Assessment of the Cytotoxicity, Mutagenicity, and Genotoxicity of Two Traditional Chinese Herbs: Aristolochia baetica and Magnolia officinalis.

Herbal remedies used in traditional medicine often contain several compounds combined in order to potentiate their own intrinsic properties. However, herbs can sometimes cause serious health troubles. In Belgium, patients who developed severe aristolochic acid nephropathy ingested slimming pills containing root extracts of an Aristolochia species, as well as the bark of Magnolia officinalis. The goal of the study was to evaluate, on a human renal cell line, Aristolochia and Magnolia extracts for their cytotoxicity by a resazurin cell viability assay, and their genotoxicity by immunodetection and quantification of the phosphorylated histone γ-H2AX. The present study also sought to assess the mutagenicity of these extracts, employing an OECD recognized test, the Ames test, using four Salmonella typhimurium strains with and without a microsomial fraction. Based on our results, it has been demonstrated that the Aristolochia-Magnolia combination (aqueous extracts) was more genotoxic to human kidney cells, and that this combination (aqueous and methanolic extracts) was more cytotoxic to human kidney cells after 24 and 48 h. Interestingly, it has also been shown that the Aristolochia-Magnolia combination (aqueous extracts) was mutagenic with a TA98 Salmonella typhimurium strain in the presence of a microsomial liver S9 fraction. This mutagenic effect appears to be dose-dependent.

[Current research situation of nephrotoxicity of Chinese herbal medicine].

To provide the basis for the future research on the nephrotoxicity of Chinese herbal medicine through systematic and comprehensive summary of all the Chinese herbal medicines which may lead to nephrotoxicity. Foreign resources included PubMed and Cochrane library， and domestic research resources was China Food and Drug Administration(CDFA) Adverse Drug Reaction Monitoring Center database. The databases were searched from establishment to January 1， 2017. There was no limitation on research type. 28 English studies were found， including 97 Chinese herbs or prescriptions with the risk of nephrotoxicity. The following six Chinese herbal medicines with the risk of nephrotoxicity had a large number of studies： aristolochic acid(5 studies)， Tripterygium wilfordii(4 studies)， Erycibe obtusifolia(2 studies)， Rheum palmatum(2 studies)， Ephedra sinica(2 studies)， and Atractylodes lances(2 studies). The remaining 91 Chinese medicines were reported with risk of nephrotoxicity in only 1 study respectively. CDFA reported 16 Chinese herbal medicines with the risk of nephrotoxicity， including Ganmaoqing Pian(capsule)， Zhenju Jiangya Pian， T. wilfordii preparation， Vc-Yinqiao Pian， Chuanhuning injection， Shuanghuanglian injection， Qingkailing injection， Lianbizhi injection， herbal decoction containing Aristolochiae Radix， Guanxin Suhe Wan， Shugan Liqi Wan， Ershiwuwei Songshi Wan， herbal decoction containing Aristolochia Fangchi， herbal granules containing root of Kaempfer Dutchmanspipe， Ganmaotong(tablets)， and Longdan Xiegan Wan. Currently， in addition to aristolochic acids， the most reported Chinese herbal medicine with the risk of nephrotoxicity is T. wilfordii preparation.

[Study and opinion on toxicity of aristolochic acid].

On October 18th, 2017, a research article named "Aristolochic acids and their derivatives are widely implicated in liver cancers in Taiwan and throughout Asia" was published on Science Translational Medicine. This article pointed out that herbs containing aristolochic acids could cause liver cancer by inducing the specific "aristolochic acids mutational signature". The public was also suggested to avoid the intake of herbs containing aristolochic acids. Since 2000, CFDA has gradually abolished the medicinal standards for herbs containing aristolochic acids such as caulis aristolochiae manshuriensis, aristolochia heterophylla and radix aristolochiae. Related drugs have been strengthened supervision since then. Chinese Pharmacopoeia has also removed the records of a series of related herbs. State Administration of Traditional Chinese Medicine held a conference on the "toxicity" of aristolochic acids as soon as the article was published. After a discussion of the studies on the toxicity of aristolochic acids, experts attending the meeting discovered several problems, including the unclearness of exposure history, tumor-producing dose and latent period, the absence of some key factors such as hepatitis B, the small sample size, miscellaneous factors, incomplete evidence chains, the missing of analyses between data with huge differences, the insufficiency of fundamental research arguments, etc. In order to understand the toxicity of aristolochic acids and the carcinogenic risks, as well as guide clinical safe medication, the experts suggested that：①Complete the systematical evaluation of aristolochic acids carcinogenicity as soon as possible. Scientifically elucidate the relationship between aristolochic acids and the genesis of liver cancer. ②Establish medication risk warnings of aristolochic acids and strengthen the supervision. ③Make an in-depth study of the toxicity of traditional Chinese medicine. Find out the adverse effects of all traditional Chinese medicine step by step.

