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Volume 38 Issue 5
Sep.  2020
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WAN Zhong, YU Dan, WANG Fei, LI Wei, ZHANG Hai. Identification of chemical components and monosaccharide assay in Nocardia rubra cell wall skeleton[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(5): 423-430. doi: 10.12206/j.issn.1006-0111.202003073
Citation: WAN Zhong, YU Dan, WANG Fei, LI Wei, ZHANG Hai. Identification of chemical components and monosaccharide assay in Nocardia rubra cell wall skeleton[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(5): 423-430. doi: 10.12206/j.issn.1006-0111.202003073

Identification of chemical components and monosaccharide assay in Nocardia rubra cell wall skeleton

doi: 10.12206/j.issn.1006-0111.202003073
  • Received Date: 2020-03-11
  • Rev Recd Date: 2020-05-25
  • Available Online: 2020-09-22
  • Publish Date: 2020-09-25
  •   Objective  To analyze and identify the chemical components in the Nocardia rubra cell wall skeleton (Nr-CWS), and to determine the contents of monosaccharides accurately.  Methods  The extract of Nr-CWS was separated and analyzed by UHPLC-Q-TOF/MS method. The chemical components were quickly identified by matching the data with the information in the Metlin database. The monosaccharide contents in the Nr-CWS extract were determined by UHPLC-MS/MS method after derivatization.  Results  A total of 64 chemical components were identified in the extract of Nr-CWS, including amino acids, monosaccharides and so on. A assay method for 8 monosaccharides by UHPLC-MS/MS was successfully established. The content of arabinose in Nr-CWS was the highest, followed by galactose, which indicated that the main polysaccharide components in Nr-CWS may be composed of these monosaccharides.  Conclusion  In this study, we analyzed the main chemical components of Nr-CWS, which are amino acids, fatty acids and so on. The content of monosaccharide after polysaccharide hydrolysis was determined by UHPLC-MS/MS. This will lay a foundation for the screening of the active components of Nr-CWS and the study of its pharmacological mechanism.
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Identification of chemical components and monosaccharide assay in Nocardia rubra cell wall skeleton

doi: 10.12206/j.issn.1006-0111.202003073

Abstract:   Objective  To analyze and identify the chemical components in the Nocardia rubra cell wall skeleton (Nr-CWS), and to determine the contents of monosaccharides accurately.  Methods  The extract of Nr-CWS was separated and analyzed by UHPLC-Q-TOF/MS method. The chemical components were quickly identified by matching the data with the information in the Metlin database. The monosaccharide contents in the Nr-CWS extract were determined by UHPLC-MS/MS method after derivatization.  Results  A total of 64 chemical components were identified in the extract of Nr-CWS, including amino acids, monosaccharides and so on. A assay method for 8 monosaccharides by UHPLC-MS/MS was successfully established. The content of arabinose in Nr-CWS was the highest, followed by galactose, which indicated that the main polysaccharide components in Nr-CWS may be composed of these monosaccharides.  Conclusion  In this study, we analyzed the main chemical components of Nr-CWS, which are amino acids, fatty acids and so on. The content of monosaccharide after polysaccharide hydrolysis was determined by UHPLC-MS/MS. This will lay a foundation for the screening of the active components of Nr-CWS and the study of its pharmacological mechanism.

WAN Zhong, YU Dan, WANG Fei, LI Wei, ZHANG Hai. Identification of chemical components and monosaccharide assay in Nocardia rubra cell wall skeleton[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(5): 423-430. doi: 10.12206/j.issn.1006-0111.202003073
Citation: WAN Zhong, YU Dan, WANG Fei, LI Wei, ZHANG Hai. Identification of chemical components and monosaccharide assay in Nocardia rubra cell wall skeleton[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(5): 423-430. doi: 10.12206/j.issn.1006-0111.202003073
  • 红色诺卡菌(Nocardia rubra,Nr)是一种放线菌,其细胞壁骨架(Nocardia rubra cell wall skeleton,Nr-CWS)具有免疫调节作用[1]。Nr-CWS作为一种治疗药物现已在国内上市并在临床使用。研究表明,Nr-CWS在增强体内巨噬细胞、T细胞和自然杀伤细胞活性的同时,还能诱导机体产生LAK细胞,提高机体内T辅助性细胞和杀伤细胞活力、增强巨噬细胞和天然杀伤细胞免疫活性,抑制肿瘤和增强免疫能力的功效[2-4]。但Nr-CWS在发挥作用的同时,往往还伴随一些副作用的发生。究其主要原因是由于红色诺卡菌细胞壁是个细胞粗提物[5],其中的成分复杂,具体哪种成分起相应的药理作用还不清楚,这极大地影响了其临床使用及后续药理作用机制的研究。本研究拟采用UHPLC-Q-TOF/MS分析方法首先对Nr-CWS提取物中化学成分进行分离分析,鉴别其中的化学成分;并对其中的多糖成分进行水解,然后对水解后的单糖进行衍生化处理,与其他分析方法相比, UHPLC-MS/MS具有较大的优越性[6-8], 其灵敏度高、专属性强、可以快速准确地测定单糖的含量。所以本研究采用UHPLC-MS/MS方法对单糖衍生化产物进行定量分析测定,这将为后期开展Nr-CWS活性成分筛选及药理作用机制的研究奠定基础。

