Genotoxicity of Asiasari Radix et Rhizoma (Aristolochiaceae) ethanolic extract in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional herbal medicines have diverse efficacy and are increasingly used worldwide. However, some of these herbal medicines have toxicities or side effects, but the scientific understanding of traditional herbal medicine toxicity has not yet been established. Asiasari Radix et Rhizoma (ARE) is known as a herbal medicine used to relieve pain, and recent studies have shown that ARE has anticancer and antimelanogenesis efficacy. AIM OF THE STUDY: Current study was conducted to assess the potential genotoxicity of an ethanolic extract of ARE. MATERIALS AND METHODS: The genotoxixity of ARE was confirmed by the bacterial reverse mutation assay (Ames test), a mammalian chromosomal aberration test, and a micronucleus test in vivo using ICR mice and comet assay using Sprague-Dawley rats. RESULTS: ARE showed no genotoxicity in a micronucleus test up to 2000 mg/kg body weight in vivo. By contrast, the chromosomal aberration test showed that ARE induced an increase in the number of chromosomal aberrations after treatment for 6 h with a metabolic activation system and for 6 and 22 h without the metabolic activation system when compared with vehicle control. In the Ames test, all strains except TA1535, with or without a metabolic activation system, showed an increase in the number of revertant mutant colonies in the ARE-treated group. In comet assay, DNA damage was observed in the stomach when ARE was administered. CONCLUSION: ARE potentially shows genotoxicity by inducing DNA damage.

Purification and characterization of the major protein isolated from Semen Armeniacae Amarum and the properties of its thermally induced nanoparticles.

From the aqueous extract of Semen Armeniacae Amarum, a major protein isolate was purified and characterized as a novel member of the 11S globulin family, which is composed of three polypeptides linked by disulfide bond. Furthermore, the feasibility of using the isolated protein for fabricating nanocarriers was investigated. The results indicate that thermal treatment of the globulin induced the rearrangement of the disulfide bond to form homodimers of acid polypeptides during the formation of nanoparticles. The harvested nanoparticles produced by heat-induced assembly are spherical in shape, with an average size of 92 nm and exhibited low cytotoxicity to L-02 and MDCK cell lines. These nanoparticles are capable to encapsulate paclitaxel, estimated the maximum encapsulation efficiency of paclitaxel loaded to the nanoparticles was 92.6% and the maximum release of paclitaxel was 57.4%. This research suggests that the screening of traditional herbal extracts could provide a novel source of protein nanocarriers.

Protective effects of Herpetospermum caudigerum extracts against liver injury induced by carbon tetrachloride in mouse.

Herpetospermum caudigerum (H. caudigerum; HC), popularly known as “Sejimeiduo” in Tibet, it is widely used in Tibetan traditional medicine for the treatment of dyspepsia, liver and colic diseases. This study was designed to evaluate the effect of H. caudigerum extract (HCE) on suppressing liver injury induced by carbon tetra chloride (CCl4). For this purpose, we used CCl4 to induce acute liver injury in mouse model. The protective effects of HCE against liver injury were evaluated by biochemical parameters, histopathological and immunohistochemical staining. The results showed that the superoxide dismutase (SOD) activity was significantly increased with the increasing dose of HCE as compared to the CCl4-treated group (p less than 0.01); while AST and ALT levels in serum, MDA and MPO in liver were reduced in a dose-dependent manner. The histopathology showed that HCE treatment promoted the recovery of histopathological changes in liver in a dose-dependent way. Meanwhile, there was a higher expression of caspase-3 and NF-κB in the nucleus of several liver cells in the CCl4-induced group, and a low expression of caspase-3 and NF-κB were found with the increasing dose of HCE. Therefore, the present study suggests that HCE is a potent hepatoprotective agent that can treat acute liver injury and this ability may be attributed towards its anti-inflammatory and antioxidant potential.

