Synthesis and phototoxicity of isomeric 7,9-diglutathione pyrrole adducts: Formation of reactive oxygen species and induction of lipid peroxidation

Pyrrolizidine alkaloids (PAs) are hepatotoxic, genotoxic, and carcinogenic in experimental animals. Because of their widespread distribution in the world, PA-containing plants are probably the most common poisonous plants affecting livestock, wildlife, and humans. Upon metabolism, PAs generate reactive dehydro-PAs and other pyrrolic metabolites that lead to toxicity. Dehydro-PAs are known to react with glutathione (GSH) to form 7-GSH-(+/−)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (7-GS-DHP) in vivo and in vitro and 7,9-diGS-DHP in vitro. To date, the phototoxicity of GS-DHP adducts has not been well studied. In this study, we synthesized 7-GS-DHP, a tentatively assigned 9-GS-DHP, and two enantiomeric 7,9-diGS-DHP adducts by reaction of dehydromonocrotaline with GSH. The two 7,9-diGS-DHPs were separated by high performance liquid chromatography (HPLC) and their structures were characterized by ¹H nuclear magnetic resonance (NMR) and ¹H–¹H correlation spectroscopy (COSY) NMR spectral analysis. Photoirradiation of 7-GS-DHP, 9-GS-DHP, and the two 7,9-diGS-DHPs as well as dehydromonocrotaline, dehydroheliotrine, and the 7-R enantiomer of DHP (DHR), by UVA light at 0 J/cm², 14 J/cm², and 35 J/cm² in the presence of a lipid, methyl linoleate, all resulted in lipid peroxidation in a light dose-responsive manner. The levels of lipid peroxidation induced by the two isomeric 7,9-diGS-DHPs were significantly higher than that by 7-GS-DHP and 9-GS-DHP. When 7,9-diGS-DHP was irradiated in the presence of sodium azide (NaN₃), the level of lipid peroxidation decreased; lipid peroxidation was enhanced when methanol was replaced by deuterated methanol. These results suggest that singlet oxygen is a product induced by the irradiation of 7,9-diGS-DHP. When irradiated in the presence of superoxide dismutase (SOD), the level of lipid peroxidation decreased, indicating that lipid peroxidation is also mediated by superoxide. These results indicate that lipid peroxidation is mediated by reactive oxygen species (ROS). These results suggest that 7,9-diGS-DHPs are phototoxic, generating lipid peroxidation mediated by ROS.     

Definitive diagnosis of hepatic sinusoidal obstruction syndrome induced by pyrrolizidine alkaloids

OBJECTIVE: Hepatic sinusoidal obstruction syndrome (HSOS) induced by a Chinese medicinal herb Tusanqi is increasingly being reported in recent years. The aim of the study was to investigate the possibility of using blood pyrrole‐protein adducts test as a confirmatory diagnostic method. METHODS: Patients with HSOS according to international diagnostic criteria associated with Tusanqi from January 2006 to August 2010 in Zhongshan Hospital Fudan University were included and clinical features were collected. Pyrrole‐protein adducts in blood sample were determined with ultra performance liquid chromatography‐mass spectrometry (UPLC‐MS) while pyrrolizidine alkaloids (PAs) in available herbal preparations were analyzed by high performance liquid chromatography‐ultraviolet (HPLC‐UV). RESULTS: Five patients (age 41–72 years, median age 54 years, all women) were included. Ascites (5/5), jaundice (5/5) and hepatomegaly (4/5) were common manifestations. The imaging features were diffused, patchy hepatic enhancement, periportal edema and ascites. Pathology ascertained that blood flow was obstructive at the site of sinusoid. PAs (Seneionine and seneciphylline) were identified in all the three available herbal preparations ingested by the HSOS patients. Pyrrole‐protein adducts were unequivocally found in all the five blood samples. Two patients recovered, two developed chronic illness and one died due to liver failure and hepatic encephalopathy. CONCLUSIONS: The detection of blood pyrrole‐protein adducts using a UPLC‐MS approach is a specific, reliable, unambiguous and confirmatory test for HSOS induced by PA, and should be used together with the conventional HSOS clinical diagnostic criteria for the definitive diagnosis of PA‐induced HSOS.     

