[Discussion on hepatic damage mechanism of Asari Radix et Rhizoma based on network pharmacology and untargeted metabolomics].

Asari Radix et Rhizoma is a common drug for relieving exterior syndrome in clinics, but its toxicity limits its use. In this study, the mechanism of hepatic damage of Asari Radix et Rhizoma was studied by network pharmacology and metabolomics. The hepatic damage-related dataset, namely GSE54257 was downloaded from the GEO database. The Limma package was used to analyze the differentially expressed genes in the dataset GSE54257. Toxic components and target genes of Asari Radix et Rhizoma were screened by TCMSP, ECTM, and TOXNET. The hepatic damage target genes of Asari Radix et Rhizoma were obtained by mapping with the differentially expressed gene of GSE54257, and a PPI network was constructed. GO and KEGG enrichment analysis of target genes were performed, and a "miRNA-target gene-signal pathway" network was drawn with upstream miRNA information. Thirty rats were divided into a blank group, a high-dose Asari Radix et Rhizoma group, and a low-dose Asari Radix et Rhizoma group, which were administered once a day. After continuous administration for 28 days, liver function indexes and liver pathological changes were detected. Five liver tissue samples were randomly collected from the blank group and high-dose Asari Radix et Rhizoma group, and small molecule metabolites were analyzed by ultra-high performance liquid chromatography-mass spectrometry(UHPLC-MS). The orthogonal partial least squares-discriminant analysis(OPLS-DA) method was used to screen differential metabolites, and enrichment analysis, correlation analysis, and cluster analysis were conducted for differential metabolites. Finally, the MetaboAnalyst platform was used to conduct pathway enrichment analysis for differential metabolites. It was found that there were 14 toxic components in Asari Radix et Rhizoma, corresponding to 37 target genes, and 12 genes related to liver toxicity of Asari Radix et Rhizoma were obtained by mapping to differentially expressed genes of GSE54257. The animal test results showed that Asari Radix et Rhizoma could significantly increase the liver function index, reduce the activity of the free radical scavenging enzyme, change the liver oxidative stress level, and induce lipid peroxidation damage in rats. The results of untargeted metabolomics analysis showed that compared with the blank group, nine metabolites were up-regulated, and 16 metabolites were down-regulated in the liver tissue of the Asari Radix et Rhizoma group. These 25 metabolites had strong correlations and good clustering. Pathway enrichment analysis showed that these differential metabolites and the 12 hepatotoxic target genes of Asari Radix et Rhizoma were mainly involved in purine metabolism, as well as the biosynthesis and metabolism of valine, leucine, glycine, serine, and threonine. The study confirmed that the hepatica damage effect of Asari Radix et Rhizoma was the result of multi-component, multi-target, and multi-signaling pathways, and its mechanism may be related to inhibiting nucleotide synthesis and affecting protein metabolism.

[Isolation and functional characterization of (iso)eugenol O-methyltransferase (IEMT) gene in Asarum sieboldii].

Methyleugenol is one of the main active constituents in the volatile oil of the traditional Chinese medicine Asari Radix et Rhizoma. It possesses various pharmacological effects such as analgesic, anesthetic, and anti-inflammatory properties. In biosynthesis, the initial precursor phenylalanine is finally converted into methyleugenol through a series of intermediate compounds including coniferyl acid, courmaryl acid, caffeic acid, ferulic acid/ferulic-CoA, coniferyl aldehyde, conferyl alcohol, cnfiferyl acetate, and eugenol/isoeugenol, which are produced through catalysis of a large number of enzymes. Eugenol O-methyltransferase(EOMT) is one of the key enzymes in the biosynthesis pathway, capable of methylating eugenol on the para-site hydroxyl group of the benzene ring, thereby generating methyleugenol. Here, an(iso)eugenol O-methyltransferase(IEMT) gene was cloned for the first time from Asarum siebo-ldii, holding an open reading frame that consisted of 1 113 bp and encoded a protein containing 370 amino acid residues. Bioinformatics analysis results showed that this protein was equipped with the characteristic structural domains of methyltransferases such as S-adenosylmethionine(SAM) binding sites and dimerization domains. The prokaryotic expression recombinant plasmid pET28a(+)-AsIEMT was constructed, and the candidate protein was induced and purified. In vitro enzyme assays confirmed that AsIEMT had dual functions. The enzyme could catalyze the production either of methyleugenol from eugenol or of methylisoeugenol from isoeugenol, although the latter was more prevalent. When isoeugenol was used as the substrate, the kinetics parameters K_m and V_(max) of catalytic reaction were(0.90±0.06) mmol·L~(-1) and(1.32±0.04)nmol·s~(-1)·mg~(-1), respectively. This study expanded our understandings of critical enzyme genes involved in phenylpropanoid metabolic pathways, and would facilitate the elucidation of quality formation mechanisms of the TCM Asari Radix et Rhizoma.