Hybrid-type carbon microcoil-chitosan composite for selective extraction of aristolochic acid I from Aristolochiaceae medicinal plants.

Aristolochic acid I is a nephrotoxic compound widely existing in many kinds of traditional Chinese medicines, especially in Aristolochiaceae medicinal plants. In this study, chitosan modified carbon microcoils were designed and prepared for the selective separation of aristolochic acid I from medicinal herbs. Successful modification of carbon microcoils was confirmed by scanning electron microscopy, Fourier-transfer infrared spectroscopy, elemental analysis, X-ray photoelectron spectroscopy, and thermogravimetric analyses. The effects of adsorption conditions were investigated and it was determined that the adsorption of aristolochic acid I was controlled by pH. Adsorption isotherms, kinetics, and selectivity tests were performed to evaluate the adsorption capacity and selectivity of the modified carbon microcoils. The chitosan modified carbon microcoils exhibited excellent binding ability (77.72 mg g-1) and satisfactory selectivity. Finally, this material was used in solid phase extraction combined with HPLC to enrich and detect aristolochic acid I from medicinal plants. The detector response for aristolochic acid I was linear from 0.5 to 150 mg L-1, and the recoveries of aristolochic acid I ranged from 73.61 to 77.73% with the relative standard deviations of less than 5%. Thus, chitosan modified carbon microcoils were ideal adsorbents for the selective extraction of aristolochic acid I from Aristolochiaceae plants.

Urotensin II enhances transforming growth factor­ß1 expression and secretion in the kidney during aristolochic acid nephropathy.

Aristolochic acid is a component of many types of Chinese medicine, which are commonly used to treat almost all human diseases. However, aristolochic acid may cause nephropathy. Urotensin II (UII) and transforming growth factor (TGF)­ß1 are important signaling factors, which are expressed at elevated levels during the development of nephropathy. However, the association between UII and TGF­ß1 expression remains unclear. In the current study, the regulatory association between UII and TGF­ß1 expression was investigated using a rat aristolochic acid nephropathy model and the NRK­52E cell line. The expression levels of UII and TGF­ß1 were identified to be constantly increased in the rat aristolochic acid nephropathy model, even 10 days after administration of Aristolochiae manshuriensis decoction was terminated. Notably, increases in the TGF­ß1 expression levels occurred later than those of UII. Furthermore, UII enhanced TGF­ß1 expression in, and secretion from, NRK­52E cells. These data indicate that UII and TGF­ß1 are important in the development of aristolochic acid nephropathy, and UII enhances TGF­ß1 expression levels and secretion during aristolochic acid nephropathy. However, the underlying mechanisms for the precise roles of UII and TGF­ß1 as well as the method by which UII regulates the expression TGF­ß1 in aristolochic acid nephropathy remain to be elucidated in future studies.

Analysis of aristolochic acids, aristololactams and their analogues using liquid chromatography tandem mass spectrometry.

More than 80 aristolochic acids (AAs) and aristololactams (ALs) have been found in plants of the Aristolochiaceae family, but relatively few have been fully studied. The present study aimed at developing and validating a liquid chromatography tandem mass spectrometry (LC/MS(n)) for the analysis of these compounds. We characterized the fragmentation behaviors of 31 AAs, ALs, and their analogues via high performance liquid chromatography coupled with electrospray ionization mass spectrometry. We summarized their fragmentation rules and used these rules to identify the constituents contained in Aristolochia contorta, Ar. debilis, Ar. manshurensis, Ar. fangchi, Ar. cinnabarina, and Ar. mollissima. The AAs and ALs showed very different MS behaviors. In MS(1) of AAs, the characteristic pseudomolecular ions were [M + NH4](+), [M + H](+), and [M + H - H2O](+). However, only [M + H](+) was found in the MS(1) of ALs, which was simpler than that of AAs. Distinct MS(n)fragmentation patterns were found for AAs and ALs, showing the same skeleton among the different substituent groups. The distribution of the 31 constituents in the 6 species of Aristolochia genus was reported for the first time. 25 Analogues of AAs and ALs were detected in this genus. A hierarchical schemes and a calculating formula of the molecular formula of these nitrophenanthrene carboxylic acids and their lactams were proposed. In conclusion, this method could be applied to identification of similar unknown constituents in other plants.

