Alkaloids from the Roots of Aristolochia Triangularis(I)

从马兜铃科植物Ａｒｉｓｔｏｌｏｃｈｉａｔｒｉａｎｇｕｌａｒｉｓ的根部分离出十五个生物碱成分。其中两种成分ｔｒｉａｎｇｕｌａｒｉｎｅ－Ａ和ｔｒｉａｎｇｕｌａｒｉｎｅ－Ｂ系新生物碱，三种系阿卟啡类季铵碱。这些化合物的结构经各种波谱（ＩＲ，ＵＶ，ＭＳ，２ＤＮＭＲ）解析和化学转化技术得以确定。     

辽细辛地下部分的化学成分(Ⅱ)

目的对辽细辛地下部分进行化学成分研究。方法采用反复硅胶柱色谱法分离纯化,通过理化性质和谱学分析鉴定化合物结构。结果分离得到6个化合物,分别鉴定为马兜铃酰胺Ⅰ(aristololactamⅠ,1)、7-甲氧基马兜铃酰胺(7-methoxy-aristololactam,2)、马兜铃酸Ⅳa(aristolochicacidⅣa,3)、对羟基苯甲酸(p-hydroxybenzoic acid,4)、5,7-二-O-β-D-吡喃葡萄糖基柚皮素(5,7-di-O-β-D-glucpyranosyl-narigenin,5)、2,6-二甲氧基-4-甲基苯基-1-O-β-D-吡喃葡萄糖苷(2,6-dime-thoxy-4-methylphenyl-1-O-β-D-glucopyranoside,6)。结论化合物4、6为细辛属内首次分离得到,化合物2、5为从该植物中首次分离得到。     

Role of mitochondrial permeability transition in human renal tubular epithelial cell death induced by aristolochic acid

Aristolochic acid (AA), a natural nephrotoxin and carcinogen, can induce a progressive tubulointerstitial nephropathy. However, the mechanism by which AA causes renal injury remains largely unknown. Here we reported that the mitochondrial permeability transition (MPT) plays an important role in the renal injury induced by aristolochic acid I (AAI). We found that in the presence of Ca(2+), AAI caused mitochondrial swelling, leakage of Ca(2+), membrane depolarization, and release of cytochrome c in isolated kidney mitochondria. These alterations were suppressed by cyclosporin A (CsA), an agent known to inhibit MPT. Culture of HK-2 cell, a human renal tubular epithelial cell line for 24 h with AAI caused a decrease in cellular ATP, mitochondrial membrane depolarization, cytochrome c release, and increase of caspase 3 activity. These toxic effects of AAI were attenuated by CsA and bongkrekic acid (BA), another specific MPT inhibitor. Furthermore, AAI greatly inhibited the activity of mitochondrial adenine nucleotide translocator (ANT) in isolated mitochondria. We suggested that ANT may mediate, at least in part, the AAI-induced MPT. Taken together, these results suggested that MPT plays a critical role in the pathogenesis of HK-2 cell injury induced by AAI and implied that MPT might contribute to human nephrotoxicity of aristolochic acid.     

Comparative study of the contents of analogues of aristolochic acid in two kinds of Aristolochiae Fructus by high-performance liquid chromatography

Aristolochiae Fructus (??Madouling??) is derived from the fruits of Aristolochia contorta and A. debilis (Aristolochiaceae). These two species contain potentially nephrotoxic constituents, but are officially used in China. Distinction of constituents and toxicity between these two species remains unclear. A high-performance liquid chromatography method was developed and validated for the simultaneous determination of seven analogues of aristolochic acid (aristolochic acids I, II, IIIa, IVa and VIIa), as well as aristololactams I and II in Aristolochiae Fructus. Chromatographic separation was achieved on a Zorbax SB-C₁₈ column with a gradient mobile phase comprising acetonitrile and 1?% acetic acid?C30?mM triethylamine (20:1, v/v) buffer. Analytes were detected with a diode array detector at 250 and 260?nm. The contents of seven constituents in samples (11 batches of A. contorta fruits, 15 batches of A. debilis fruits and 33 commercial samples of Madouling) were determined. The content of aristolochic acid IVa was higher than that of aristolochic acid VIIa in A. contorta fruits, whereas the opposite was true in A. debilis fruits. This feature can be used to distinguish the two species from each other and identify the resource plant of Madouling. Through a morphological method and a newly found principle based on the ratio AA-IVa/AA-VIIa, we found that the 33 commercial samples collected from 12 provinces in China were all derived from the fruits of A. contorta.     