  • 安捷伦-1290 Infinity高效液相色谱系统-电喷雾离子源(安捷伦,Palo Alto, CA, USA)串联安捷伦6538四级杆-高分辨飞行时间质谱(UHPLC-Q-TOF/MS),安捷伦-1290 Infinity高效液相色谱系统-电喷雾离子源(安捷伦,Palo Alto, CA, USA)串联安捷伦6460三重四级杆质谱(UHPLC-MS/MS),AMIDE色谱柱(3.0 mm×100 mm,3.5 µm,Waters,USA),Waters Xbridge C18色谱柱(2.1 mm×100 mm,1.7 μm),Milli-Q50 SP纯水制备系统制备(Millipore Corporation, MA, USA),电热干燥箱(上海恒科仪器有限公司,DHG-9145AZ)。

  • Nr-CWS提取物(辽宁格瑞士特生物科技有限公司,按照产品工艺提取);Sephadex G-100(国药集团化学试剂有限公司);D-甘露糖、D-核糖、L-鼠李糖、D-果糖、D-葡萄糖、D-木糖、D-半乳糖、D-阿拉伯糖(纯度>98%,国药集团化学试剂有限公司);三氟乙酸(TFA)、1-苯基-3-甲基-5-吡唑啉酮(PMP,国药集团化学试剂有限公司);HPLC色谱级甲醇、乙腈(默克公司,Darmstadt, Germany);乙醇、甲酸(Fluka公司,Buchs, Switzerland)。

  • UHPLC-Q-TOF/MS分析在安捷伦1290 Infinity 液相色谱系统和安捷伦6538四极杆-高分辨飞行时间串联质谱仪(Agilent,USA)上进行,色谱分离在Amide色谱柱上进行(3.0 mm×100 mm,3.5 µm,Waters,USA),柱温40 ℃,流动相A为0.1 %甲酸水溶液,流动相B为乙腈溶液,流速0.4 ml/min,流动相采用梯度洗脱,洗脱条件为:0~1 min,5 % A;1~5 min,5 %~20 % A;5~20 min,20%~45 % A;20~30 min,45 % A,进样量为5 µl,自动进样器温度保持在25 ℃。电喷雾离子源(ESI)采用正、负离子模型。Q-TOF/MS质谱参数如下:毛细管电压,正离子模式下4 kV,负离子模式下3.5 kV;干燥器流速11 L/min;气体温度350 ℃;雾化器压力45 psig;碎片电压120 eV;Skimmer电压60 eV。质谱的采集范围m/z 50~1100,分析碰撞能量10~40 eV。

  • 取Nr-CWS提取物溶于1 ml纯水中,充分涡旋溶解后制成Nr-CWS提取物母液,取100 µl于1.5 ml EP管中,加入3倍量的乙醇进行蛋白沉淀。然后4 ℃下13 000 r/min离心15 min,吸取200 µl上清液,于进样瓶中用于UHPLC-Q-TOF/MS分析。

  • 采用UHPLC-Q-TOF/MS分析方法对Nr-CWS提取物样品溶液进行快速地分离与分析,获得正、负离子模式下的总离子流图,通过与Metlin数据库中代谢物信息比对分析,可对Nr-CWS提取物中化学成分进行快速分析与鉴别,结果如图1表1所示。