Aristolochic acid I is a substrate of BCRP but not P-glycoprotein or MRP2.

ETHNOPHARMACOLOGICAL RELEVANCE: Aristolochic acid nephropathy is a severe kidney disease caused by the administration of aristolochic acid, which is widely existed in plants of the Aristolochiaceae family. Aristolochic acid I (AAI) is the main toxic component in aristolochic acid. AIM OF THE STUDY: The roles of intestinal efflux drug transporters in the transport of AAI are unclear. This study investigates the interaction between AAI and main intestinal efflux transporters. MATERIALS AND METHODS: Firstly, bidirectional transport of AAI in Caco-2 cell monolayers was investigated. Then, MDCK-MDR1 (gene of P-glycoprotein (P-gp)), MDCK-MRP2 and LLC-PK1-BCRP cell lines were used for further investigation. RESULTS: In this study, we observed that the efflux ratio of AAI in Caco-2 cell monolayers was 5.8, which indicated that efflux transporters might be involved in the transport of AAI. AAI did not inhibit Rho123 efflux by P-gp and calcein efflux by MRP2, and intracellular accumulation of AAI in P-gp or MRP2 overexpressing cells was not different from their parental cells. These results indicated that AAI was not a substrate of P-gp or MRP2. In contrast, intracellular accumulation of AAI in LLC-PK1-BCRP cells was significantly lower than in their parental cells. The presence of GF120918, a BCRP inhibitor, significantly increased AAI accumulation in BCRP overexpressing cells but not in their parental cells. In addition, bidirectional transport assay of AAI in LLC-PK1-BCRP monolayers showed that the net efflux ratios of AAI were 13.8, 8.0 and 7.0 at 20, 40 and 80 µM AAI, respectively, and decreased to 3.0, 1.9 and 2.0 by the addition of 10 µM GF120918. CONCLUSIONS: These results indicated that AAI was a substrate of BCRP but not P-gp or MRP2.

Aza-polycyclic aromatic hydrocarbons from Saruma henryi.

A new azafluoranthene alkaloid, named sarumine (1), along with six known N-containing derivatives of phenanthrenes, 2-7, were isolated from the whole herb of Saruma henryi. Their structures were elucidated on the basis of extensive spectroscopic analysis. Moreover, antimicrobial activities of all compounds were evaluated.

Herbs and hazards: risk of aristolochic acid nephropathy in Iran.

Herbs are usually considered as inherently harmless products. Nonetheless, various renal injuries have been reported in association with several herbs. The best-known herb-induced chronic kidney disease is aristolochic acid nephropathy. Aristolochic acid is found in Chinese slim herbs. Balkan endemic nephropathy is nowadays considered as an aristolochic acid nephropathy. Plants of Aristolochiaceae (also known as birthwort, dutchman's pipe, and somersworth) is named zaravand or chopoghak in Persian and it grows in different mountainous and rural areas of Iran. The fruit and the steam of the Aristolochiacae are named zaravand gerd (nokhod alvand) and zaravand dearaz, respectively, and have different usage in Iranian teadirional such as treatment of headache, back pain, and anxiety. Some patients with end-stage renal disease and bilateral small kidneys have a history of exposure to some herbal remedies. We need to consider the possibility of environmental toxins and even Aristolochia nephrotoxicity as a potential danger in Iran.

Localization of aristolochic acid in mouse kidney tissues by immunohistochemistry using an anti-AA-I and AA-II monoclonal antibody.