Gremlin-mediated decrease in bone morphogenetic protein signaling promotes aristolochic acid-induced epithelial-to-mesenchymal transition (EMT) in HK-2 cells

Ingestion of aristolochic acid (AA) is associated with the development of aristolochic acid nephropathy (AAN), which is characterized by progressive tubulointerstitial fibrosis, chronic renal failure and urothelial cancer. Our previous study showed that bone morphogenetic protein-7 (BMP-7) could attenuate AA-induced epithelial-to-mesenchymal transition (EMT) in human proximal tubule epithelial cells (PTEC). However, how gremlin (a BMP-7 antagonist) antagonizes the BMP-7 action in PTEC remained unsolved. The aim of the current study was to investigate the role of gremlin in AA-induced EMT in PTEC (HK-2 cells). HK-2 cells were treated with AA (10 μmol/L) for periods up to 72 h. Cell viability was determined by tetrazolium dye (MTT) assay. Morphological changes were assessed by phase-contrast microscopy. Markers of EMT, including E-cadherin and α-smooth muscle actin (α-SMA) were detected by indirect immunofluorescence stains. The BMP-7 and gremlin mRNA and protein expression in HK-2 cells were analyzed by quantitative real-time PCR (real-time RT-PCR) and western blotting after exposure to AA. The level of phosphorylated Smad1/5/8, a marker of BMP-7 activity, was also determined by western blot analysis. Cells were transfected with gremlin siRNA to determine the effects of gremlin knockdown on markers of EMT following treatment with AA. Our results indicated that AA-induced EMT was associated with acquisition of fibroblast-like cell shape, loss of E-cadherin, and increases of alpha-SMA and collagen type I. Interestingly, exposure of HK-2 cells to 10 μmol/L AA increased the mRNA and protein expression of gremlin in HK-2 cells. This increase was in parallel with a decrease in BMP-7 expression and a down-regulation of phosphorylated Smad1/5/8 protein levels. Moreover, transfection with siRNA to gremlin was able to recover BMP-7 signaling activity, and attenuate EMT-associated phenotypic changes induced by AA. Together, these observations strongly suggest that gremlin plays a critical role in the modulation of reno-protective action of BMP, and that inhibition of gremlin will be a promising means of developmenting novel treatments for AAN.     

Toxicities of aristolochic acid I and aristololactam I in cultured renal epithelial cells

Aristolochic acid nephropathy, a progressive tubulointerstitial renal disease, is primarily caused by aristolochic acid I (AA-I) intoxication. Aristololactam I (AL-I), the main metabolite of AA-I, may also participate in the processes that lead to renal damage. To investigate the role and mechanism of the AL-I-mediated cytotoxicity, we determined and compared the cytotoxic effects of AA-I and AL-I on cells of the human proximal tubular epithelial (HK-2) cell line. To this end, we treated HK-2 cells with AA-I and AL-I and assessed the cytotoxicity of these agents by using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, flow cytometry, and an assay to determine the activity of caspase 3. The proliferation of HK-2 cells was inhibited in a concentration- and time-dependent manner. Cell-cycle analysis revealed that the cells were arrested in the S-phase. Apoptosis was evidenced by the results of the annexin V/propidium iodide (PI) assay and the occurrence of a sub-G1 peak. In addition, AA-I and AL-I increased caspase 3-like activity in a concentration-dependent manner. These results also suggested that the cytotoxic potency of AL-I is higher than that of AA-I and that the cytotoxic effects of these molecules are mediated through the induction of apoptosis in a caspase 3-dependent pathway.     

