Targeted metabolomics assisted rapid screening and characterization of aristolochic acids and their DNA adducts in aristolochia plants by ultra-high performance liquid chromatography coupled with ion mobility quadrupole time-of-flight mass spectrometry.

Aristolochic acids are one of the major compounds in aristolochia plants, which are nephrotoxic and carcinogenic. A method was established for the detection and identification of aristolochic acids and their DNA adducts in four different herbs using ultra-high performance liquid chromatography-ion mobility quadrupole time-of flight mass spectrometry. Solid phase extraction conditions were optimized to improve the sensitivity of the experiment by using 40 mg of C18 as adsorbent and 100 µL ethanol as elution solvent. At a collision energy of 10-40 eV, these compounds and cleavage patterns were precisely identified and analyzed by secondary fragmentation and collision cross section values. The obtained mass spectrometry data were then analyzed by targeted metabolomics, including principal component analysis, partial least squares-discriminant analysis and hierarchical clustering analysis, and importing the samples in the established model, the confidence values can reach 0.61 and 0.76. All in all, this method can provide a useful tool for the detection of aristolochic acids and deoxyribonucleic acid adducts. In conclusion, this method was successfully used for the detection and identification of aristolochic acids and their DNA adducts.

Combustion-Derived Pollutants Linked with Kidney Disease in Low-Lying Flood-Affected Areas in the Balkans.

Tens of thousands of people in southern Europe suffer from Balkan endemic nephropathy (BEN), and four times as many are at risk. Incidental ingestion of aristolochic acids (AAs), stemming from the ubiquitousAristolochia clematitis(birthwort) weed in the region, leads to DNA adduct-induced toxicity in kidney cells, the primary cause of BEN. Numerous cofactors, including toxic organics and metals, have been investigated, but all have shown small contributions to the overall BEN relative to non-BEN village distribution gradients. Here, we reveal that combustion-derived pollutants from wood and coal burning in Serbia also contaminate arable soil and test as plausible causative factors of BEN. Using a GC-MS screening method, biomass-burning-derived furfural and coal-burning-derived medium-chain alkanes were detected in soil samples from BEN endemic areas levels at up to 63-times and 14-times higher, respectively, than in nonendemic areas. Significantly higher amounts were also detected in colocated wheat grains. Coexposure studies with cultured kidney cells showed that these pollutants enhance DNA adduct formation by AA, - the cause of AA nephrotoxicity and carcinogenicity. With the coincidence of birthwort-derived AAs and the widespread practice of biomass and coal burning for household cooking and heating purposes and agricultural burning in rural low-lying flood-affected areas in the Balkans, these results implicate combustion-derived pollutants in promoting the development of BEN.

Genome-Wide Identification and Characterization of miRNAs and Natural Antisense Transcripts Show the Complexity of Gene Regulatory Networks for Secondary Metabolism in Aristolochia contorta.

Aristolochia contorta Bunge is an academically and medicinally important plant species. It belongs to the magnoliids, with an uncertain phylogenetic position, and is one of the few plant species lacking a whole-genome duplication (WGD) event after the angiosperm-wide WGD. A. contorta has been an important traditional Chinese medicine material. Since it contains aristolochic acids (AAs), chemical compounds with nephrotoxity and carcinogenicity, the utilization of this plant has attracted widespread attention. Great efforts are being made to increase its bioactive compounds and reduce or completely remove toxic compounds. MicroRNAs (miRNAs) and natural antisense transcripts (NATs) are two classes of regulators potentially involved in metabolism regulation. Here, we report the identification and characterization of 223 miRNAs and 363 miRNA targets. The identified miRNAs include 51 known miRNAs belonging to 20 families and 172 novel miRNAs belonging to 107 families. A negative correlation between the expression of miRNAs and their targets was observed. In addition, we identified 441 A. contorta NATs and 560 NAT-sense transcript (ST) pairs, of which 12 NATs were targets of 13 miRNAs, forming 18 miRNA-NAT-ST modules. Various miRNAs and NATs potentially regulated secondary metabolism through the modes of miRNA-target gene-enzyme genes, NAT-STs, and NAT-miRNA-target gene-enzyme genes, suggesting the complexity of gene regulatory networks in A. contorta. The results lay a solid foundation for further manipulating the production of its bioactive and toxic compounds.