不同商品规格吴茱萸有效成分含量和肝细胞毒性的研究

目的评价市售吴茱萸EuodiaeFructus的质量并研究不同商品规格吴茱萸中化学成分与肝细胞毒性的相关性,为吴茱萸全面质量控制提供依据。方法按吴茱萸药材商品规格收集小花吴茱萸7批,中花吴茱萸22批,大花吴茱萸10批,建立了UPLC法同时测定吴茱萸中柠檬苦素(limonin,LIM)、吴茱萸碱(evodiamine,EVD)、吴茱萸次碱(rutaecarpine,RUT)、 1-甲基-2-壬基-4-(1H)-喹诺酮[1-methyl-2-nonyl-4(1H)-quinolone, MNQ]、 1-甲基-2-十一烷基-4(1H)-喹诺酮[1-methyl-2-undecyl-4(1H)-quinolone,MUQ]和吴茱萸新碱(evocarpine,EVC)的含量;采用CCK-8法检测吴茱萸对L02肝细胞的生长抑制作用。运用SIMCA-P 14.1统计软件对吴茱萸的细胞存活率进行主成分分析(principal component analysis,PCA)。结果有19批商品符合《中国药典》2020年版规定,不符合药典要求的20批样品中有10批为药材基原不符,有1批EVD和RUT的总量不符合药典规定,9批LIM含量不符合药典规定。7批小花吴茱萸中1批不符合药典要求,22批中花吴茱萸中10批不符合药典要求,10批大花吴茱萸中9批不符合药典要求。同时发现小花吴茱萸中LIM含量高于中花和大花吴茱萸,而生物碱含量中花和大花吴茱萸中较高。其中S33～S39样品按果实大小属于中花吴茱萸但不含LIM,将其称为中花样吴茱萸劣品。基于建立的UPLC方法同时测定吴茱萸中LIM和5种生物碱含量;PCA分析模型将小花吴茱萸、中花吴茱萸、大花吴茱萸和中花样吴茱萸劣品的毒性很好地区分为4个区域,对L02肝细胞的生长抑制作用为中花样吴茱萸劣品大花吴茱萸中花吴茱萸小花吴茱萸,且LIM与5种生物碱的比值和吴茱萸对L02肝细胞的半数抑制率(IC_(50))总体上呈正相关。结论吴茱萸的毒性与LIM和5种生物碱比例有关,LIM与5种生物碱含量和的比值越大,吴茱萸样品毒性越小。提示大花吴茱萸不适合作为商品流通,同时吴茱萸质量控制应考虑药材中LIM和生物碱类成分含量的比例。     

含吡咯里西啶生物碱中成药潜在风险评估

吡咯里西啶生物碱(pyrrolizidine alkaloids,PAs)是一种双环吡咯啶生物碱,因多数具有肝毒性,又称肝毒性吡咯里西啶生物碱(hepatotoxic PAs)。PAs广泛存在于菊科、豆科、紫草科的多种药用植物中。《中国药典》2020年版共收载含有PAs的药材8种,包括千里光Senecionis Scandentis Herba、款冬花Farfarae Flos、佩兰Eupatorii Herba、紫草Arnebiae Radix等,涉及成方制剂数十种。此外,《卫生部药品标准》《药品注册标准》等收载含PAs制剂尚有200余种。除千里光药材外,其他含PAs中药及其制剂均没有关于PAs类成分的安全限量标准,其临床用药安全存在极大的风险。对含PAs中成药的研究概况进行了系统总结、分析,以期为此类中成药的临床用药安全提供警示和参考。     

