus information are identified. The new strategy can further exclude about 41% of the irrelevant ionic information on the basis of MTSF technology, and also provide accurate structural type information of 553 candidate compounds. Finally 71 compounds were identified. Compounds. Based on the MTSF technique, we identified 13 prototype components and 150 metabolites in plasma, urine, feces and bile of rats after administration of Erxian decoction. Secondly, we established a high-throughput UHPLC-MS/MS quantitative analysis method for steroid hormones of different functional groups. In one analysis, the derivatization of carbonyl and phenolic steroid hormones was performed simultaneously. The established method requires only 500 mu L serum samples and can quantitatively analyze 20 steroid hormones within 28 minutes of a single run. The method first uses liquid-liquid extraction to extract steroid hormones from biological samples, followed by off-line extraction. Gillard P derivatization and automatic sampling procedure were used to automatically absorb two samples: first, Gillard P derivatized carbonyl steroid hormones were loaded onto the column; second, steroid hormones were used in the sample needle for on-line derivatization of sulfonyl chloride, and then the phenolic hydroxyl groups derived from sulfonyl chloride were used after the reaction. Steroid hormones were loaded onto chromatographic columns. Meanwhile, the MS switching valve cooperated closely with the sampling procedure to introduce the derived steroid hormones and remove the redundant derivative reagents. Then the two types of steroid hormone derivatives were analyzed by MS in sequence. The results showed that the linear range was 2-400. The relative standard deviation (RSD) of each compound was less than 20%, and the accuracy was between 80% and 120%. It could completely meet the requirements of steroid hormone analysis in biological samples. Thirdly, Erxian decoction was carried out bilaterally in different menopausal stages based on lipomics. To study the regulating effect of ovarian rats, sham operation group, bilateral ovariectomy model group, Erxian Decoction group and nilestriol group were set up. Body weight, blood lipid index (cholesterol, high density lipoprotein, low density lipoprotein and triglyceride) and liver oil red O staining were used as pharmacodynamic indexes to evaluate the effect of Erxian decoction. Pharmacodynamics at different time points after ovariectomy. At the same time, based on the established analysis platform of hormones, lipids and sphingolipids, combined with statistical methods, lipid metabolism of ovariectomized rats was studied; the characteristics of lipid metabolism in plasma and liver tissue disorders and plasma of ovariectomized rats at different stages after intervention of Erxian Decoction were studied. Based on the pathway of lipid synthesis and metabolism, the mechanism of Erxian Decoction in regulating lipid metabolism disorder was systematically interpreted, and the pathological changes in different menopausal stages and the characteristics of Erxian Decoction in treatment were studied. The results showed that Erxian Decoction could effectively regulate the synthesis and substitution of steroid hormones and lipids induced by bilateral ovariectomy. Among the metabolic disorders, the largest number of potential biomarkers, including 15 glycerides in plasma, 10 phosphatidylglycerides and 4 steroid hormones, 8 glycerides in the liver and 10 phosphatidylglycerides, were found four weeks after administration, indicating both lipid metabolism disorders and the regulatory effects of Erxian Decoction in bilateral ovariectomized rats. According to the research on the quantity, species and repeated biomarkers of potential biomarkers found in different menopausal stages, it is found that the regulating effect of Erxian Decoction is closely related to time. Finally, based on the method of reverse molecular docking, the main components of Erxian Decoction were docked by reverse molecular docking, and the metabolic pathways were screened out. The relationship between endogenous metabolites and exogenous components of traditional Chinese medicine was constructed. Western-blot and ELISA methods were used to verify the role of Erxian Decoction in regulating the synthesis pathway of triglycerides in plasma and liver, further revealing the regulation of Erxian Decoction on lipid metabolism disorders in bilateral ovariectomized rats. The material foundation and mechanism of action are further revealed in the mechanism of Erxian Decoction.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R285
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本文編號(hào)：2208835