Inhibition of Naja naja venom hyaluronidase: role in the management of poisonous bite

Hyaluronidase is present virtually in all snake venoms and has been known as a "spreading factor." The enzyme damages the extracellular matrix at the site of the bite, leading to severe morbidity. In this study, the benefits of inhibiting the hyaluronidase activity of Indian cobra (Naja naja) venom have been investigated. Anti-NNH1 and aristolochic acid both inhibited the in vitro activity of the purified hyaluronidase, (NNH1) and the hyaluronidase activity of whole venom in a dose-dependent manner. Both anti-NNH1 and aristolochic acid abolished the degradation of hyaluronan in human skin tissue sections by NNH1 and by whole venom. Aristolochic acid quenched the fluorescent emission of NNH1. A non-competitive mechanism of NNH1 inhibition was observed with aristolochic acid. NNH1 potentiates the toxicity of Daboia russellii VRV-PL-VIII myotoxin and hemorrhagic complex-I. However, the potentiation of toxicity was inhibited dose-dependently by anti-NNH1 and aristolochic acid. Further, mice injected with whole venom which had been preincubated with anti-NNH1/aristolochic acid, showed more than a two-fold increase in survival time, compared to mice injected with venom alone. A more moderate increase in survival time was observed when mice were injected with anti-NNH1/aristolochic acid 10 min after whole venom injection. This study illustrates the significance of venom hyaluronidase in the pathophysiology of snake venom poisoning and the therapeutic value of its inhibition.     

The effects of the phospholipase A2 inhibitors aristolochic acid and PGBx on A23187-stimulated mobilization of arachidonate in human neutrophils are overcome by diacylglycerol or phorbol ester.

Aristolochic acid and PGBx, two structurally unrelated, protein-targeted inhibitors of isolated phospholipases A2, are effective antagonists of calcium ionophore A23187-stimulated mobilization of [3H]arachidonate from human neutrophils. We now report that preincubation of neutrophils with oleoylacetylglycerol (OAG, 15 microM) substantially reverses the inhibitory effect of 200 microM aristolochic acid (from 70 to 24% inhibition). Similarly, OAG increases the IC50 for PGBx from 2.5 to greater than 20 microM. The effects of OAG on inhibition by either aristolochic acid or PGBx are dose-dependent, with an ED50 of 2.5 microM. Protection against inhibition by either aristolochic acid or PGBx is also observed with phorbol myristate acetate (PMA, ED50 3 nM), but not 4-alpha-phorbol didecanoate. Aristolochic acid and PGBx do not inhibit PMA-stimulated superoxide generation, and are thus not protein kinase C inhibitors. Furthermore, neither aristolochic acid nor PGBx inhibit diglyceride generation through the phospholipase D/phosphatidate phosphohydrolase pathway. A23187-stimulated [3H]arachidonate mobilization is increased by 20-50% when neutrophils are preincubated with OAG or PMA. The present results indicate that OAG and PMA also modulate the A23187-stimulated [3H]arachidonate mobilization so as to render it less sensitive to inhibitors of phospholipase A2.     

Inhibition of <Emphasis Type="Italic">Naja naja</Emphasis> Venom Hyaluronidase by Plant-Derived Bioactive Components and Polysaccharides

The inhibitory effect of several bioactive compounds on the activity of hyaluronidase enzyme purified from Naja naja venom was investigated in vitro. Compounds were found to inhibit the hyaluronidase activity dose dependently. Among glycosaminoglycans, heparin, heparan sulfate, and dermatan sulfate showed maximum inhibition compared to chondroitin sulfates. Different molecular forms of chitosan inhibit the enzyme, and inhibition appears to depend on the chain length. In addition, plant-derived bioactive compounds also inhibited the activity of hyaluronidase dose dependently. Among those tested, aristolochic acid, indomethacin, quercetin, curcumin, tannic acid, and flavone exhibited inhibition, with aristolochic acid and quercetin completely inhibiting the enzyme activity. It is concluded that the inhibitors of hyaluronidase could be used as potent first aid agents in snakebite therapy. Furthermore, these inhibitors not only reduce the local tissue damage but also retard the easy diffusion of systemic toxins and hence increase survival time.     