Aristolochic acids (AAs) are found in herbal medicines of Aristolochiaceae plants, including Aristolochia and Asarum species. AAs are associated with a rapidly progressive interstitial nephritis, which is called aristolochic acid nephropathy (AAN). However, the in-situ localization of AAs in the target organ, the kidney, has not been investigated yet. In the present study, the accumulation of aristolochic acid I (AA-I) in mouse kidney was revealed by immunoperoxidase light microscopy as well as colloidal gold immunoelectron microscopy (IEM) based on an anti-AA-I and AA-II monoclonal antibody (mAb). Male BALB/c mice were treated with 1.25 or 2.50 mg kg(-1) of AA-I per day for 5 days. Paraffin sections and ultra-thin sections of kidney tissue were respectively prepared. Under light microscopy, the apical surface of proximal tubules was strongly stained for AA-I, whereas no obvious immunostaining was found in the distal tubules and glomerulus, which remained relatively intact. Under electron microscopy, epithelial cells of the proximal tubules, distal tubules and collecting tubules were broken to various degrees. Gold labeling in the proximal and distal tubules was stronger than that in the collecting tubules. In renal tubules, immunogold signals of AA-I tended to accumulate in the mitochondria and peroxisomes, though the signals could be observed all over the cell. Gold signals were also found in the erythrocytes of glomeruli. The MAb against AA-I and AA-II provides a clue for the identification of proteins or factors which might interact with AA-I and thus induce targeted damage of kidney.

Cloud point extraction-HPLC method for the determination and pharmacokinetic study of aristolochic acids in rat plasma after oral administration of Aristolochiae Fructus.

Based on cloud-point extraction (CPE), a high performance liquid chromatography method (HPLC) was developed and validated for the determination of aristolochic acids (AAs) in rat plasma after oral administration of Aristolochiae Fructus (AF). Non-ionic surfactant Genapol X-080, an environmentally friendly solvent, was used for the micelle-mediated extraction. Various influencing factors on CPE process were investigated and optimized. AAs were extracted from rat plasma after adding 1ml of 4.5% (v/v) surfactant in the presence of 0.2mol/l HCl and 20mg NaCl, and the incubation temperature and time were 50°C and 10min, respectively. Base-line separation was obtained for the AAs in rat plasma with the optimized chromatography conditions. The detection limits (LOD) reached downward 10ng/ml. The intra-day and inter-day precisions were less than 7.8%, the accuracies were within ±5.5%, and the average recovery factors were in the range of 94.5-105.4%. In comparison with liquid-liquid extraction, the CPE method has a considerable LOD and higher recoveries. The proposed CPE-HPLC method was specific, sensitive and reliable, and could be an effective tool for the determination of AAs in biological matrixes. With the method the pharmacokinetics of AAs were investigated successfully after oral administration of AF by rats.

Spectroscopic and quantum chemical study of an alkaloid aristolochic acid I.

Aristolochic acids (AAs) (Aristolochiaceae) are used in the traditional Chinese herb medicine. We have presented the geometry optimization, electrostatic potential surface, frontier orbital energy gap and vibrational wavenumbers of aristolochic acid I (AA I) using ab initio Hartree-Fock (HF) and density functional theory (DFT/B3LYP) method employing 6-311G(d,p) basis set. A complete vibrational assignment has been done on the basis of calculations on monomer and dimer of AA I. The UV-vis absorption spectrum has been recorded in ethanol solvent and compared with the calculated one in the gas phase as well as in solvent environment (integral-equation formalism polarizable continuum model; IEF-PCM) using TD-DFT/6-31G basis set. A short outline of the NBO analysis segment with their structural meaning has been presented. The variation of thermodynamic properties with temperature was calculated theoretically and the thermal response of the compound has been recorded with the help of differential scanning calorimetry (DSC) in N2 environment.

Association of blood lead and mercury with estimated GFR in herbalists after the ban of herbs containing aristolochic acids in Taiwan.

