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.

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.

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 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.

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.

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.

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.

Constituents of Asarum sieboldii with inhibitory activity on lipopolysaccharide (LPS)-induced NO production in BV-2 microglial cells.

Bioassay-guided fractionation of the root extract of Asarum sieboldii led to the isolation of the four active compounds (-)-sesamin (1), (2E,4E,8Z,10E)-N-(2-methylpropyl)dodeca-2,4,8,10-tetraenamide (2), kakuol (3), and '3,4,5-trimethoxytoluene' (=1,2,3-trimethoxy-5-methylbenzene; 4), in terms of inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) production. Compounds 1-4 showed potent inhibition of NO production, with IC(50) values in the low nanomolar-to-micromolar range. Also isolated were the known compounds methylkakuol (5), '3,5-dimethoxytoluene', safrole, asaricin, methyleugenol, and (-)-asarinin, which were found to be inactive in the above assay. Among the ten known isolates, compounds 1, 2, and 5 were found for the first time in this plant.

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.

Aryltetralol and aryltetralone lignans from Holostylis reniformis.

Aryltetralol and aryltetralone lignans were isolated from the hexane extracts of the roots of Holostylis reniformis. Their structures were determined by spectroscopic methods and chemical transformations.

Subchronic toxicity studies of the aqueous extract of Aristolochiae fructus in Sprague-Dawley rats.

The subchronic toxicity of Aristolochiae fructus containing aristolochic acids (AAs), a natural component in the Aristolochiaceae family, was investigated. The A. fructus was daily administered by gavage to male and female rats for 90 d at dose levels of 21.35, 213.5, and 2135 mg/kg (equivalent to 0.05, 0.5, and 5 mg/kg as AAs, respectively). During the test period, clinical signs, mortality, body weights, food and water consumption, hematology, serum biochemistry, organ weights, and histopathology were examined. Significant decreases in body weight gain were noted in the high-dose group receiving both the aqueous extract of A. fructus and AAs. Decreases in food consumption were noted beginning at 50 d and did not recover in the high-dose group of aqueous extract of A. fructus and AAs. Irrespective of dose, water consumption was not affected. There was no mortality or adverse clinical signs, hematology, or serum biochemistry in the treatment groups versus control. Nephrotoxicity and hyperplasia of epithelial cells in the forestomach were observed in rats receiving the highest dose of aqueous extract of A. fructus and at doses of >or= 0.5 mg/kg/day AAs. For both genders, the no-observed-adverse-effect level (NOAEL) for A. fructus based on this subchronic study in rats was considered to be 21.3 mg/kg/d.

Suppression of MeIQ-induced SOS response by allylbenzenes from Asiasarum heterotropoides in the Salmonella typhimurium OY1001/1A2 umu test.

Three allylbenzenes from Asiasarum heterotropoides, methyleugenol (1), elemicin (2) and gamma-asaron (3) showed suppressive effects on umu gene expression of the SOS response in the Salmonella typhimurium OY1001/1A2 umu test against the mutagen 2-amino-3,4-dimethylimidazo[4,5-f ]quinoline (MeIQ). Gene expression was suppressed 70.0, 75.9 and 81.7% at a concentration of 0.4 mM, respectively. The ID50 values (50% inhibition dose) of these compounds were 0.125, 0.098 and 0.059 mM, respectively. On the other hand, compounds 1-3 showed weak suppressive effects of the SOS-inducing activity on activated MeIQ.

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.

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.

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.

Aryltetralone lignans and 7,8-seco-lignans from Holostylis reniformis.

Aryltetralone lignans and two 7,8-seco-lignans were isolated from the acetone and hexane extracts of the roots of Holostylis reniformis, together with (-)-galbacin. Their structures were determined by spectroscopic methods.

Volatile constituents of Aristolochia argentina.

The essential oils from leaves, aerial stems and underground organs of Aristolochia argentina Gris., a medicinal plant popularly known as "charrúa", were obtained by hydrodistillation and analyzed by GC and GC/MS. Forty-three components were identified in the oils. All parts of the plant afforded volatile oils characterized by high levels of argentilactone (57-89%) and the presence of undecatriene isomers (0.3-4.0%), these latter compounds providing the essential oils and extracts with an intense particular odor. Terpenes account for the remaining portion of the essential oils (5-29%). Bicyclogermacrene predominates in the aerial parts of the plant, whereas ishwarane is the main terpene of the subterranean organs. Argentilactone, a suspected carcinogenic compound, was also identified in a medicinal commercial tincture of A. argentina.