高效液相色谱法测定马蹄香中马兜铃酸A含量

用高效液相分析方法,测定了马蹄香植物体不同部位中马兜铃酸A的含量。结果表明,马兜铃酸A主要存在于马蹄香的地下根及根茎部分,地上茎中含量极少,叶中不含马兜铃酸A。在根及根茎中马兜铃酸A的含量为0.165%～0.198%,茎中含量为0.012%～0.023%。与马兜铃科其他植物比较,马蹄香植物体中马兜铃酸A的含量较高。     

马兜铃酸与钙调素相互作用的荧光光谱分析——一种非钙离子依赖性钙调素拮抗剂的研究

本文报道我室近来发现的一种天然钙调素(Calmodulin,CaM)拮抗剂马兜铃酸(Aristolochic acid,ATA)的研究。利用丹磺酰标记的CaM(D-CaM)对马兜铃酸的研究表明,马兜铃酸是一种非钙离子依赖性钙调素拮抗剂,实验测得马兜铃酸与D-CaM结合的解离常数,有Ca~(2+)和无Ca~(2+)情况下分别为70μmol/L、77μmol/L。两种状况下马兜铃酸对D-CaM荧光强度的抑制分别为40％、41％。暗示马兜铃酸主要作用于CaM上Ca~(2+)诱导的疏水区之外。三氟啦嗪(TFP)引起的D-CaM荧光增强可被马兜铃酸明显降低,而TFP在达到马兜铃酸浓度的15倍以上仍未能逆转马兜铃酸对D-CaM荧光强度的降低作用,这为马兜铃酸主要作用于CaM上Ca~(2+)诱导的疏水区以外提供了又一佐证。     

Phytochemical and biological investigation of Aristolochia maurorum L

Aristolochia maurorum L. of Jordanian origin has been investigated phytochemically, quantitatively, and biologically. Three atypical alkaloids, namely aristolochic acid I (1), aristolochic acid II (2) and aristolochic acid IIIa (3), have been isolated and identified. Of these known 1-phenanthrenecarboxylic acids, 2 and 3 are reported for the first time from this species. The identified compounds 1-3 were first evaluated biologically as cytotoxic agents against the brine shrimp lethality test (BST), in which compound 1 was found to be the most potent (LC50, 4.9 microg/mL). The antiplatelet activity of the methanolic extracts, the acidic fractions of aerial and root parts, and the identified compounds 1-3 were evaluated using an automatic platelet aggregometer and coagulation tracer (APACT 2). Using external reference standards, and a reverse-phase isocratic method, the distribution of aristolochic acid I and aristolochic acid II in different plant parts of Aristolochia maurorum L. during flowering stage was analyzed by PDA-HPLC. A quantitative comparison between two previously reported extraction methods was also made. Roots were found to be the main storage of aristolochic acid I and aristolochic acid II during flowering stage with about 0.22 and 0.108% (w/w), respectively.     

Induction of structural chromosome aberrations and sister chromatid exchanges in human lymphocytes in vitro by aristolochic acid

The medicinal use of Aristolochia clematitis has been known for some time. The main active agent of this medicinal plant is aristolochic acid, a nitrophenanthrenecarbonic acid. Very recently, however, the Federal Health Office withdrew the licence for all drugs containing aristolochic acid, because of the well-founded suspicion that aristolochic acid may be a very potent carcinogen. We investigated the induction of structural chromosome aberrations and sister chromatid exchanges (SCEs) by aristolochic acid in human lymphocytes in vitro. Cells were treated with the agent tested throughout culture time and during the G0 phase of the cell cycle. We tested concentrations over a range of 1 to 20 micrograms/ml. Both treatment conditions resulted in an increased aberration frequency. The induction of gaps and breaks as well as the induction of SCEs showed a dose-dependent increase. The number of SCEs per metaphase was enhanced by a factor of 2 to 3. If conventional cytogenetic methods had been applied in time, one would have recognized the mutagenic risk of aristolochic acid earlier.     

Phenanthrene derivatives from Aristolochia argentina

Aristolochic acid Ia, aristolochic acid I methyl ester and aristolochic acid II methyl ester were identified in the roots of Aristolochia argentina.     