Long-term oral administration of Kelisha capsule does not cause hepatorenal toxicity in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Kelisha capsules (KLS) are often used to treat acute diarrhoea, bacillary dysentery, heat stroke, and other diseases. One of its components, Asarum, contains aristolochic acid I which is both nephrotoxic and carcinogenic. However, the aristolochic acid (AA) content in KLS and its toxicity remain unclear. AIM OF THE STUDY: The aims of this study were to quantitatively determine the contents of five aristolochic acid analogues (AAAs) in Asarum and KLS, and systematically evaluate the in vivo toxicity of KLS in rats. MATERIALS AND METHODS: Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to determine the content of the five AAAs in Asarum and KLS. Sprague-Dawley rats were administered KLS at 0, 0.75, 1.5, and 3.0 g/kg respectively, and then sacrificed after 4 weeks of administration or after an additional 2 weeks of recovery. The endpoints assessed included body weight measurements, serum biochemistry and haematology indices, and clinical and histopathological observations. RESULTS: The AAAs content in Asarum sieboldii Miq. (HB-ESBJ) were much lower than those of the other Asarums. The contents of AA I, AA IVa, and aristolactam I in KLS were in the ranges of 0.03-0.06 µg/g, 1.89-2.16 µg/g, and 0.55-1.60 µg/g, respectively, whereas AA II and AA IIIa were not detected. None of the rats showed symptoms of toxic reactions and KLS was well tolerated throughout the study. Compared to the control group, the activated partial thromboplastin time values of rats in the 1.5 and 3.0 g/kg groups significantly reduced after administration (P < 0.05). In addition, the serum triglycerides of male rats in the 0.75 and 1.5 g/kg groups after administration, and the 0.75, 1.5, 3.0 g/kg groups after recovery were significantly decreased (P < 0.01 or P < 0.001). No significant drug-related toxicological changes were observed in other serum biochemical indices, haematology, or histopathology. CONCLUSIONS: The AA I content in KLS met the limit requirements (<0.001%) of the Chinese Pharmacopoeia. Therefore, it is safe to use KLS in the short-term. However, for safety considerations, attention should be paid to the effects of long-term KLS administration on coagulation function and triglyceride metabolism.

Phytochemical investigation of Asarum sieboldii var. seoulense and the phenotypic profiles of its constituents against a Parkinson's Disease olfactory cell line.

Asarum sieboldii var. seoulense is a plant species under the family Aristolochiaceae and has been used for centuries as an ingredient in a well-known Traditional Chinese medicine (TCM), "Xixin", to treat symptoms of the neurodegenerative condition Parkinson's Disease (PD). Although there have been studies on the neuroprotective effect of this TCM, the phenotypic profiles of its chemical constituents against PD-implicated cellular organelles have not been reported. This research investigated the chemistry of A. sieboldii var. seoulense extract to identify the active small molecules that exhibited perturbation to the cellular compartments related to PD, potentially supporting its traditional application in treating this condition. 1H NMR-guided chemical investigation of this plant yielded twenty secondary metabolites which belong to isobutylamides, lignans and phenolics. The compounds were evaluated against an olfactory cell line derived from a PD patient using phenotypic assay. Several isolates, 2, 3, 7, 11, 13-16 and 18-20, were found to induce moderate perturbation to the staining of mitochondria, autophagosome and α-tubulin of the cells. Considering that PD pathogenesis is closely related to these cellular compartments, the results provided a rationale for the traditional application of Xixin in the treatment of PD.

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.