Effects of bioconversion by Battus polydamas on the chemical composition of Aristolochia spp. and evaluation of antimicrobial activity and biocompatibility.

Aristolochia plants are emblematic from an ethnopharmacological viewpoint and are know to possess numerous biological properties, including antiseptic. However, the medicinal potential of these species is debatable because of their representative chemical constituents, aristolochic acids (AAs) and aristolactams (ALs), which are associated, for instance, with nephropathy and cancer. These contrasting issues have stimulated the development of approaches intended to detoxification of aristoloquiaceous biomasses, among which is included the bioconversion method using larvae of the specialist phytophagous insect Battus polydamas, previously shown to be viable for chemical diversification and to reduce toxicity. Thus, eleven Aristolochia spp. were bioconverted, and the antimicrobial activities of the plant methanolic extracts and its respective bioconversion products were evaluated. The best results were found for Aristolochia esperanzae, Aristolochia gibertii, and Aristolochia ringens against Bacillus cereus, with MIC ranging from 7.8 to 31.25 µg/mL. These three species were selected for chemical, antioxidant, cytotoxic, hemolytic, and mutagenic analyses. Chemical analysis revealed 65 compounds, 21 of them possible bioconversion products. The extracts showed potential to inhibit the formation and degradation of B. cereus biofilms. Extracts of A. gibertii and its bioconverted biomass showed antioxidant activity comparable to dibutylhydroxytoluene (BHT) standard. Bioconversion decreased the hemolytic activity of A. esperanzae and the cytotoxicities of A. esperanzae and A. gibertii. None of the extracts was found to be mutagenic. The bioactivities of the fecal extracts were maintained, and biocompatibility was improved. Therefore, the results obtained in this study reveal positive expectations about the natural detoxification process of the Aristolochia species.

Paradigm Shift in Gastric Cancer Prevention: Harnessing the Potential of Aristolochia olivieri Extract.

Gastric cancer, particularly adenocarcinoma, is a significant global health concern. Environmental risk factors, such as Helicobacter pylori infection and diet, play a role in its development. This study aimed to characterize the chemical composition and evaluate the in vitro antibacterial and antitumor activities of an Aristolochia olivieri Colleg. ex Boiss. Leaves' methanolic extract (AOME). Additionally, morphological changes in gastric cancer cell lines were analyzed. AOME was analyzed using HPLC-MS/MS, and its antibacterial activity against H. pylori was assessed using the broth microdilution method. MIC and MBC values were determined, and positive and negative controls were included in the evaluation. Anticancer effects were assessed through in vitro experiments using AGS, KATO-III, and SNU-1 cancer cell lines. The morphological changes were examined through SEM and TEM analyses. AOME contained several compounds, including caffeic acid, rutin, and hyperoside. The extract displayed significant antimicrobial effects against H. pylori, with consistent MIC and MBC values of 3.70 ± 0.09 mg/mL. AOME reduced cell viability in all gastric cancer cells in a dose- and time-dependent manner. Morphological analyses revealed significant ultrastructural changes in all tumor cell lines, suggesting the occurrence of cellular apoptosis. This study demonstrated that AOME possesses antimicrobial activity against H. pylori and potent antineoplastic properties in gastric cancer cell lines. AOME holds promise as a natural resource for innovative nutraceutical approaches in gastric cancer management. Further research and in vivo studies are warranted to validate its potential clinical applications.

Enhanced Production of Nitrogenated Metabolites with Anticancer Potential in Aristolochia manshuriensis Hairy Root Cultures.