竹叶马兜铃化学成分的研究

<正> 竹叶马兜铃Aristolochia bambusifolia.C.F Liang sp.nov.是最近在广西隆林首次发现的马兜铃科马兜铃属植物新种。该属植物大部分为药用或民间有名草药。文献曾报导广西马兜铃属植物有12种,但无此种,民间亦未使用。其化学成分尚未研究过,因此我们对其块根化学成分进行系统研究是很有意义的。     

The effects of qualitative and quantitative variation of aristolochic acids on preference and performance of a generalist herbivore

Nearly all plants possess chemicals that are inferred to play a role in anti‐herbivore defense or resistance. The effects of various chemical defenses can vary among herbivores. Often, plant defensive compounds are examined in broad, inclusive categories, with an emphasis on total quantity, which might ignore qualitative variation in activity. Aristolochic acids are alkaloids characteristic of plants of the genus Aristolochia (Aristolochiaceae). Although aristolochic acids have been documented as effective herbivore deterrents, it remains unknown whether different kinds of aristolochic acid vary in their efficacy as defense against herbivores. We manipulated the aristolochic acid content of artificial diet to examine the effects of four aristolochic acids on larval preference and performance of the generalist herbivore Spodoptera exigua Hübner (Lepidoptera: Noctuidae). Using choice tests, we observed that the four aristolochic acids tested varied in their deterrent effectiveness, with AA‐I having the strongest effect and AA‐II having the weakest effect. No‐choice tests were used to examine larval performance. The effect on performance varied among the aristolochic acids tested. Higher concentrations of aristolochic acid were generally associated with reduced larval developmental rate and larger size at pupation. These results indicate that various forms of aristolochic acid can vary in their effect on herbivores and that simple aggregate measures of total concentration might not reflect the chemical defensive phenotype of the plant.     

Arachidonic acid causes cell death through the mitochondrial permeability transition. Implications for tumor necrosis factor-alpha aopototic signaling

We have investigated the effects of arachidonic and palmitic acids in isolated rat liver mitochondria and in rat hepatoma MH1C1 cells. We show that both compounds induce the mitochondrial permeability transition (PT). At variance from palmitic acid, however, arachidonic acid causes a PT at concentrations that do not cause PT-independent depolarization or respiratory inhibition, suggesting a specific effect on the PT pore. When added to intact MH1C1 cells, arachidonic acid but not palmitic acid caused a mitochondrial PT in situ that was accompanied by cytochrome c release and rapidly followed by cell death. All these effects of arachidonic acid could be prevented by cyclosporin A but not by the phospholipase A(2) inhibitor aristolochic acid. In contrast, tumor necrosis factor alpha caused phospholipid hydrolysis, induction of the PT, cytochrome c release, and cell death that could be inhibited by both cyclosporin A and aristolochic acid. These findings suggest that arachidonic acid produced by cytosolic phospholipase A(2) may be a mediator of tumor necrosis factor alpha cytotoxicity in situ through induction of the mitochondrial PT.     

High-performance liquid chromatographic determination for aristolochic acid in medicinal plants and slimming products

A HPLC procedure with a silica gel RP-18 reversed-phase column for the determination of aristolochic acids I, II in medicinal plants and slimming products was developed. The mobile system 0.3% ammonium carbonate solution-acetonitrile (75:25, v/v) with pH 7.5 was the optimal buffer to clearly separate aristolochic acids I, II within 20 min. The recovery of aristolochic acids I, II in medicinal plants and slimming products was better than 90% by extracting with methanol and purifying through a PHP-LH-20 column. The major component was aristolochic acid I in Aristolochia fangchi and the level ranged from 437 to 668 ppm. Aristolochic acid II was the major component for Aristolochia contorta and its range was <1-115 ppm. Twelve out of 16 samples of slimming pills and powders contained aristolochic acids I and/or II. The major component in most slimming products was aristolochic acid II and the level ranged from <1 to 148 ppm. It may indicate that slimming products were not mainly made of A. fangchi.     