    序号化合物名称[M+X]分子式m/z保留时间(t/min)
    12-(S-glutathionyl)acetyl glutathioneM-HC22H34N6O13S2654.162 50.91
    22-(S-glutathionyl)acetyl glutathioneM+HC22H34N6O13S2654.668 00.91
    3estradiol-17alpha 3-D-glucuronosideM-HC24H32O8448.209 71.12
    4tyrosineM+HC9H11NO3181.190 01.13
    5cer(d18∶0/23∶0)M+HC41H83NO3637.637 31.30
    6palmitic acidM-HC16H32O2256.424 11.31
    7stearic acidM-HC18H36O2284.271 51.31
    8L-threonineM+HC4H9NO3119.120 01.33
    9D-xyloseM-HC5H10O5150.130 01.34
    10N-acetyl-D-glucosamineM-HC8H15NO6221.210 01.39
    11meso-2,6-diaminopimelic acidM+HC7H14N2O4190.197 11.39
    12leinoleic acidM+HC18H32O2280.445 51.41
    13cholineM+HC5H15NO104.170 81.56
    14L-rhamnose monohydrateM-HC6H12O5164.160 01.64
    15docosapentaenoic acidM-HC22H34O2330.255 91.67
    16prolineM-HC5H9NO2115.130 01.93
    17fructose 1,6-bisphosphateM+HC6H14O12P2339.996 02.22
    18glucose 6-phosphateM+HC6H13O9P260.135 82.28
    19fucoseM-HC6H12O5164.150 02.34
    20L-prolineM-HC5H9NO2115.063 32.34
    21PG(18∶0/20∶3(8Z,11Z,14Z))M-HC44H81O10P800.556 75.96
    22tetracosanoic acidM-HC24H48O2368.636 77.07
    23orotidylic acidM-HC10H13N2O11P368.190 87.65
    24sphinganineM+HC18H39NO2301.507 87.72
    25valineM-HC5H11NO2117.150 07.74
    26cervonoyl ethanolamideM-HC24H36O3372.540 87.94
    27phytosphingosineM+HC18H39NO3317.507 27.94
    28tryptophanM+HC11H12N2O2204.230 07.97
    29trihexosylceramide (d18∶1/12∶0)M-HC48H89NO18967.608 08.10
    30histidineM+HC6H9N3O2155.160 08.45
    31L-asparagineM-HC4H8N2O3132.120 09.65
    32galactinol dihydrateM-HC12H26O13378.327 09.66
    333-hydroxydodecanoyl carnitineM+HC19H37NO5359.500 89.70
    34L-cysteineM+HC3H7NO2S121.120 09.84
    35cysteinyl-threonineM-HC7H14N2O4S222.067 49.98
    36D-mannoseM-HC6H12O6180.160 010.10
    37MG (0∶0/24∶1(15Z)/0∶0)M-HC27H52O4440.386 610.11
    384-(methylnitrosamino)-1-(3-pyridyl)-1-butanol glucuronideM-HC16H23N3O8385.369 111.32
    39D-glucoseM-HC6H12O6180.160 011.34
    40desmosineM+HC24H40N5O8526.603 111.38
    41L-methionineM+HC5H11O2NS149.210 011.59
    42glycogenM-HC24H42O21666.577 711.90
    43levanM-HC18H32O16504.437 111.90
    44D-galactoseM-HC6H12O6180.160 011.92
    45L-beta-aspartyl-L-aspartic acidM+HC8H12N2O7248.190 112.00
    46muramic acidM-HC9H17NO7251.233 812.16
    471-pyrroline-2-carboxylic acidM-HC5H7NO2113.114 612.25
    48ADP-glucoseM-HC16H25N5O15P2589.341 712.25
    49DG(42∶10)M+HC45H68O5688.506 712.27
    50isoleucineM-HC6H13NO2131.170 012.55
    51leucineM-HC6H13NO2131.170 012.79
    52PGP(16∶1(9Z)/18∶0)M-HC40H78O13P2828.491 812.99
    53PGP(16∶0/20∶4)M+HC42H76O13P2850.992 613.01
    54TG(62∶6)M-HC65H114O6990.861 513.60
    55ganglioside GM3 (d18∶1/16∶0)M-HC57H104N2O211152.713 214.15
    56valyl-methionineM+HC10H20N2O3S248.342 014.18
    57DG(18∶2n6/0∶0/22∶6n3)M-HC44H70O5678.522 315.27
    58PS(16∶0/18∶2)M-HC40H74NO10P759.990 015.90
    59PGP(18∶1/22∶6)M-HC46H78O13P2900.491 816.62
    60CDP-DG(16∶0/18∶0)M-HC46H85N3O15P2981.545 617.41
    61TG(22∶6(4Z,7Z,10Z,13Z,16Z,19Z)/24∶0/22∶6(4Z,7Z,10Z,13Z,16Z,19Z))M-HC71H114O61 062.861 518.28
    62TG(24∶0/24∶0/24∶0)M-HC75H146O61 143.111 919.25
    63serineM-HC3H7NO3105.090 027.23
    64alanineM-HC3H7NO289.090 027.28
  • 准确称取D-甘露糖、D-核糖、L-鼠李糖、D-果糖、D-葡萄糖、D-木糖、D-半乳糖、D-阿拉伯糖各10 mg,分别置10 ml 容量瓶,各加水定容,即得1 mg/ml浓度的各单糖标准品溶液。分别精密取上述单糖标准品溶液,混合后加重蒸水稀释,制成100 μg/ml的单糖混合标准溶液。