Long-term oral administration of Asarum heterotropoides f. mandshuricum (Maxim.) Kitag. decoction and its aristolochic acid analogs do not cause renal toxicity in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Asarum heterotropoides f. mandshuricum (Maxim.) Kitag. (AH) is widely used to treat influenza, COVID-19, allergic rhinitis, headache, toothache, rheumatoid arthritis, and peptic ulcer. However, its clinical use is controversial due to the concern of aristolochic acid nephropathy (AAN) caused by its component aristolochic acid analogs (AAs). AIM OF THE STUDY: The chronic toxicity of AH decoction and its main components AA IVa (AA-IVa) and aristolactam I (AL-I) was evaluated in mice. MATERIALS AND METHODS: AAs contents in AH were quantitated by liquid chromatography-mass spectrometry. A parallel design was employed to examine the potential chronic toxicity of AH decoction at doses equivalent to 0.5, 1.6, and 5.0 g/kg AH (approximately 10-100 times the clinical doses for humans) and its major AA components at doses equivalent to that in 5.0 g/kg AH to mice after consecutive daily oral administration for 12 and 24 weeks, and at 32 weeks after withdrawal for 8 weeks. RESULTS: AH crude herb contained 2.18 µg/g of AA-I, 48.49 µg/g of AA-IVa, and 14.0 µg/g of AL-I. AH decoction contained 5.45 µg/g of AA-IVa and 2.71 µg/g of AL-I. None of AA-II and AA-IIIa were detected in AH. After long-term administration of AH decoction and its major components AA-IVa and AL-I, mice showed no signs of illness or body weight changes. In addition, biochemical and pathohistological examinations showed that long-term administration of AH decoction and its major components AA-IVa and AL-I did not alter 1) serum levels of glutamic-pyruvic transaminase, glutamic oxalacetic transaminase, alkaline phosphatase, creatinine, and urea nitrogen, 2) renal tissue mRNA expression of kidney injury molecule 1 and neutrophil gelatinase-associated lipocalin, and 3) pathological morphology in the mouse liver, kidney, stomach, and bladder. CONCLUSIONS: AH has no obvious toxicity to mice and is relatively safe when it is used in the form of decoction. AA-IVa and AL-I, the two major AAs in AH, are not toxic to mice at the dose equivalent to that in the high dose of AH decoction. Considering the limited toxicological data on AH, we recommend that AH decoction medication should not overdose and the duration should not be too long.

Anti-Inflammatory Lignans from the Roots of Asarum heterotropoides var. mandshuricum and Their Mechanism of Action.

Bioassay-guided fractionation of Asarum heterotropoides var. mandshuricum F. Maekawa (Aristolochiaceae) root extract led to the isolation and characterization of one new ferulic acid glucose ester (1) and nine known lignans (2-10). Their structures were elucidated using extensive spectroscopic methods, including 1D and 2D NMR, and MS spectra. The anti-inflammatory effects of the isolated compounds were investigated via their inhibition against nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 mouse macrophage cells. Among them, compound 7 ((1R,2S,5R,6R)-5'-O-methylpluviatilol) showed the most effective inhibitory activity against NO production and expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein in an exceedingly dose-dependent manner. In addition, further study revealed that the mechanism of anti-inflammatory activity of the most active lignan (7) might be associated with the inhibition of extracellular-signal-regulated kinase (ERK) and nuclear factor kappa B (NF-κB) phosphorylation.

The genus Asarum: A review on phytochemistry, ethnopharmacology, toxicology and pharmacokinetics.

ETHNOPHARMACOLOGICAL RELEVANCE: In essentially every quadrant of the globe, many species of genus Asarum are used as a common herbal medicine and appear in many formulas or Kampo. Crude drug from several medicinal plants of genus Asarum (MA) known as Asari Radix et Rhizoma (ARR) has been proven to have the functions of dispelling cold, relieving pain, and reducing phlegm according to Traditional Chinese Medicine (TCM) theory for thousands of years. AIM OF THE STUDY: This article reviews the ethnopharmacology, phytochemistry, pharmacology, toxicology and metabolic kinetics related research of genus Asarum to evaluate its ethnopharmacology use and future opportunities for research. MATERIALS AND METHODS: Information on relevant studies of the genus Asarum was gathered via the Internet using Baidu Scholar, Web of Science, Elsevier, ResearchGate, ACS, Pudmed and Chinese National Knowledge Infrastructure (CNKI). Additionally, information was also obtained from some local books, PhD, MS's dissertations and Pharmacopeias. RESULTS: The genus Asarum has played an important role in herbal treatment. At present, more than 277 compounds have been isolated or identified from genus Asarum. Among them, volatile oil and lignans are the major active constituents and important chemotaxonomic markers. Modern pharmacological studies indicated that genus Asarum and its active compounds possess a wide range of pharmacological effects, especially analgesic, anti-inflammatory, neuroprotective, cardiovascular protection, antitussive, immunosuppressive, anti-tumor, and microbicidal activities. CONCLUSIONS: Based on this review, therapeutic potential of genus Asarum has been demonstrated with the pharmacological effects on inflammation, CNS, respiratory regulation, cardiovascular diseases, cancer and microbial infection. The available literature showed that the major activities of the genus Asarum can be attributed to the active lignans and essential oils. Further in-depth studies on the aspects of the genus for mechanism of actions, metabolism, pharmacokinetics, toxicology, drug interactions, and clinical trials are still limited, thereby intensive research and assessments should be performed.