Mutational signature of aristolochic acid: Clue to the recognition of a global disease.

Mutational signatures associated with specific forms of DNA damage have been identified in several forms of human cancer. Such signatures provide information regarding mechanisms of tumor induction which, in turn, can reduce exposure to carcinogens by shaping public health policy. Using a molecular epidemiologic approach that takes advantage of recent advances in genome sequencing while applying sensitive and specific analytical methods to characterize DNA damage, it has become increasingly possible to establish causative linkages between certain environmental mutagens and disease risk. In this perspective, we use aristolochic acid, a human carcinogen and nephrotoxin found in Aristolochia herbs, to illustrate the power and effectiveness of this multidisciplinary approach. The genome-wide mutational signature for this toxin, detected initially in cancers of the upper urinary tract, has subsequently been associated with cancers of the liver and kidney. These findings have significant implications for global public health, especially in China, where millions of individuals have used Aristolochia herbal remedies as part of traditional Chinese medicine and, thus, are at risk of developing aristolochic acid nephropathy and/or upper urinary tract carcinomas. The studies reported here set the stage for research into prevention and early detection, both of which will be required to manage a potentially devastating global disease.

Discovery of GABA(A) receptor modulator aristolactone in a commercial sample of the Chinese herbal drug "Chaihu" (Bupleurum chinense roots) unravels adulteration by nephrotoxic Aristolochia manshuriensis roots.

In a two-microelectrode voltage clamp assay using Xenopus laevis oocytes, a petroleum ether extract prepared from a commercial sample of the traditional Chinese herbal drug labelled as " Chaihu" (Bupleurum chinense DC. roots) enhanced the I(GABA) by 156 % ± 22 % when tested at 100 µg/mL. By means of HPLC-based activity profiling combined with high-resolution LC-MS and microprobe NMR, the germacranolide aristolactone was identified as one of the main active compounds (EC50 56.02 µM ± 5.09 µM). However, aristolactone has been previously reported only from the genus Aristolochia (Aristolochiaceae), suggesting a possible adulteration. With the aid of a validated HPTLC protocol for detection of aristolochic acids and with reference samples, the commercial sample was confirmed to be a mixture of Aristolochia manshuriensis root and Bupleurum chinense root. This finding was corroborated by macroscopic inspection of the drug. This case of adulteration with a highly nephrotoxic drug raises concerns about adequate quality control of TCM drugs commercialized in Europe.

[The determination of aristolochic acid A in different processed Aristolochia manshuriensis and the test of influence about renal function in rats].

OBJECTIVE: To study and approach the processing methods and mechanism which can markedly reduce the content of aristolochic acid in Aristolochia manshuriensis and lighten the nephrotoxicity of aristolochic acid. METHODS: A traditional "attenuation" processing method was used and 30 types of samples which contain one crude and 29 types of processed sample were obtained. The contents of aristolochic acid A in every sample were determined by HPLC. According to the Rat's acute renal injury test, the influence of animal's renal function was investigated for representative samples. RESULTS: The content of aristolochic acid in six types of samples depressed markedly (30% or more depressed) which processing with boiling in the limewater, steaming with limewater, boiling in the juice of liquorice, boiling in the decoction of black soybean, boiling in the soda water and stir-baked with talcum powder, the content of aristolochic acid in other processed samples also depressed with a large discrepancy. The toxicology test results showed that the above-mentioned 6 samples all can relieve renal injury of rats. There could be some associativity between the degree of renal injury relieving and the content of aristolochic acid A in the samples. CONCLUSION: The content of aristolochic acid can be reduced and the nephrotoxicity for animals can be lightened with some eligible processing methods for the traditional Chinese medicines containing aristolochic acid with the representative of Aristolochia manshuriensis.