  • 精密称取1-苯基-3-甲基-5-吡唑啉酮(PMP)1.0 g,置10 ml量瓶中,加甲醇溶解并定容,摇匀,制得0.5 mol/L的PMP 甲醇溶液。

  • 精密吸取单糖及混合标准品溶液100 μl置2 ml EP管中,添加100 μl氨水,再加入100 μl 0.5 mol/L的PMP甲醇溶液,涡旋30~45 s,于70 ℃烘箱中加热30 min,取出放冷至室温。加入100 μl乙酸,涡旋30 s中和,加重蒸水至1 ml,再加500 μl氯仿,涡旋30 s,混匀,弃去下层溶液,重复3 次,12 000 r/min离心10 min,水层经0.22 μm微孔滤膜滤过,取次滤液用于UHPLC-MS/MS分析,见表2

    序号化合物名称保留时间(t/min)分子式单糖分子量衍生物分子量母离子子离子碰撞电压(eV)
    1D-甘露糖2.012C6H12O6180.1512.1511.1175.0, 217.134
    2D-核糖2.216C5H10O5150.1482.1481.1175.0, 217.131
    3L-鼠李糖2.423C6H14O6164.1496.1495.1175.0, 217.131
    4D-果糖2.504C6H12O6180.1512.1511.1175.0, 217.134
    5D-葡萄糖3.725C6H12O6180.1512.1511.1175.0, 217.134
    6D-木糖3.917C5H10O5150.1482.1481.1175.0, 217.131
    7D-半乳糖3.918C6H12O6180.1512.1511.1175.0, 217.134
    8D-阿拉伯糖4.086C5H10O5150.1482.1481.1175.0, 217.131
  • UHPLC-MS/MS分析采用安捷伦1290 Infinity 液相色谱系统和安捷伦6460三重四极杆串联质谱仪(Agilent,USA)。色谱分离在Waters Xbridge C18色谱柱(2.1 mm×100 mm,1.7 μm),柱温40 ℃,流动相A为20 mmol/L的乙酸铵水溶液(氨水调pH 8.0),流动相B为乙腈溶液,流速0.4 ml/min,流动相采用梯度洗脱,洗脱条件为:0~2 min,15%~20% B;2~4 min,20%~25% B;4~5 min,25%~95 % B,5~6 min,15 % B,样品分析时间5 min,柱后平衡时间1 min。进样量为2 µl,自动进样器温度保持25 ℃。三重四级杆质谱条件为:电喷雾离子源(ESI)采用负离子,多级反应选择离子监测模式:毛细管电压3.5 kV;干燥器流速11 L/min;气体温度350 ℃;雾化器压力40 psig;碎片电压80 eV;Skimmer电压60 eV。

  • 精密吸取Nr-CWS提取物溶液1 ml,加入2 mol/L的TFA 2.0 ml,放入110 ℃的真空干燥箱中,酸性条件下水解6 h,冷却至室温,4 ℃条件下离心干燥,挥发三氟乙酸,加重蒸水1 ml复溶,用于衍生化处理。

  • 精密吸取Nr-CWS提取物的水解液100 μl,按照前述单糖衍生化方法进行前处理后,精密吸取前处理后的样品溶液2 μl进UHPLC-MS/MS分析,混合标准品和Nr-CWS样品衍生化后的UHPLC-MS/MS色谱图见图2

  • 精密吸取单糖混合标准品溶液,按前述单糖衍生化方法进行前处理后,精密吸取前处理后的样品溶液2 μl用UHPLC-MS/MS法分析,以各单糖峰面积与相应浓度进行线性回归分析,计算回归方程和相关系数,逐步稀释后,按照信噪比S/N=10和3,分别计算其定量限和检测限,结果见表3