Protective effect of BMP-7 against aristolochic acid-induced renal tubular epithelial cell injury

Aristolochic acid nephropathy (AAN) is regarded as a kind of rapidly progressive renal fibrosis caused by the ingestion of herbal remedies containing aristolochic acid (AA). Recent studies showed that bone morphogenetic protein-7 (BMP-7) exerts beneficial effects on acute and chronic kidney injuries induced by different pathological conditions. We examined whether BMP-7 protects human renal tubular epithelial cells (HK-2) against AA-induced injury in vitro. HK-2 cells were cultured with different concentrations of AA and BMP-7 for 48 h. Cell viability was determined by Cell Counting Kit-8 assay and lactate dehydrogenase (LDH) release. The apoptosis rate and the activity of caspase 3 protease were also examined. Epithelial-to-mesenchymal transition (EMT) was determined by cell morphology, E-cadherin and α-smooth muscle actin (α-SMA) protein expression, and TGF-β₁ and collagen III secretion. Additionally, the effect of anti-TGF-β1 antibody on AA-induced EMT was assessed. Our results indicated that BMP-7 significantly increased cell proliferation, decreased apoptosis rate and attenuated activation of caspase-3, resulting in the protection of HK-2 cells from AA-induced cytotoxicity. In addition, studies on EMT revealed that BMP-7 could inhibit AA-induced myofibroblast phenotype and restored the epithelial morphology in a dose-dependent manner. It was partially through reducing the activation of a myofibroblast phenotype and production TGF-β1. Treatment with neutralizing anti-TGF-β1 antibody also blocked AA-induced EMT and collagen III secretion. Together, these observations strongly suggest that BMP-7 is a potent inhibitor of AA-induced renal tubular epithelial cell injury and might be a promising agent for aristolochic acid-induced kidney damage.     

单叶细辛中一个新的马兜铃酸类化合物

为了研究单叶细辛(Asarum himalaicum)的化学成分,利用溶剂提取、硅胶柱色谱、凝胶(SephadexLH-20)柱色谱和半制备型高效液相色谱(semi-preparative high performance liquid chromatography,HPLC)等手段进行分离、纯化,从单叶细辛全草中共分离鉴定了15个化合物。其结构经1H NMR、13C NMR、HR-ESI-MS等谱学方法分别鉴定为4-去甲氧基马兜铃酸BII(4-demethoxyaristolochic acid BII,1)、马兜铃酸I(aristolochicacid I,2)、马兜铃酸Ia(aristolochic acid Ia,3)、7-羟基马兜铃酸I(7-hydroxyaristolochic acid I,4)、马兜铃酸IV(aristolochic acid IV,5)、马兜铃次酸II(aristolic acid II,6)、青木香酸(debilic acid,7)、马兜铃内酰胺I(aristololactam I,8)、9-羟基马兜铃内酰胺I(9-hydroxyaristololactam I,9)、7-...     

Coumarin congeners as antidepressants.

3-Carboethyl coumarin (I) was converted to coumarin 3-acid hydrazide (II). This on reaction with appropriate aldehyde yielded 3-arylidino amino coumarin (III). Compound III on diazotisation and reaction with ferric chloride yielded the corresponding formazans viz. 3-substituted phenyl azoarylidino, amido coumarins (IVa1-a10) and oxadiazoles viz. 2-aryl-5-(3-coumarinyl)-1,3,4-oxadiazoles (Va1-a3), respectively. Simultaneously 3-carboethyl coumarin on hydrolysis gave 3-carboxy coumarin (VI) which on reaction with aryl amine in methylene chloride yielded 3-(N-aryl)amido coumarin (VIIa1-a3). The compounds were screened for their antidepressant activity against a tricyclic antidepressant (imipramine). Compounds IVa4, IVa5 and IVa9 exhibited activity better than imipramine with no toxicity (ALD50 greater than 1000 mg/kg) but IVa5 showed some side effects.     