[Nephrotoxicity study of Aristolochia fangchi in rats by metabonomics].

OBJECTIVE: To study the changes of metabolites in rat urine after treatment of Aristolochia fangchi decoction by metabonomic method. METHODS: Sixty-four male SD rats were divided into Aristolochia fangchi group and normal control group. Rats in the Aristolochia fangchi group were orally administered with 8.1 g/(kg.d) of Aristolochia fangchi and the normal control group was administered with equal volume of distilled water for 4 weeks. Twenty-four hour urine was collected at different time points (before, after 2- and 4-week administration and 2 weeks after administration) and their H nuclear magnetic resonance (NMR) spectra were acquired and subjected to data process, including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) or orthogonal signal correction (OSC). The contents of blood urea nitrogen (BUN) and serum creatinine (SCr) and histopathological changes of the renal tissues were also detected. RESULTS: The content of BUN of the Aristolochia fangchi group was markedly higher than that of the normal control group after 2-week administration (P<0.05), and cellular edema in tubular endothelial cells, structure damage of glomeruli and inflammatory cell infiltration were found in the kidney. Along with the lasting of administration to 4-week, the renal injury in the Aristolochia fangchi group became more serious, and the contents of BUN and SCr were all significantly higher as compared with the normal control group (P<0.05). Two weeks after administration, the content of BUN in the Aristolochia fangchi group was still higher than that in the normal control group (P<0.05), and the pathological changes in renal tissues were not different from those on the 4th week. The urine of the Aristolochia fangchi group could be readily discriminated from the normal control group at every time point based on PCA. During the whole administration period, compared with the normal control group, the concentration of urinary taurine was increased time-dependently, while the citrate was decreased in the Aristolochia fangchi group. The concentration of hippurate was increased at the 2nd week and the 6th week (2 weeks after drug withdrawal) but decreased at the 4th week; the concentration of creatinine/creatine was increased at the 4th week but decreased at the 6th week; the concentration of 2-oxo-glutarate was decreased and the concentration of trimethylamine oxide was increased at the 4th and 6th week. CONCLUSION: High-dose Aristolochia fangchi can induce renal lesion and its seriousness is correspondent to the lasting of administration. Aristolochia fangchi may also have toxicity on liver.

[Hepatic and renal injury induced by Radix Aristolochiae or Guanxin Suhe Wan for a long-term in rats].

OBJECTIVE: To evaluate the toxicity of Radix Aristolochiae supplied experimental evidence of rational use of drug in clinic. METHOD: After treatment with small dose Radix Aristolochiae, Guanxin Suhe Wan (with Radix Aristolochiae) and Guanxin Suhe Wan (without Radix Aristolochiae) in different group for a long- term, respectively, the biochemical indicator of PT, ALT, AST, ALB, ALP, Crea and BUN were detected, and the kidney, liver, stomach and urinary bladder were examined by pathologic assaying. RESULT: In Radix Aristolochiae group and Guanxin Suhe Wan (with Radix Aristolochiae) group, all of biochemical indicator were changed significantly, and hepatonecrosis, renal tubular necrosis, gastric carcinoma and bladder carcinoma were discovered. CONCLUSION: Radix Aristolochiae and Guanxin Suhe Wan (with Radix Aristolochiae) can damage kidney and liver, and cause gastric carcinoma and bladder carcinoma by intensive toxicity.