OBJECTIVE: This study was undertaken to explore the association of estimated glomerular filtration rate (GFR) with exposure to aristolochic acids (ALAs) and nephrotoxic metals in herbalists after the ban of herbs containing ALAs in Taiwan. METHODS: This cross-sectional study recruited a total of 138 herbalists without end-stage renal disease or urothelial carcinoma from the Occupational Union of Chinese Herbalists in Taiwan in 2007. Aristolochic acid I (ALA-I) was measured by ultra-high-pressure liquid chromatography/ tandem mass spectrometry (UHPLC-MS/MS) and heavy metals in blood samples were analysed by Agilent 7500C inductively coupled plasma-mass spectrometry. Renal function was assessed by using a simplified Modification of Diet in Renal Disease Study equation to estimate GFR. RESULTS: Blood lead was higher in herbal dispensing procedures (p=0.053) and in subjects who self-prescribe herbal medicine (p=0.057); mercury was also higher in subjects living in the workplace (p=0.03). Lower estimated GFR was significantly associated with lead (ß=-10.66, 95% CI -18.7 to -2.6) and mercury (ß=-12.52, 95% CI -24.3 to -0.8) with a significant interaction (p=0.01) between mercury and lead; however, estimated GFR was not significantly associated with high ALA-I level groups, arsenic and cadmium after adjusting for other confounding factors. CONCLUSIONS: We found that lower estimated GFR was associated with blood lead and mercury in herbalists after the ban of herbs containing ALAs in Taiwan. The ALA-I exposure did not show a significant negative association of estimated GFR, which might due to herbalists having known how to distinguish ALA herbs after the banning policy. Rigorous monitoring is still needed to protect herbalists and the general population who take herbs.

Evaluation of the cytotoxicity and genotoxicity of aristolochic acid I - a component of Aristolochiaceae plant extracts used in homeopathy.

The medicinal plants Aristolochia clematitis L. as well as Asarum europaeum L., representatives of the plant family Aristolochiaceae and mentioned in the German Homeopathic Pharmacopeia, contain aristolochic acid. We found that the mother tinctures of A. clematitis and A. europaeum inhibited DNA synthesis in human hepatoma HepG2 cells in a dose-dependent manner. One of the components of the plant extract, aristolochic acid I (AAI), is linked to the development of nephropathy and urothelial cancer in humans. Therefore, we also evaluated the cytotoxicity and genotoxicity of AAI in HepG2 cells. Cell proliferation was inhibited concentration-dependently by AAI using BrdU-ELISA and colony forming assay. AAI formed DNA adducts (measured by (32)P-postlabeling), induced chromosomal aberrations (micronuclei) and DNA strand breaks. DNA damage induced by AAI led to an arrest of cells in the S-phase which was associated with the increased expression of p53 and p21 proteins. The results are discussed under consideration of former studies.

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-C(18) column with a gradient mobile phase comprising acetonitrile and 1 % acetic acid-30 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.

4,5-seco-guaiane and a nine-membered sesquiterpene lactone from Holostylis reniformis.

Root extracts of Holostylis reniformis (Aristolochiaceae) yielded three new natural sesquiterpenes, a sesquiterpene with an unusual carbon skeleton, 4,5-seco-guaiane (7-epi-11-hydroxychabrolidione A, 1), a nine-membered lactone with new carbon skeleton (holostylactone, 2), and a new megastigmane [(6S,7E)-6,9-dihydroxy-10-(2'-hydroxy-ethoxy)-4,7-megastigmadien-3-one, 3], together with bulnesol and sitosterol-3-O-β-D-glucopyranoside. The structures of these compounds were determined by spectroscopic analyses and B3LYP/STO-3G** theoretical studies.

Evaluation of microwave-assisted extraction for aristolochic acid from Aristolochiae Fructus by chromatographic analysis coupled with nephrotoxicity studies.