Aristolochia manshuriensis is a relic liana, which is widely used in traditional Chinese herbal medicine and is endemic to the Manchurian floristic region. Since this plant is rare and slow-growing, alternative sources of its valuable compounds could be explored. Herein, we established hairy root cultures of A. manshuriensis transformed with Agrobacterium rhizogenes root oncogenic loci (rol)B and rolC genes. The accumulation of nitrogenous secondary metabolites significantly improved in transgenic cell cultures. Specifically, the production of magnoflorine reached up to 5.72 mg/g of dry weight, which is 5.8 times higher than the control calli and 1.7 times higher than in wild-growing liana. Simultaneously, the amounts of aristolochic acids I and II, responsible for the toxicity of Aristolochia species, decreased by more than 10 fold. Consequently, the hairy root extracts demonstrated pronounced cytotoxicity against human glioblastoma cells (U-87 MG), cervical cancer cells (HeLa CCL-2), and colon carcinoma (RKO) cells. However, they did not exhibit significant activity against triple-negative breast cancer cells (MDA-MB-231). Our findings suggest that hairy root cultures of A. manshuriensis could be considered for the rational production of valuable A. manshuriensis compounds by the modification of secondary metabolism.

Rapid simultaneous determination of thirteen aristolochic acids analogs in Aristolochiaceae plants by Ultra-High-Performance liquid Chromatography- tandem mass spectrometry in dynamic multiple reaction monitoring mode.

Asarum and Aristolochia are two large genera of Aristolochiaceae plants containing typical toxicant aristolochic acid analogs(AAAs), AAAs can be deemed as toxicity markers of Aristolochiaceae plants. Based on the least AAAs in dry roots and rhizomes of Asarum heterotropoides, Asarum sieboldii Miq and Asarum sieboldii var, all of which are enrolled in the Chinese pharmacopeia up to now. AAAs distribution in Aristolochiaceae plants, especially Asarum L. plants, is still obscure and controversial due to few AAAs measured, unverified species of Asarum, and complicated pretreatment in analytical samples making the results more challenging to reproduce. In the present study, a simple ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method in dynamic multiple reaction monitoring mode for simultaneous determination of thirteen AAAs was developed for evaluating the distribution of toxicity phytochemicals in Aristolochiaceae plants. The sample was prepared by extracting Asarum and Aristolochia powder with methanol, and the supernatant was analyzed using the Agilent 6410 system on an ACQUITY UPLC HSS PFP column with gradient elution of water and acetonitrile, containing 1% v/v formic acid (FA) each, at a flow rate of 0.3 mL/min. The chromatographic condition provided good peak shape and resolution. The method was linear over the specific ranges with the coefficient of determination (R2) > 0.990. Satisfactory intra- and inter-day precisions were achieved with RSD less than 9.79%, and the average recovery factors obtained were in the range of 88.50%～105.49%%. The proposed method was successfully applied for simultaneous quantification of the 13 AAAs in 19 samples from 5 Aristolochiaceae species, especially three Asarum L. species enrolled in the Chinese Pharmacopoeia. Except Asarum heterotropoides, the results supported that the Chinese Pharmacopoeia (2020 Edition) adopting the root with rhizome as medicinal parts of Herba Asari instead of the whole herb for drug safety by providing scientific data.

Study on the difference and correlation between the contents and toxicity of aristolochic acid analogues in Aristolochia plants.

ETHNOPHARMACOLOGICAL RELEVANCE: The nephrotoxicity and carcinogenicity induced by traditional Chinese medicines (TCMs) containing aristolochic acids (AAs) and related compound preparations have greatly limited their clinical application. While the toxicity of AA-I and AA-II is relatively clear, there are marked differences in the toxic effects of different types of aristolochic acid analogues (AAAs). Thus, the toxicity of TCMs containing AAAs cannot be evaluated based on the toxicity of a single compound. AIM OF THE STUDY: To systematically investigate the toxicity induced by Zhushalian (ZSL), Madouling (MDL) and Tianxianteng (TXT) as representative TCMs derived from Aristolochia. MATERIALS AND METHODS: AAA contents in ZSL, MDL and TXT were determined using HPLC. Subsequently, mice were treated for 2 weeks with high (H) and low (L) dosages of TCMs containing total AAA contents of 3 mg/kg and 1.5 mg/kg, respectively. Toxicity was evaluated using biochemical and pathological examination and was based on organ indices. Correlations between AAA contents and induced toxicity were analysed using multiple methods. RESULTS: Of the total AAA content, ZSL contained mainly AA-I and AA-II (>90%, of which AA-I accounted for 49.55%). AA-I accounted for 35.45% in MDL. TXT mainly contained AA-IVa (76.84%) and other AAAs accounted for <10%. Short-term toxicity tests indicated that ZSL and high-dose MDL induced obvious renal interstitial fibrosis and gastric injury, whereas TXT (high and low dosages) caused only slight toxicity. Correlation analysis suggested that AA-I might be the critical hazard factor for toxicity. CONCLUSIONS: The toxicity of TCMs containing AAAs cannot be generalised. The toxicity of TXT is relatively low compared with those of ZSL and MDL. The toxicity of Aristolochia depends mainly on the AA-I content; therefore, control of AA-I levels in TCMs and related compound preparations is required to reduce the risk of toxicity associated with the use of Aristolochia herbs in clinical settings.