Comparative 28-day repeated oral toxicity of Longdan Xieganwan, Akebia trifoliate (Thunb.) koidz., Akebia quinata (Thunb.) Decne. and Caulis aristolochiae manshuriensis in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Longdan Xieganwan, which contains Aristolochia species, is a traditional Chinese prescription. It has been used for thousands of years to "enhance liver". However, many cases of Longdan Xieganwan induced nephropathy were reported recently. AIM OF THE STUDY: This study was designed to compare the possible toxic effects of Longdan Xieganwan and three different Aristolochia species, i.e. Akebia trifoliate (Thunb.) koid (Akebia trifoliate), Akebia quinata (Thunb.) Decne. (Akebia quinata) and Caulis aristolochiae manshuriensis (Aristolochia manshuriensis). MATERIALS AND METHODS: Mice were orally administered these drugs for 28 days. Clinical signs, body weights, serum biochemistry, organ weights and histopathology were examined. RESULTS: Significantly decreased body weights and obvious nephropathy were noticed in the Aristolochia manshuriensis groups at doses higher than 0.24 g/kg/d. A few endothelial cell degenerations in renal glomerulus were observed in the Akebia trifoliate group at a high-dose of 2.00 g/kg/d. No significant changes were observed in the other groups. CONCLUSIONS: The no-observed-adverse-effect levels (NOAELs) for Aristolochia manshuriensis, Akebia trifoliate, Akebia quinata and Longdan Xieganwan in this study for mice were 0.06 g/kg/d, 0.40 g/kg/d, higher than 3.00 g/kg/d and higher than 10.00 g/kg/d, which were equivalent to 0.25 times, 5 times, 25 times and 10 times of normal human dose in clinical prescription, respectively.

Aristolochic acid nephropathy: a worldwide problem.

Aristolochic acid nephropathy (AAN), a progressive renal interstitial fibrosis frequently associated with urothelial malignancies, was initially reported in a Belgian cohort of more than 100 patients after the intake of slimming pills containing a Chinese herb, Aristolochia fangchi. Although botanicals known or suspected to contain aristolochic acid (AA) were no longer permitted in many countries, several AAN cases were regularly observed all around the world. The incidence of AAN is probably much higher than initially thought, especially in Asia and the Balkans. In Asian countries, where traditional medicines are very popular, the complexity of the pharmacopoeia represents a high risk for AAN because of the frequent substitution of the botanical products by AA-containing herbs. In the Balkan regions, the exposure to AA found in flour obtained from wheat contaminated with seeds of Aristolochia clematitis could be responsible for the so-called Balkan-endemic nephropathy. Finally, despite the Food and Drug Administration's warnings concerning the safety of botanical remedies containing AA, these herbs are still sold via the Internet.

Efectos tóxicos de cuatro plantas amazónicas sobre Chironomus calligraphus Goeldi 1905 (Diptera: Chironomidae) y Artemia franciscana Kellog 1906 (Anostraca: Artemiidae) / Toxic effects of four amazonian plants on Chironomus calligraphus Goeldi 1905 (Diptera: Chironomidae) and Artemia franciscana Kellog 1906 (anostraca: artemiidae)

The aim of current research was to evaluate the ecotoxic effect of four Amazonian plants of ethnobotanical importance in Pucallpa, Peru: Dutchman’s Pipe Aristolochia pilosa Kunth (Aristolochiaceae) in stem and leaves, Soapberry Paullinia clavigera Simpson (Sapindaceae) in cortex, Wandering Jew Tradescantia zebrina Hort ex Bosse (Commelinaceae) in all plant and Curare Chondrodendron tomentosum Ruiz & Pavon corr. Miers (Menispermaceae) in cortex and leaves on first instars’ insect larvae of Chironomus calligraphus Goeldi 1905 and second instars’ nauplii of brine shrimp Artemia franciscana Kellog, 1906. Standard procedures were employed to detect semiqualitative phytochemistry of hexane, chloroform and hydroalcohol extracts, for each plant evaluated. The LC50 mean values of hexane and chloroform extracts of all plants on A. franciscana and hydroalcoholic extract on C. calligraphus were lower than values of chloroform extracts on C. calligraphus. Leaves hexane extract and stem chloroform extract of A. pilosa, and hydroalcohol extract of P. clavigera had higher activity on C. calligraphus. However, hexane and chloroform extracts of T. zebrina, and stem hydroalcoholic extract of A. pilosa had higher activity on A. franciscana.