In this paper, a microwave-assisted extraction (MAE) method was established for aristolochic acid-I from Aristolochiae Fructus, and the advantage of MAE was evaluated by chromatographic analysis coupled with nephrotoxicity studies. The experimental parameters of MAE for aristolochic acid-I in Aristolochiae Fructus were investigated and MAE was compared with Soxhlet extraction and ultrasound-assisted extraction in terms of extraction yields and extraction conditions. Under the optimum conditions, MAE could provide higher extraction yields of aristolochic acid-I (1.10 mg/g) than ultrasound-assisted extraction (0.82 mg/g) and Soxhlet extraction (0.95 mg/g), in addition to using less solvent and having a shorter extraction time. Furthermore, the nephrotoxicities of the extracts of Aristolochiae Fructus from different extraction procedures were investigated in Sprague-Dawley rats. The results of nephrotoxicity studies of, for example, general conditions, biochemistry parameters and histopathology examination showed no significantly differences in the nephrotoxicity levels of the extracts from MAE and that from Soxhlet extraction. These results indicated that MAE technique is a simple, rapid and effective extraction method, and the microwave irradiation during MAE procedure did not have any influence on the nephrotoxicity of Aristolochiae Fructus compared with Soxhlet extraction.

Asiasari sieboldii suppresses inflammatory mediators through the induction of hemeoxygenase-1 expression in RAW264.7 cells.

OBJECTIVE: Asiasari sieboldii is widely used in Korean traditional medicine. In this study, we examined the anti-inflammatory effects of A. sieboldii ethanolic extract (ASEE) in a lipopolysaccharide (LPS)-induced murine macrophage RAW264.7 cell, and then sought to understand the mechanism(s) underlying the observed effects. MATERIALS AND METHODS: The production levels of nitrite oxide (NO), prostaglandin E2 (PGE2) and cytokines were measured using the Griess reagent and enzyme-linked immunosorbent assays (ELISA), while the cell protein expression levels were monitored by Western blot analysis. RESULTS: Our results revealed that ASEE had prominent inhibitory effects on NO, PGE2, interleukin (IL)-6 and tumor necrosis factor (TNF)-α production, as well as the expression of inducible nitrite oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in LPS-induced RAW264.7 cells. Mechanistically, ASEE upregulated the expression of hemeoxygenase-1 (HO-1), and inhibited the nuclear translocation of nuclear factor (NF)-κB by preventing degradation of inhibitor κB-α (IκB-α). CONCLUSION: These results indicate that the anti-inflammatory activity of ASEE occurs at least partially through the induction of HO-1 and subsequent suppression of the NF-κB pathway.

5-methoxyaristololactam I, the first natural 5-substituted aristololactam from Asarum ichangense.

A new aristololactam, 5-methoxyaristololactam I (1), was isolated from an ethanol extract of the whole plant of Asarum ichangense C. Y. Cheng et C. S. Yang, together with twenty known compounds (2-21). The structure of 1 was elucidated by spectroscopic methods. Compounds 11, 13, 17, and 19 were isolated from Aristolochiaceae for the first time. Compounds 2, 14, and 15 are considered as common constituents of the genus Asarum.

Subacute toxicological evaluation of Asiasari radix methanol extract.

Asiasari radix, a traditional herbal medicine commonly used to treat various diseases, currently has a lack of information about adverse effects. Safety information of A. radix and its extract is limited to its historical use. The safety of A. radix methanol extract was tested in an oral subacute 28-day toxicity study in both male and female Sprague-Dawley (SD) rats at doses of 50, 250, and 500 mg/kg/day. No mortality and significant signs of toxicity were observed in either the control or treated groups of both sexes. There were no significant differences in the body and organ weights or in food and water consumption. Hematological and biochemical parameters showed no changes in either the control or treated groups of both sexes. Pathologically, neither gross abnormalities nor histopathological changes were observed. Therefore, methanolic extract of A. radix appears to be safe and nontoxic in these studies, and a no observed adverse effect level in rats is established at 500 mg/kg/day, the highest dose tested.