苎叶蒟中1个新的含氮木脂素

目的对苎叶蒟Piper boehmeriifolium的枝叶部位进行化学成分研究。方法采用硅胶、RP-C_(18)柱色谱、Sephadex LH-20凝胶柱色谱和半制备HPLC等方法进行分离和纯化,并综合运用IR、HR-ESI-MS、~1H-NMR、~(13)C-NMR、DEPT、HSQC、HMBC等方法鉴定化合物的结构。结果从苎叶蒟乙醇提取物中分离得到15个化合物,分别鉴定为3-(3,4-methylenedioxybenzyl)-4-(3,4-dimethoxybenzyl)-1H-pyrrole-2,5-dione(1)、2E,4E-decadienoylpiperidide(2)、2E-decenoylpiperidide(3)、墙草碱(4)、piperchabamide B(5)、假蒟亭碱(6)、piperdardine(7)、胡椒新碱(8)、胡椒碱(9)、二氢荜茇明宁碱(10)、chingchengenamide A(11)、假荜茇酰胺B(12)、guineensine(13)、荜茇宁(14)和kusunokinin(15)。结论化合物1为新化合物,命名为胡椒木脂酰亚胺,化合物2、3、7、8、10和15为首次从该植物中分离得到。     

蜈蚣中1个喹啉类新化合物

综合运用硅胶正相色谱、反相中低压制备色谱、高压半制备液相色谱等分离和纯化方法,从蜈蚣乙酸乙酯部位中分离得到3个化合物,分别为蜈蚣素丙(1)、尿嘧啶(2)、次黄嘌呤(3),应用NMR,MS等波谱方法,并结合文献,鉴定了其化学结构,其中化合物1为喹啉类新化合物。     

狗牙花总生物碱的身体依赖性实验研究

目的评价狗牙花总生物碱的身体依赖性潜力。方法采用小鼠催促戒断和大鼠自然戒断模型对狗牙花总生物碱进行身体依赖性实验。结果在小鼠催促实验中,给予狗牙花总生物碱低剂量(140mg/kg)、高剂量(430mg/kg),连续注射7d,而后经纳洛酮激发,实验小鼠未出现明显的戒断症状、跳跃次数增加和体重下降现象。在大鼠自然戒断实验中,给予狗牙花总生物碱低剂量(210mg/kg)、高剂量(775mg/kg),连续注射30d,停药后未见大鼠出现戒断反应和体重下降现象。结论狗牙花总生物碱无身体依赖性。     

HPLC同时测定苦黄注射液中生物碱和大黄蒽醌类成分的含量

目的 建立同时测定苦黄注射液中生物碱和大黄蒽醌类成分的定量分析方法。方法 采用Venusil MP C₁₈(2)(4.6 mm×250 mm,5 μm)色谱柱为分析柱,甲醇(A)-0.4%二乙胺(B),梯度洗脱(A为5%→5%→40%→60%→80%→5%,相应时间周期为0→5→20→30→55→60 min),流速为1.0 mL·min^-1,紫外检测波长为254 nm,柱温位30 ℃。结果 在选定色谱条件下,芦荟大黄素、大黄酸、苦参碱、大黄素、大黄酚、氧化苦参碱、大黄素甲醚、槐定碱、槐果碱分别在0.012 3~0.369?μg、0.023~0.69 μg、0.138~4.14 μg、0.056~1.68 μg、0.065~1.95 μg、0.145~4.35 μg、0.012?5~0.375 μg、0.146~4.38 μg和0.101~ 3.03 μg内线性关系良好(r=0.999 3,0.999 6,0.999 6,0.999 6,0.999 4,0.999 3,0.999 5,0.999 4,0.999 4),加样回收率分别为98.38%,100.79%,98.03%,99.81%,98.62%,99.28%,98.90%,101.75%,100.75%,RSD分别为1.60%,2.65%,2.12%,2.78%,2.08%,2.45%,2.08%,2.35%,1.72%。结论 该方法操作简便易行,重复性好,结果准确可靠,可为苦黄注射液的质量控制提供定量评价方法。     

Three new monoterpenoid indole alkaloids from Vinca major

Three new monoterpenoid indole alkaloids, 19-hydroxyl-10-methoxy-19, 20-dihydrovinorine (1), 19-O-acetyl-10-methoxy-19, 20-dihydrovinorine (2), and 19, 21α-dihydroxyl-10-methoxy-19, 20-dihydrovinorine (3), along with five known analogues (4–8), were isolated from the whole plants of Vinca major. The new structures were elucidated by extensive NMR and MS analysis and comparison with known compounds. In addition, compounds 1–3 were evaluated for their cytotoxicities against five human cancer cell lines.