Studies on morphology and aristolochic acid analogue constituents of <Emphasis Type="Italic">Asarum campaniflorum</Emphasis> and a comparison with two official species of Asari Radix et Rhizoma

Dried whole herbs or roots and rhizomes of Asarum campaniflorum have been sold under the trade name Xixin and used as folk remedies in its producing areas for a long time. In order to avoid the misuse of A. campaniflorum as official species of Xixin (Asari Radix et Rhizoma), a comparative study based on the morphological and phytochemical analysis of the aerial and underground parts was carried out. The usual morphological methods and a microscopic imaging system were used. The results show that A. campaniflorum could be easily distinguished from two official species (Asarum sieboldii and A. heterotropoides var. mandshuricum) by the diameter of thicker roots (1.3–2.7 mm), distinct large parenchymatous cells in phloem of roots, and the size of oil cells in upper leaf epidermises [(40)80–140(174) μm in diameter, where numbers in parentheses are for rare cases], etc. Nine aristolochic acid analogues (AAAs) were identified and estimated by high-performance liquid chromatography–diode array detection (HPLC-DAD). The aerial and underground parts of A. campaniflorum contained 3–4 AAAs, i.e., aristololactam-II-N-β-d-glucoside (AL-II-Glc), aristololactam I (AL-I), aristololactam II (AL-II), and aristolochic acid I (AA-I), while only AL-I was detected in underground parts of A. sieboldii and no AAAs were detected in underground parts of A. heterotropoides var. mandshuricum. The respective contents (in mg/g) in aerial and underground parts of A. campaniflorum, were as follows: AL-I, 0.06–0.12, 0.05–0.10; AL-II, 0.03–0.04, 0.01–0.03; AA-I, 0.01–0.02, 0.0–0.0. These data suggest A. campaniflorum has a high risk of causing aristolochic acid nephropathy. All these discoveries can contribute to not only the better understanding of this new resource species, but also the safe use of the crude drug Xixin.     

A new biflavonoid from Aristolochia contorta

From the fruits of Aristolochia contorta Bge, beside the known aristolochic acids IVa and VII, aristolactam-N-beta-D-glucopyanoside, aristoloctam Ia N-beta-D-glucopyanoside, pinitol and daucosterol, a new biflavonoid was isolated. Its structure was determined as (+/-)-2"R,3"R-dihydro-3"-hydroxyamentoflavone (1) by means of spectral methods including 1D-, 2D-NMR and HR-ESIMS and the known compounds were identified on the basis of comparing their NMR data with those of the corresponding compounds in the literature.     

Endoplasmic reticulum stress mediates aristolochic acid I-induced apoptosis in human renal proximal tubular epithelial cells

Aristolochic acid (AA), derived from the Aristolochia species, has been associated with aristolochic acid nephropathy (AAN), which has emerged as a worldwide disease. Aristolochic acid I (AAI) is the main ingredient of AA, and the underlying mechanisms for AAI-induced nephrotoxicity are still unclear. In this study, we investigated whether endoplasmic reticulum (ER) stress was involved in AAI-induced nephrotoxicity. The results showed that treatment of HK-2 cells (a human proximal tubular epithelial cell line) with AAI caused an increase in eukaryotic initiation factor-2α (eIF2α) phosphorylation, X-box binding protein 1 (XBP1) mRNA splicing and the expression of glucose-regulated protein (GRP) 78 and CAAT/enhancer-binding protein-homologous protein (CHOP). These events represent typical markers of the ER stress-related signaling pathway. Pretreatment with 4-phenylbutyrate (4-PBA) or salubrinal (Sal) significantly inhibited AAI-induced apoptosis, indicating the role of ER stress in AAI-induced apoptosis. In addition, AAI-induced cell death followed an increase of reactive oxygen species (ROS) formation in HK-2 cells. Pretreatment with N-acetyl cysteine (NAC) or glutathione (GSH) significantly inhibited AAI-induced ER stress proteins and cell death, suggesting that ROS mediate AAI-induced ER stress. Taken together, these results suggest that the ER stress response is involved in apoptosis induced by AAI in HK-2 cells, thus offering a new insight into the nephrotoxicity of AAI.     

Determination of aristolochic acid I and II in North American species of Asarum and Aristolochia

Wild ginger, Asarum canadense, which has folk uses as a medicinal and food plant, has been reported to contain aristolochic acid I. Rhizomes of North American species of Aristolochiaceae were surveyed for the presence of aristolochic acids by HPLC. Aristolochic acid I (1) and aristolochic acid II (2) were present in Aristolochia species and Hexastylis; 1 alone was detected in multiple accessions of A. canadense and Asarum caudatum, though not in Asarum wagneri. Concentrations in A. canadense were highly variable, reaching as much as 0.037 percent of dry weight.     