Characterization of two putative norlaudanosoline methyltransferases from Aristolochia debilis.

In view of the nephrotoxicity, hepatotoxicity, and carcinogenicity of aristolochic acids (AAs), the removal of AAs from plants becomes an urgent priority for ensuring the safety of Aristolochia herbal materials. In this study, based on the root-predominant distribution of aristolochic acid I (AAI) in Aristolochia debilis, transcriptome sequencing, in combination with phylogenetic analyses, and gene expression pattern analysis together provided five candidate genes for investigating AAI biosynthesis. Comprehensive in vitro and in vivo enzymatic assays revealed that Ab6OMT1 (6-O-methyltransferase) and AbNMT1 (N-methyltransferase) exhibit promiscuity in substrate recognition, and they could act in a cooperative fashion to achieve conversion of norlaudanosoline, a predicted intermediate in AAI biosynthetic route, into 3'-hydroxy-N-methylcoclaurine through two different methylation reaction sequences. These results shed light on the molecular basis for AAI biosynthesis in Aristolochia herbs. More importantly, Ab6OMT1 and AbNMT1 may be employed as targets for the metabolic engineering of AAI biosynthesis to produce AAs-free Aristolochia herbal materials.

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.

Comparative Analysis of Aristolochic Acids in Aristolochia Medicinal Herbs and Evaluation of Their Toxicities.

Aristolochic acids (AAs) are a group of nitrophenanthrene carboxylic acids present in many medicinal herbs of the Aristolochia genus that may cause irreversible hepatotoxicity, nephrotoxicity, genotoxicity and carcinogenicity. However, the specific profile of AAs and their toxicity in Aristolochia plants, except for AAs Ι and ΙΙ, still remain unclear. In this study, a total of 52 batches of three medicinal herbs belonging to the Aristolochia family were analyzed for their AA composition profiles and AA contents using the UPLC-QTOF-MS/MS approach. The studied herbs were A. mollissima Hance (AMH), A. debilis Sieb.etZucc (ADS), and A. cinnabaria C.Y.Cheng (ACY). Chemometrics methods, including PCA and OPLS-DA, were used for the evaluation of the Aristolochia medicinal herbs. Additionally, cytotoxicity and genotoxicity of the selected AAs and the extracts of AMH and ADS were evaluated in a HepG2 cell line using the MTT method and a Comet assay, respectively. A total of 44 AAs, including 23 aristolochic acids and 21 aristolactams (ALs), were detected in A. mollissima. Moreover, 41 AAs (23 AAs and 18 ALs) were identified from A. debilis Sieb, and 45 AAs (29 AAs and 16 ALs) were identified in A. cinnabaria. Chemometrics results showed that 16, 19, and 22 AAs identified in AMH, ADS, and ACY, respectively, had statistical significance for distinguishing the three medicinal herbs of different origins. In the cytotoxicity assay, compounds AL-BΙΙ, AAΙ and the extract of AMH exhibited significant cytotoxicities against the HepG2 cell line with the IC50 values of 0.2, 9.7 and 50.2 µM, respectively. The results of the Comet assay showed that AAΙ caused relatively higher damage to cellular DNA (TDNA 40-95%) at 50 µM, while AAΙΙ, AMH and ADS extracts (ranged from 10 to 131 µM) caused relatively lower damage to cellular DNA (TDNA 5-20%).