    序号化合物回归方程r浓度范围(μg/ml)定量限(ng/ml)检测限(ng/ml)
    1D-甘露糖Y = 383.2 X − 92.40.9960.05~10205
    2D-核糖Y = 656.8 X − 109.50.9940.10~205020
    3L-鼠李糖Y = 1025.3 X − 386.40.9920.50~100205
    4D-果糖Y = 902.3 X − 133.10.9940.10~2010050
    5D-葡萄糖Y = 2875.3 X − 342.90.9930.50~10010050
    6D-木糖Y = 2391.1 X − 1004.60.9950.05~105020
    7D-半乳糖Y =3482.4 X − 1093.50.9981.00~20010050
    8D-阿拉伯糖Y = 5436.8 X − 2102.30.9925.00~10005020
  • 取经水解和衍生化反应后的单糖混合标准溶液,按照前述UHPLC-MS/MS分析测定条件,连续进样6次,计算其测定结果的RSD为3.13%,结果表明该方法的精密度良好,符合分析测定的要求。

  • Nr-CWS提取物溶液经水解和衍生化反应后,室温下放置,分别于0、5、l5、30、60、120 min进样,进行UHPLC-MS/MS分析,测定其浓度,对样品的稳定性进行评价,结果6次测定结果的RSD为7.63%,表明样品在2 h内稳定性良好,符合分析测定的要求。

  • 取同一批样品,重复测定6次,计算其测定结果RSD为4.67%,表明该方法的重复性良好,符合分析测定的要求。

  • 精密吸取已知含量的Nr-CWS提取物加入接近等量的单糖混合标准品溶液,经水解和衍生化反应后,进行UHPLC-MS/MS分析,计算其平均回收率为86.93%,RSD为9.15%,符合分析测定的要求。

  • 精密吸取Nr-CWS提取物样品溶液6份,经水解衍生化后,按照前述UHPLC-MS/MS分析条件对衍生化后的样品进行分离分析,测定其中单糖的含量,结果见表4,可以看出8种单糖成分都能被检出,其中阿拉伯糖和半乳糖的含量最高。

    提取物编号单糖成分(μg/ml)
    D-甘露糖D-核糖D-鼠李糖D-果糖D-葡萄糖D-木糖D-半乳糖D-阿拉伯糖
    10.95.312.218.955.120.9320.8456.1
    22.14.621.412.448.319.2205.6504.8
    33.23.530.59.861.923.1223.0327.3
    45.56.619.815.445.827.8305.4462.8
    51.54.923.414.739.112.1311.7489.1
    64.77.625.122.552.727.2289.2510.8
    平均值3.05.422.115.650.521.7276.0458.5
  • Nr-CWS提取物中化学成分的分析鉴别,采用UHPLC-Q-TOF/MS分析方法对Nr-CWS提取物样品溶液进行快速地分离分析[15-16],获得正、负离子模式下的总离子流图,通过与Metlin数据库中代谢物信息快速地比对分析,共鉴别出Nr-CWS提取物中64个化学成分,其中包含氨基酸、脂肪酸、单糖、胞壁酸、粘肽等成分,说明Nr-CWS提取物中含有很多的细胞代谢产物,且以氨基酸、脂肪酸和糖类成分为主,这些成分就是Nr-CWS提取物发挥作用的主要成分。

    由于Nr-CWS提取物中成分复杂,采用UHPLC-Q-TOF/MS方法直接对提取物进行分析,仅能检测到个别单糖成分,如木糖、半乳糖、葡萄糖等,说明Nr-CWS提取物中主要含有多糖成分,经水解衍生化后[17-20],采用UHPLC-MS/MS方法进行检测,8个单糖都能被检测到,且阿拉伯糖和半乳糖的含量最高,说明Nr-CWS中的多糖主要由阿拉伯糖和半乳糖组成,这将为进一步的多糖成分分析鉴别奠定基础。

    对单糖进行分析测定的方法有很多,我们通过比较衍生化和不衍生化方法的检测限和定量限,发现单糖衍生化后,其定量限和检测限更低,测定干扰更少,分析方法学验证结果表明,其精密度、重现性、稳定性和回收率均符合分析测定的要求。

  • 本研究采用UHPLC-Q-TOF/MS方法对Nr-CWS提取物中化学成分进行了快速地分离与分析,通过与Metlin代谢物成分数据库比对,共鉴别出Nr-CWS提取物中64个化学成分,主要包括氨基酸、糖类、脂肪酸等成分;对Nr-CWS提取物中糖类成分衍生化后,采用UHPLC-MS/MS分析方法对Nr-CWS提取物中8种单糖成分进行含量测定。结果表明,Nr-CWS中阿拉伯糖的含量最高,其次为半乳糖,说明Nr-CWS中发挥作用的主要多糖类成分可能主要由这几种单糖组成。通过本研究为后期开展Nr-CWS活性成分筛选及药理作用机制的研究奠定了基础。

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