A two-step strategy for quality control of Xin-Yu-Tie-Pian, an ointment patch.

Xin-Yu-Tie-Pian, an ointment patch which is composed of Tetradium ruticarpum and Asarum sieboldii Miquel, can be used for curing recurrent oral ulcers owing to its good bioactivities. Currently, the lack of a method for its quality evaluation hinders the development and clinical application of Xin-Yu-Tie-Pian. Thus, it is necessary to perform research on quality control. The chromatographic fingerprint, as an identification method, and the simultaneous determination method for two bioactive constituents, evodiamine and rutecarpine, can be used to evaluate the quality of traditional medicine. In this study, a two-step strategy including fingerprint analysis for identification and a simultaneous determination method for two bioactive constituents was performed for Xin-Yu-Tie-Pian quality control. The fingerprint analysis was validated by stability, precision and repeatability tests and a similarity evaluation was performed with 10 selected characteristic fingerprint peaks of 10 batches of Xin-Yu-Tie-Pian patch. Meanwhile, the simultaneous determination method was evaluated by methodological experiments, including linearity, accuracy, repeatability, stability and feasibility. Finally, the results indicate that this two-step strategy, including HPLC fingerprint analysis and simultaneous determination method, can be successfully applied for the assessment of the quality and quantity of Xin-Yu-Tie-Pian.

Salvia miltiorrhiza-asarum ointment combined with Chinese medical massage alleviates symptoms of osteoarthritis in a rat model through the Notch1/ matrix metalloproteinase-13 signaling pathway.

OBJECTIVE: This study investigated the effect of salvia miltiorrhiza-asarum ointment (SMAO) plus Chinese medical massage on knee osteoarthritis in a rat model. METHODS: Hulth's method was used to establish a Sprague-Dawley rat model of knee osteoarthritis (OA). The levels of matrix metalloproteinase-13 (MMP-13), collagen-II, aggrecan, interleukin (IL)-1ß, tumor necrosis factor-α (TNF-α), and IL-6 were measured by enzyme-linked immunosorbent assays. The joint space was assessed by a Perlove X-ray system. Histopathology was examined by hematoxylin-eosin and Safranin O staining. The mRNA and protein expression levels of Notch1, MMP-13, collagen-II, and aggrecan were measured by quantitative reverse transcription-polymerase chain reaction and Western blotting, respectively. RESULTS: SMAO plus Chinese medical massage significantly decreased the levels of MMP-13, IL-1ß, TNF-α, and IL-6, and increased serum collagen-II and aggrecan levels. Pathological injury of the knee joint was improved by SMAO treatment. mRNA and protein expression of Notch1 and MMP-13 was remarkably downregulated, but collagen-II and aggrecan levels were significantly upregulated in cartilage tissues. CONCLUSION: SMAO combined with Chinese medical massage effectively relieves OA symptoms, which may involve inhibiting inflammation through the Notch1/MMP-13 signaling pathway.

Rapid characterization and pharmacokinetic study of aristolochic acid analogues using ion mobility mass spectrometry.

Aristolochic acid analogues (AAAs), naturally existing in herbal Aristolochia and Asarum genera, were once widely used in traditional pharmacopeias because of their anti-inflammatory properties, but lately they were identified as potential nephrotoxins and mutagens. A method for rapid characterization of AAAs in serum was developed using ion mobility spectrometry coupled with mass spectrometry (IMS-MS). Five AAAs, containing four aristolochic acids and one aristolactam, were separated and identified within milliseconds. AAAs were separated in gas phase based on the difference of their ion mobility (K0), and then identified based on their K0 values, m/z, and product ions from MS/MS. Quantitative analysis of AAAs was performed using an internal standard with a satisfactory sensitivity. Limits of detection (signal-to-noise = 3) and quantification (signal-to-noise = 10) were 1-5 ng/mL and 3-8 ng/mL, respectively. The method was validated and successfully applied to the pharmacokinetics study of AAAs in rats, offering a promising way for fast screening and evaluation of AAAs in biological samples.