Antimycobacterial,antibacterial and antifungal activities of the methanol extract and compounds from Thecacoris annobonae (Euphorbiaceae)

This study was designed to evaluate the antimycobacterial, antibacterial and antifungal activities of the methanol extract from the stem bark of Thecacoris annobonae Pax & K. Hoffm, that of aristolochic acid I (1) and other isolated compounds. The microplate alamar blue assay (MABA) and the broth microdilution method were used to determine the minimal inhibitory concentration (MIC) and minimal microbicidal concentration (MMC) of the above samples. The H⁺-ATPase-mediated proton pumping assay was used to evaluate a possible mechanism of action for both the methanol extract and aristolochic acid I. The results of the MIC determinations showed that the methanol extract and aristolochic acid I prevent the growth of all studied organisms. The results obtained in this study also showed that the methanol extract as well as aristolochic acid I inhibited the H⁺-ATPase activity. The overall results provided evidence that the methanol extract of T. annobonae might be a potential source of new antimicrobial drug against tuberculosis, and some bacterial and fungal diseases, but should be consumed with caution, bearing in mind that the main active component, aristolochic acid I is a potentially toxic compound.     

Inhibition of aquaporin-1 expression by RNAi protects against aristolochic acid I-induced apoptosis in human proximal tubular epithelial (HK-2) cells

The aim of this study was to investigate the protective effect of inhibition of aquaporin-1 (AQP1) expression against aristolochic acid I (AA-I)-induced apoptosis. HK-2 cells impaired by AA-I were used in this study as the cell model of aristolochic acid nephropathy. Apoptosis was studied by different methods, including 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assays, flow cytometry, and caspase 3 activity assays. We compared AA-I-mediated apoptosis in HK-2 cells with or without knockdown of AQP1 expression by RNA interference. MTT assays showed that AA-I inhibited the viability of HK-2 cells in a time- and concentration-dependent manner. Apoptosis was evidenced by the results of the Annexin V/propidium iodide assay and the occurrence of a sub-G1 peak in cell-cycle analysis. The activity of caspase 3 was found to have been increased by AA-I in a concentration-dependent manner. However, AQP1 RNA interference provided protection against injury in cells treated with AA-I (40 μM) for 24 h and attenuated the number of apoptotic cells. These results suggested that AQP1 plays an important role in AA-I-induced apoptosis and that inhibition of AQP1 expression may protect HK-2 cells from AA-I-induced apoptotic damage.     

Competitive inhibition of phospholipase A2 in vesicles

Kinetic studies with phospholipase A2 are complicated by the fact that binding of the enzyme to the interface precedes catalytic turnover. This difficulty can be overcome by monitoring interfacial catalysis in the scooting mode where the enzyme does not leave the interface. The kinetics of inhibition by transition-state analogues shows that specific competitive inhibition is the result of competition between inhibitor and substrate for the binding to the active site of the enzyme in the interface. Several lipophilic compounds, including alkanols, substituted butyrophenones, aristolochic acid, and mepacrine apparently reduce the rate of lipolysis by promoting the desorption of phospholipase A2 from the interface.     

Family matters: effect of host plant variation in chemical and mechanical defenses on a sequestering specialist herbivore

Insect herbivores contend with various plant traits that are presumed to function as feeding deterrents. Paradoxically, some specialist insect herbivores might benefit from some of these plant traits, for example by sequestering plant chemical defenses that herbivores then use as their own defense against natural enemies. Larvae of the butterfly species Battus philenor (L.) (Papilionidae) sequester toxic alkaloids (aristolochic acids) from their Aristolochia host plants, rendering larvae and adults unpalatable to a broad range of predators. We studied the importance of two putative defensive traits in Aristolochia erecta: leaf toughness and aristolochic acid content, and we examined the effect of intra- and interplant chemical variation on the chemical phenotype of B. philenor larvae. It has been proposed that genetic variation for sequestration ability is ??invisible to natural selection?? because intra- and interindividual variation in host-plant chemistry will largely eliminate a role for herbivore genetic variation in determining an herbivore??s chemical phenotype. We found substantial intra- and interplant variation in leaf toughness and in the aristolochic acid chemistry in A. erecta. Based on field observations and laboratory experiments, we showed that first-instar larvae preferentially fed on less tough, younger leaves and avoided tougher, older leaves, and we found no evidence that aristolochic acid content influenced first-instar larval foraging. We found that the majority of variation in the amount of aristolochic acid sequestered by larvae was explained by larval family, not by host-plant aristolochic acid content. Heritable variation for sequestration is the predominant determinant of larval, and likely adult, chemical phenotype. This study shows that for these highly specialized herbivores that sequester chemical defenses, traits that offer mechanical resistance, such as leaf toughness, might be more important determinants of early-instar larval foraging behavior and development compared to plant chemical defenses.     