Studies on antioxidant, anti-diabetic and GC-MS analyses of methanol extract of Aristolochia bracteolata root bark.

Aristolochia bracteolatais utilized in confronting multiple and complicated disease conditions such as cancer, lung inflammation, dysentery, syphilis, gonorrhea, arthritis, skindiseases, snake bite and oxidative stress relating to humans due to their acceptability, affordability and proximity. This investigation seeks to determine the antioxidant and anti-diabetic effects of methanol extract of A. bracteolate root bark in vitro. The phytochemical screening, antioxidant, and enzymes inhibitory (alpha-amylase and alpha-glucosidase) properties of root bark extract were evaluated by standard procedures. The methanol extract indicated the presence of diverse phytochemicals (tannins, saponins, flavonoids, alkaloids, phenols, glycosides and terpenoids) and contained a remarkable amount of saponins (8.20±0.03%), phenols (6.82±0.01%), alkaloids (4.71±0.03%) and flavonoids (3.50±0.12%). The extract showed not only strong antioxidant properties against DPPH, FRAP and TBARS radicals with IC50 value of 57.87, 54.64 and 47.54 mg/ml, respectively but also anti-diabetic activity by inhibiting alpha-amylase (IC50=53.70 mg/ml) and alpha-glucosidase (IC50=49.18 mg/ml). GC-MS chromatogram identified a diverse array of active metabolites in the methanol extract of A. bracteolate root bark. This study suggested that the methanol extract of A. bracteolate root bark possessed anti-oxidative and anti-diabetic activities.

Uptake, Translocation, and Fate of Carcinogenic Aristolochic Acid in Typical Vegetables in Soil-Plant Systems.

When Aristolochia plants wilt and decay, aristolochic acids (AAs) are released into the soil, causing soil contamination. It has been demonstrated that aristolochic acid can be accumulated and enriched in crops through plant uptake. However, there is a lack of systematic studies on the migration and accumulation of AAs in a realistic simulated soil environment. In this study, Aristolochia herbal extracts were mixed with soil for growing three typical vegetables: lettuce, celery, and tomato. The contents of AAs in the above-mentioned plants were determined by an established highly sensitive LC-MS/MS method to study the migration and accumulation of AAs. We found that AAs in the soil can be transferred and accumulated in plants. AAs first entered the roots, which were more likely to accumulate AAs, and partially entered the above-ground parts. This further confirms that AAs can enter the food chain through plants and can have serious effects on human health. It was also shown that plants with vigorous growth and a large size absorbed AAs from the soil at a faster rate. The more AAs present in the soil, the more they accumulated in the plant.

[Risk assessment, safe medication and scientific supervision of traditional Chinese medicine containing aristolochic acids--toxicity is different among aristolochic acids, and detection and control of aristolochic acid â /â ¡ is critical].

The safety problem of traditional Chinese medicine containing aristolochic acid is of great concern in China and abraod, which poses a challenge in clinical application and supervision. There are many types of aristolochic acid analogues(AAAs) and 178 have been reported. According to the structure, they are classified into aristolochic acids(AAs) and aristololactams(ALs). The toxi-city is remarkably different among AAAs of different types. For example, AA-Ⅰ has strong nephrotoxicity and carcinogenicity, and the toxicity of AA-Ⅱ is lower than that of AA-Ⅰ. Besides, AA-Ⅳa and AA-Ⅰa are considered to have no obvious nephrotoxicity and carcinogenicity. The types and content of AAAs are significantly different among traditional Chinese medicines derived from different Aristolochiaceae species. For example, Asari Radix et Rhizoma and Aristolochiae Herba mainly consist of AAAs without obvious toxicity(such as AA-Ⅳa). The content of AAAs in compound preparations is related to the proportions of the medicinals and the processing method. The content of AA-Ⅰ in some compound preparations is very low or below the detection limit. Therefore, the author concludes that AAAs of different types have different toxicity, but not all AAAs has nephrotoxicity and carcinogenicity. Moreover, the toxicity of traditional Chinese medicines containing AAAs should not be generalized and AA-Ⅰ and AA-Ⅱ should be emphasized. In this paper, it is suggested that traditional Chinese medicine containing AAAs should be used rationally and research, analysis, and toxicological study of AAAs species and content should be strengthened. In addition, limit standards of AA-Ⅰ and AA-Ⅱ should be formulated and science-based supervision should be performed.