Detection of aristolochic acid in Chinese phytomedicines and dietary supplements used as slimming regimens.

Over the last 10 years, numerous cases of intoxications, leading for the most part to end-stage renal failure, have been reported after consumption of slimming regimens made of Chinese herbal preparations. These intoxications were associated with species of the Aristolochia genus, such as Aristolochia fangchi (Aristolochiaceae), known to contain very nephrotoxic and carcinogenic metabolites named aristolochic acids. Several commercial dietary supplements, teas and phytomedicines used as slimming regimens were analysed for their aristolochic acid I content. A preliminary detection of this toxic compound was made by thin-layer chromatography. The presence of aristolochic acid I in these preparations was confirmed by a HPLC/UV-DAD/MS analysis. A quantitative determination of aristolochic acid I was also achieved in the incriminated preparations using both UV and MS detection. Out of 42 analysed preparations, four were found to contain aristolochic acid I and two were suspected to contain aristolochic acid derivatives. Immediate removal of these products from the Swiss market was called for.     

穆坪马兜铃化学成分的研究

自穆坪马兜铃(Aristolochia moupinensis Franch)根、茎中分得十三个化合物,其中化合物Ⅰ～Ⅻ经鉴定分別为马兜铃酸Ⅰ(aristolochic acid Ⅰ)(Ⅰ),尿囊素(allantoin)(Ⅱ),紫丁香酸(syringic acid)(Ⅲ),香豆酸(P-coumaric acid)(Ⅳ),马兜铃酸Ⅳ(aristolochic acid Ⅳ)(Ⅴ),β-谷甾醇(Ⅵ),木兰碱(magnoflorine)(Ⅶ),马兜铃酸Ⅳ甲醚aristolochic acid Ⅳmethyl ether(Ⅵ),棕榈酸(Ⅸ),马兜铃酸Ⅱ(aristolochic acid Ⅱ)(Ⅹ),N(Phydroxyphenethy1)P-coumaramide(Ⅺ),马兜铃酸Ⅳ甲醚甲酯(aristolochic acid Ⅳmethyl ether methyl ester)(Ⅻ),化合物ⅩⅢ为新化合物,经光谱分析和化学合成等方法证明其结构为N(P-hydroxyphenethyl)-ferulamide,命名为穆坪马兜铃酰胺(moupinamide)。初步药理试验表明穆坪马兜铃酰胺体外有抑制血小板聚集和影响血小板内前列腺素合成的作用。     

Pharmacokinetics and nephrotoxicity of aristolochic acid in rabbits

Aristolochic acids (AAs) which exist in plants of the genus Aristolochia are the toxins responsible for aristolochic acid nephropathy (AAN). To investigate the pharmacokinetics and nephrotoxicity of AAs, rabbits were used in this study. The plasma concentrations of the main components of AAs, aristolochic acid I (AA I) and aristolochic acid II (AA II), were determined by a validated high-performance liquid chromatographic method. After intravenous administration of different doses (0.25, 0.5, 1.0, and 2.0 mg/kg) of aristolochic acid sodium (AANa) to 4 respective groups of rabbits (n=6 for each dose), linear relationships between the doses of AA I and AA II and the area under the plasma concentration curve (AUC) were found to exist (p<0.001). AANa was also given in escalating doses (0.5, 1.0, and 2.0 mg/kg) to the same rabbits at 7-day intervals. The clearance rates of both AA I and AA II significantly decreased with the escalating dose (p<0.001). A nonlinear relationship between the dose and AUC was obtained. Kidney specimens of rabbits were obtained to observe morphological changes on days 1 and 7 after AANa administration. The renal lesions caused by AAs consisted of progressive and dose-dependent tubular damage. However, no remarkable changes in the morphology of glomeruli were observed.     