Aristoloside,an aristolochic acid derivative from stems of Aristolochia manshuriensis

Aristoloside, a new companion aristolochic acid derivative isolated from stems of Aristolochia manshuriensis has been shown to be 6-O-β-d-glucopyranoside of aristolochic acid-D on chemical and physicochemical evidence. Three known acids, aristolochic acids I, IV (both as their corresponding methyl esters), and -D have also been characterized from stems of the plant.     

Antagonistic, stage-specific selection on defensive chemical sequestration in a toxic butterfly

Larvae of the pipevine swallowtail ( Battus philenor ) sequester toxic alkaloids called aristolochic acids from their Aristolochia host plants, rendering both larvae and adults chemically defended against most predators. Using a chemically controlled artificial diet, we observed substantial among-family variation in sequestration ability and larval developmental rate in a population occurring in central Texas. Early instar larvae from families that sequester greater amounts of aristolochic acid showed increased survivorship in a field experiment in which cohorts from each family were exposed to natural predators, whereas among-family variation in growth rate did not predict survivorship. Conversely, the aristolochic acid content of adult butterflies was negatively correlated with adult fat content, a fitness correlate. Sequestration ability positively affects the probability of larval survivorship, but at the cost of adult fat content. The costs and benefits of aristolochic acid sequestration vary during the course of the butterfly's development, and these antagonistic selection pressures may explain why variation in sequestration ability persists in wild populations.     

Constituents of Aristolochia cinnabarina

Nine compounds, aristolochic acid (1), aristolochic acid- Ⅱ (2), aristolochic acid- Ⅲ a (3), aristoloside (4), aristolactam-N-β-D-glucoside (5), aristolactam-β-D-glucoside (6), tuberosinone (7), tuberosinone N-β-D-glucoside (8) and 6-O-p-coumaroyl-D-glucopyranose (9), were isolated from methanol extracts of fresh roots of Aristolochia cinnabarina. Among them, compound 9 was reported in Aristolochiaceae for the first time.     

Essential fatty acid deficiency impairs macrophage spreading and adherence. Role of arachidonate in cell adhesion

Dietary polyunsaturated fatty acid manipulation exerts a strikingly protective effect in models of tissue inflammation and injury. A critical element of this effect appears to revolve around leukocyte trafficking but underlying mechanisms are ill understood. In the current study it was observed that essential fatty acid (EFA) deficiency markedly impaired the capacity of resident macrophages to spread and adhere. This effect was not a simple function of the alteration of membrane fatty acid composition. Elicited EFA-deficient macrophages were equally adherent to elicited control cells, despite the fact that they were equally EFA-deficient relative to resident EFA-deficient cells. With respect to the mechanism underlying defective macrophage adherence in EFA deficiency, no change in the expression of cell surface adherence molecules (Fc receptor, Mac-1, or LFA-1) was noted with the deficiency state. Also, an adherence defect could not be induced in normal cells pharmacologically with cyclooxygenase blockade, lipoxygenase blockade, or a platelet-activating factor receptor antagonist. In contrast, phospholipase inhibition was able to induce a spreading and adherence defect in resident macrophages similar to that seen with EFA deficiency. Using several phospholipase inhibitors, a correlation between phospholipase inhibition and impairment of adherence was observed. Adding back exogenous fatty acids to cells after phospholipase inhibition demonstrated that normal adherence was reconstituted with arachidonate. This alteration in macrophage spreading and adherence with EFA deficiency may be an important component of the anti-inflammatory effect of dietary polyunsaturated fatty acid manipulation. Additionally, these results suggest that arachidonate may be an intracellular mediator of leukocyte adherence.