A PCR-lateral flow immunochromatographic assay (PCR-LFA) for detecting Aristolochia species, the plants responsible for aristolochic acid nephropathy.

Aristolochic acids (AAs), which are strong carcinogens, have caused dietary supplements with Aristolochia plants to be discontinued worldwide. Therefore, the development of a method to identify these herbs is critical for customer safety. To support the regulation of Aristolochia-free products, a PCR coupled with lateral flow immunochromatographic assay (PCR-LFA) that is specific to the nucleotide signature in plastid rbcL gene region of Aristolochia species was developed to detect Aristolochia plants and related herbal products. Triplex primers (A397F, C357F and R502) were designed based on specific nucleotides observed exclusively in the rbcL sequences of Aristolochia. Positive results for Aristolochia occur when the three pink lines are clearly developed on the developed lateral flow strip and can be seen by the naked eye. In this study, the lateral flow strip has sensitivity for detecting amplicons amplified from genomic DNA at the concentrations as low as 0.01 ng. Various kinds of samples, including purchased crude drugs and polyherbal samples, have been investigated, and the results showed that Aristolochia crude drugs and Aristolochia-containing products are still present in dispensaries. In conclusion, with the goal of protecting consumers from the health risks associated with Aristolochia contamination, PCR-LFA was developed and demonstrated to be efficient for detecting plants belonging to Aristolochia in various kinds of samples.

Biological Activities of Organic Extracts of the Genus Aristolochia: A Review from 2005 to 2021.

Different ethnomedicinal studies have investigated the relationship between various phytochemicals as well as organic extracts and their bioactive aspects. Studies on biological effects are attributed to secondary metabolites such as alkaloids, phenolic compounds, and terpenes. Since there have been no reviews in the literature on the traditional, phytochemical, and ethnomedicinal uses of the genus Aristolochia so far, this article systematically reviews 141 published studies that analyze the associations between secondary metabolites present in organic extracts and their beneficial effects. Most studies found associations between individual secondary metabolites and beneficial effects such as anticancer activity, antibacterial, antioxidant activity, snake anti-venom and anti-inflammatory activity. The aim of this review was to analyze studies carried out in the period 2005-2021 to update the existing knowledge on different species of the genus Aristolochia for ethnomedicinal uses, as well as pharmacological aspects and therapeutic uses.

Validation of Unani concept of Abadal-i-Adwiya (drug substitution) by physicochemical standardization and hepatoprotective activity of Aristolochia rotunda Linn. and its substitute Curcuma Zedoaria Rosc. in albino Wistar rats.

OBJECTIVES: To validate the concept of abadal-i-adwiya (drug substitution) by evaluation of physicochemical standardization and hepatoprotective activity of Aristolochia rotunda & its substitute, Curcuma Zedoaria in albino Wistar rats. METHODS: Physicochemical standardization by estimation of moisture content, ash values and extractive values were carried out using standard methods. Hepatotoxicity was induced in albino Wistar rats using CCl4 1 mL/kg s. c. on alternate day for 14 days. Group I was served as Plain control and Group II as Negative control. Group III was administered silymarin 50 mg/kg p. o. while Group IV received HAE of A. rotunda 89.64 mg/kg p. o., and Group V was administered HAE of C. Zedoaria 45.73 mg/kg p. o. At the end of the study, serum bilirubin, AST (SGOT), ALT (SGPT) and ALP were estimated. The histopathology of liver was also carried out. RESULTS: The physicochemical parameters of both test drugs viz. moisture content, total ash, acid insoluble ash and water soluble ash were found within normal limit. The total serum bilirubin, direct bilirubin, AST (SGOT), ALT (SGPT) levels were significantly decreased in Test groups A and B when compared to the Negative and Standard controls. The microscopic examination of liver collected from animals of Group IV and Group V revealed significant recovery from hepatic toxicity compared to the Negative control. CONCLUSIONS: The study experimentation has revealed that C. Zedoaria may be used as a substitute for A. rotunda in the treatment of liver diseases. However, the outcome has to be further corroborated with phytochemical evaluation and clinical trials of both the drugs. Furthermore, the concept of drug substitute in Unani system of medicine is also validated in the light of above study.