El objetivo del presente trabajo fue evaluar el efecto ecotoxico de cuatro plantas amazónicas de importancia etnobotánica en Pucallpa, Perú: “Huancahuisacha” Aristolochia pilosa Kunth (Aristolochiaceae) en talloy hoja, “Sacha Yoco” Paullinia clavigera Simpson (Sapindaceae) en corteza, “Oreja de Tigre” Tradescantia zebrina Hort ex Bosse (Commelinaceae) toda la planta y “Curare” Chondrodendron tomentosum Ruiz & Pavon corr. Miers (Menispermaceae) en corteza y hoja sobre las larvas de primer estadio de Chironomus calligraphus Goeldi 1905 y sobre los nauplios de segundo estadio del camarón salino Artemia franciscana Kellog, 1906. Se emplearon procedimientos estándares para la detección fitoquímica semicualitativa de los extractos hexánicos, clorofórmicos e hidroalcohólicos de las plantas evaluadas...

[The nephrotoxicity in rats caused by Longdan Xiegan decoction].

OBJECTIVE: To observe the renal injury in rats induced by Longdan Xiegan decoction (LDXGD) containing different dosages Aristolochia manshriensis. METHOD: SD rats were divided into four groups at random, and were fed with three kinds of LDXGD 13, 14.5, 17.5 g x kg(-1) (containing respectively A. manshriensis 1.5, 3, 6 g x kg(-1)) and distilled water respectively for 12 weeks. Renal functional parameters on 4,8,12 w were determined and changes of histomorphology in rats on the end of experiment were observed. RESULT: The LDXGD containing low dose (1.5 g x kg(-1)) A. manshriensis did't induce significantly renal injury in rats during 12 weeks; the LDXGD containing midst dose(3 g x kg(-1)) A. manshriensis induced light damage of proximal convoluted tubule epithelial cells in rats during 12 weeks; the LDXGD containing high dose(6 g x kg(-1)) A. manshriensis induced significantly renal injury in rats after administed 4 weeks. Along with the lasting of administration, the degree of injury became more seriously. The main renal injury location was in proximal convoluted tubule. CONCLUSION: The renal toxicity of LDXGD is correlated with the dose of A. manshriensis and the time of administration. The LDXGD containing low dose A. manshriensis has relative security. However, the LDXGD containing high dose A. manshriensis can induce renal injury.

[Experimental study of chronic renal tubular-interstitial injury induced by Radix Aristolochiae Fangchi Extract in rats].

OBJECTIVE: Following the former report, we continue to observe the chronic renal tubular-interstitial injury induced by Radix Aristolochiae Fangchi Extract(RAFE) in rats in order to understand whether RAFE in different doses causes the renal tubular-interstitial injury or not. METHOD: RAFE at the dose of 25.0 mg x kg(-1) x d(-1), 120.0 mg kg(-1) x d(-1) and 200.0 mg x kg(-1) x d(-1) and aristolochic acid (AA, 10.0 mg x kg(-1) d(-1)) was interruptedly administrated by gastric tube for 22 w and 4 w durg withdrawal. Blood, urine and kidney were taken out respectively in 17 w, 22 w and 26 w to measure the indexes of renal function. The morphology of kidney was observed, and Masson staining of kidney were made respectively to compare RAFE groups with AA group. RESULT: Pathological changes of renal tissue forms were as follows: All RAFE groups and AA group could develop the pathological process of renal tubular injury-chronic renal interstitial fibrosis. The pathologic changes of RAFE were similar with AA. CONCLUSION: RAFE at all doses administrated interruptedly by gastric tube above 13 w caused chronic renal tubulo-interstitium fibrosis. The renal injury in functions and tissue forms in rats were similar with AA closely. The results showed that AA was the main toxic composition of RAFE.

[Experimental study of chronic renal tubular-interstitial injury induced by radix aristolochiae fangchi extract in rats].