Protective effect of erythropoietin against aristolochic acid-induced apoptosis in renal tubular epithelial cells

The goal of this research was to investigate the renoprotective effect of erythropoietin against aristolochic acid-induced apoptosis in cultured LLC-PK1 cells as well as underlying mechanism. LLC-PK1 cells impaired by aristolochic acid were used in this study as the cell model of aristolochic acid nephropathy. Apoptosis was studied by different methods (transmission electron microscopy, fluorescence-activated cell sorting, TUNEL, caspase-3 activation). Cells showed apoptotic morphology when exposed to 10 mug/ml aristolochic acid for 24 h, and the apoptotic index was increased (37.67%) compared with the control (6.09%). The presence of recombinant human erythropoietin (10, 20 U/ml, 24 h) significantly lowered the apoptotic index (22.41%, 14.63%) and the damage of cytoskeleton was also ameliorated. Studies on the apoptotic signaling showed that recombinant human erythropoietin inhibited the activation of caspase-3 and upregulated the expression of anti-apoptotic gene, Bcl-XL. Moreover, recombinant human erythropoietin (10, 20 U/ml, 24 h) promoted the expression of proliferating cell nuclear antigen in renal tubular cells stimulated by 10 mug/ml aristolochic acid (46.34%, 48.11% vs. 28.46%). Together, our data provide in vitro evidence that recombinant human erythropoietin mediated renoprotective effect against aristolochic acid injury in renal tubular cells by ameliorating the damage of cytoskeleton, reducing the number of apoptotic cells and promoting cell regeneration. So a possibility is suggested that recombinant human erythropoietin may be beneficial in preventing aristolochic acid-induced apoptosis and could be a new promising therapeutic strategy for aristolochic acid-induced kidney damage.     

Studies on the metabolism of aristolochic acids I and II

1. After oral administration of aristolochic acid I (AAI) and aristolochic acid II (AAII) to rats, the following metabolites were isolated from the urine and their structures elucidated: aristolactam I, aristolactam Ia, aristolochic acid Ia, aristolic acid I and 3, 4-methylenedioxy-8-hydroxy-1-phenanthrenecarboxylic acid (metabolites of AAI); or aristolactam Ia, aristolactam II and 3,4-methylenedioxy-1-phenanthrenecarboxylic acid (metabolites of AAII). A further metabolite of AAII having a lactam structure has not yet been isolated in pure form.

2. The metabolic pathways have been elucidated by administration of various metabolites.

3. The principal metabolite of AAI in rats was aristolactam Ia; 46% of the dose was excreted in the urine in form of this metabolite and 37% in the faeces. The other substances were minor metabolites. Those metabolites of AAII whose structures have been elucidated were minor metabolites; the largest proportion consisted of aristolactam II, which accounted for 4.6% in the urine and 8.9% in the faeces.

4. The mouse was the only animal which had the same metabolite patterns of AAI and AAII as those found in the rat. Not all the metabolites listed above were found in urine from guinea pigs, rabbits, dogs and man.     

Studies on the metabolism of aristolochic acids I and II

1. After oral administration of aristolochic acid I (AAI) and aristolochic acid II (AAII) to rats, the following metabolites were isolated from the urine and their structures elucidated: aristolactam I, aristolactam Ia, aristolochic acid Ia, aristolic acid I and 3,4-methylenedioxy-8-hydroxy-1-phenanthrenecarboxylic acid (metabolites of AAI); or aristolactam Ia, aristolactam II and 3,4-methylenedioxy-1-phenanthrenecarboxylic acid (metabolites of AAII). A further metabolite of AAII having a lactam structure has not yet been isolated in pure form. 2. The metabolic pathways have been elucidated by administration of various metabolites. 3. The principal metabolite of AAI in rats was aristolactam Ia; 46% of the dose was excreted in the urine in form of this metabolite and 37% in the faeces. The other substances were minor metabolites. Those metabolites of AAII whose structures have been elucidated were minor metabolites; the largest proportion consisted of aristolactam II, which accounted for 4.6% in the urine and 8.9% in the faeces. 4. The mouse was the only animal which had the same metabolite patterns of AAI and AAII as those found in the rat. Not all the metabolites listed above were found in urine from guinea pigs, rabbits, dogs and man.