Aristolochic acid I: an investigation into the role of food crops contamination, as a potential natural exposure pathway.

Aristolochic acid I (AAI) is a potent nephrotoxic and carcinogenic compound produced by plants of the Aristolochiaceae family and thoroughly investigated as a main culprit in the etiology of Balkan endemic nephropathy (BEN). So far, the AAI exposure was demonstrated to occur through the consumption of Aristolochia clematitis plants as traditional remedies, and through the contamination of the surrounding environment in endemic areas: soil, food and water contamination. Our study investigated for the first time the level of AAI contamination in 141 soil and vegetable samples from two cultivated gardens in non-endemic areas, A. clematitis being present in only one of the gardens. We developed and validated a simple and sensitive ultra-high-performance liquid chromatography-ion trap mass spectrometry method for qualitative and quantitative AAI analysis. The results confirmed the presence of AAI at nanogram levels in soil and vegetable samples collected from the non-endemic garden, where A. clematitis grows. These findings provide additional evidence that the presence of A. clematitis can cause food crops and soil contamination and unveil the pathway through which AAI could move from A. clematitis to other plant species via a common matrix: the soil. Another issue regarding the presence of AAI, in a non-endemic BEN area from Romania, could underlie a more widespread environmental exposure to AAI and explain certain BEN-like cases in areas where BEN has not been initially described.

Two New Aristolochic Acid Analogues from the Roots of Aristolochia contorta with Significant Cytotoxic Activity.

Twelve compounds, including two new aristolochic acid analogues with a formyloxy moiety (9-10) and 10 known aristolochic acid derivates (1-8 and 11-12), were obtained from the roots of Aristolochiacontorta. Their structures were elucidated using extensive spectroscopic methods. Their cytotoxic activity in human proximal tubular cells HK-2 was evaluated by the MTT method, which has been widely used to assess cell viability. Among these molecules, compounds 3 and 9 were found to be more cytotoxic. Furthermore, molecular modeling was used to evaluate, for the first time, the interactions of compounds 3 and 9 with the target protein organic anionic transporter 1 (OAT1) that plays a key role in mediating aristolochic acid nephropathy. Structure-activity relationships are briefly discussed.

Determination of Aristolochic Acids in Vegetables: Nephrotoxic and Carcinogenic Environmental Pollutants Contaminating a Broad Swath of the Food Supply and Driving Incidence of Balkan Endemic Nephropathy.

Balkan endemic nephropathy (BEN) is a slowly progressive interstitial fibrotic disease affecting numerous people living along the Danube River in the Balkan Peninsula, of which aristolochic acids (AAs) produced naturally in Aristolochia plants are key etiological agents. However, the exposure biology of the disease remains poorly understood. Initially, the high incidence of BEN in the Balkan Peninsula was thought to occur through ingestion of bread prepared from flour made with wheat grains comingled with the seeds of Aristolochia clematitis L., an AA-containing weed that grows abundantly in the wheat fields of the affected areas. In this study, by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, we show for the first time that vegetables, in particular root vegetables of endemic areas, are extensively contaminated with AAs taken up through root absorption from the AA-tainted soil. Furthermore, we found a pH dependence of the n-octanol/water partition coefficient (Kow) of AAs, which resulted in a dramatically higher hydrophobicity-driven plant uptake efficiency of AAs into food crops in endemic areas, characterized by higher acidity levels, compared to non-endemic areas. We believe the results of this study have significantly unraveled the mystery surrounding the uneven distribution of BEN incidence.