OBJECTIVE: To observe the acute and chronic renal toxicity induced by Radix Aristolochiae Fangchi Extract (RAFE) in different doses in rats. METHOD: The conventional method of acute toxicity was used. RAFE at the dose of 25.0 mg x kg(-1) x d(-1), 120.0 mg x kg(-1) x d(-1) and 200.0 mg x kg(-1) x d(-1) and aristolochic acid (AA, 10.0 mg x kg(-1) x d(-1)) were interruptedly administrated to rats for 13 week by gastric tube, and the sample of blood, urine and kidney were collected at 4 week, 8 week and 13 week respectively. The indexes of renal function were measured and the morphology of kidney was observed. RESULT: LD50 of RAFE was 36.8 g x kg(-1) (the crude drug) and the 95% confidence limit was 38.8 - 28.9 g x kg(-1). The changes of renal functions were azotemia, massive proteinuria and the increase of urinary NAGase (beta-N-acetylglucosaminidase) in the earlier period of administration with RAFE in rats. Pathological changes of renal tissue were as follows: acute renal tubular necrosis mainly in the boundary of cortex and medulla was observed in the earlier period, and with the elongation of administration, the pathological process of renal interstitial fibrosis observed in the middle and high groups of RAFE and AA group. CONCLUSION: RAFE at middle and high doses administrated by interrupted gavage above 13 week can cause the injury of renal tubular functions in rats. NAGase can be used as one of observation targets in the earlier period of renal injury.

Studies on the toxicity of Aristolochia manshuriensis (Guanmuton).

AIMS: (1) To study the acute and chronic toxicity of stem of Aristolochia manshuriensis (AMA Guanmuton) which is a Chinese medicinal herb in order to provide basis for safe clinical use. (2) To investigate the possibility of reducing toxicity of the herb combined with Rhizoma Coptidis (Huanglian). METHODS: The 70% ethanol extract of the herb was fed to mice via gastric tube for 8 weeks. The blood was collected to assess liver and renal functions. The tissue samples of the liver, kidney and bladder were collected from executed animals for pathology examination. RESULTS: The LD50 with a 95% average trustable probability (P=0.95) of AMA from Hanzhong (HZ) is 29.2+/-3.71 g/kg. The weight of animals in the treatment group at a dose of 4 g/kg raw drug, equivalent to 40 times of normal human dose in clinical prescription, remained the same as the control group (P>0.05). On pathological examination, there were no morphological changes under light microscope in the liver and bladder. For the kidney, the renal toxicity was significantly reduced after using ethanol extract of R. coptidis to process HZ AMA in that there were no interstitial inflammation, formation of hyaline cast or regeneration of renal tubules.

Cytotoxic effect of Argentine medicinal plant extracts on human hepatocellular carcinoma cell line.

Methanolic extracts from Achyrocline satureioides (Dc.) Lam, Aristolochia macroura Gomez, Lithraea molleoides (Vell.) Engl., Schinus molle L., unlike those from Celtis spinosa Spreng, Chenopodium ambrosioides L., Petiveria alliacea L., and Plantago major L. showed cytotoxic activity against a human hepatocellular carcinoma cell line, Hep G2. Schinus molle L. was the most active (IC50=50+/-7 microg/ml). These results call for further studies of these extracts.

[Comparison among families of Mutong].

OBJECTIVE: To distinguish families of Mutong correctly and direct effective and safe clinical administration. METHOD: Comparison among families of Mutong on Herbs, Taxology, Clinic, Pharmacology and Toxicology. RESULT: 1. There are mainly three families of Mutong: Lardizabalaceae, Ranunculaceae, Aristolochiaceae, which were all included in China Pharmacopeia in 1963. However only Mutong of Ranunculaceae and Aristolochiaceae family have been included in China Pharmacopeia since 1977, but Mutong of Lardizabalaceae family has not been included in China Pharmacopeia ever since. 2. It was Mutong of Lardizabalaceae family that was used mainly through the ages without toxic records, and Mutong of Aristolochiaceae e.g. Caulis Aristolochia manshuriensis (CAM) was not put down in writing of past ages but is mainly used today with toxicity repeatedly. 3. CAM contain aristolochic acid and aristololactam with high toxicity, which plays an uncertain role in diuresis with poor bactericidal power. Mutong of Lardizabalaceae family e.g. Akebia trifoliata (Thunb.) Koidz. var. australis (Diels) Rehd (ATKV) don't contain aristolochic acid and aristololactam, which has low toxicity and plays a certain role in diuresis with high bactericidal power. CONCLUSION: It may be quite safe to use ATKV instead of CAM in clinics. So we suggest that ATKV should be reused as first Mutong in China Pharmacopeia revised edition in order to ensure a correct understanding of the facts and reveal Mutong in its true colors, and CAM should be used as second Mutong strictly according to the rules in China Pharmacopeia revised edition.