Pharmacokinetic Study of Safrole and Methyl Eugenol after Oral Administration of the Essential Oil Extracts of Asarum in Rats by GC-MS.

Asarum is a traditional medicine and has been widely used as remedies for inflammatory diseases, toothache, headache, local anesthesia, and aphthous stomatitis in China, Japan, and Korea. Our previous research found that safrole and methyl eugenol were vital compounds that contribute to distinguish the different species and raw Asarum and its processed products apart. The pharmacokinetics of safrole and methyl eugenol after oral administration of Asarum extract has not been reported yet. In this study, a rapid and simple gas chromatography-mass spectroscopy (GC-MS) method that has a complete run time of only 4.5 min was developed and validated for the simultaneous determination and pharmacokinetic study of safrole and methyl eugenol in rat plasma after administration of Asarum extracts. The chromatographic separation was realized on a DB-17 column (30 m × 0.25 mm × 0.25 µm). And detection was carried out under selected ion monitoring (SIM) mode. Plasma samples were pretreated by n-hexane. The pharmacokinetic parameters provided by this study will be beneficial for further developments and clinical applications of Asarum.

A systematic strategy for uncovering quality marker of Asari Radix et Rhizoma on alleviating inflammation based chemometrics analysis of components.

Asari Radix et Rhizoma (Asarum), a traditional Chinese medicine (TCM), has been applied in clinical generally. However, due to the lack of valid methods for Asarum quality control, inhomogenous quality and therapy issues have become severe with each passing day. In this study, we aimed to establish a comprehensive multi-system to explore the quality control markers underlying pharmaceutical effects based on chemometrics analysis on the total ingredients of Asarum. In brief, DNA barcoding technology was used to screen out the unadulterated herbs in the 15 batches Asarum collected from different origins. Then, the chemical profiles of volatile/nonvolatile components of 10 batches Asarum with definite resource were obtained by HPLC Q-TOF/MS and GC/MS. Combination with chemometrics methods, 14 characteristic ingredients and 4 qualitative and quantitative markers were figured out preliminarily. Moreover, correlation analysis between the characteristic ingredients and the cytokines integrating the virtual targets prediction of network pharmacology, 3 potential bioactive substance were ascertained. In conclusion, l-asarinin, 2-Methoxy-4-vinylphenol and safrole were considered as the potent candidates for quality control markers based on the comprehensive understanding for therapeutic effects and the chemical information of Asarum, which provided a novel perspective of the development for the quality control of TCM.

Fumigant Toxicity and Oviposition Deterrent Activity of Volatile Constituents from Asari Radix et Rhizoma against Phthorimaea operculella (Lepidoptera: Gelechiidae).

Phthorimaea operculella (Zeller) is a worldwide pest of potato. Plant-borne chemicals would be potential alternatives of synthetic chemical fumigants against P. operculella in the storage. Asari Radix et Rhizoma is derived from the dry roots and rhizomes of Asarum heterotropoides Fr. Schmidt var. mandshuricum, A. sieboldii Miq. var. seoulense, or A. sieboldii. In this study, fumigant toxicity and oviposition deterrent of volatile constituents from ARR, δ-3-carene, γ-terpinene, terpinolene, eucarvone, 3,5-dimethoxytoluene, and methyleugenol were tested against P. operculella. The preliminary verification of preventive and control effects of eucarvone, 3,5-dimethoxytoluene and methyleugenol on P. operculella was carried out by simulating warehouse experiments. The results indicated that the six compounds above had fumigation toxic effects on the adults and eggs of P. operculella. Among them, δ-3-carene, γ-terpinene, and terpinolene had weaker fumigation effects than those of eucarvone, 3,5-dimethoxytoluene, and methyleugenol. The LC50 values of eucarvone, 3,5-dimethoxytoluene, and methyleugenol against adult P. operculella were 1.01, 1.78, 1.51 mg/liter air, respectively. The LC50 values against egg P. operculella were 1.09, 0.55, 0.30 mg/liter air, respectively. The oviposition deterrent experiment showed that only methyleugenol (at 5 and 1 mg/ml) and eucarvone (5 mg/ml) had a substantial oviposition deterrent effect. The simulated warehouse experiment verified that methyleugenol, eucarvone, and 3,5-dimethoxytoluene protected potatoes from P. operculella and demonstrated that methyleugenol had the best preventive and control effects. It was concluded that methyleugenol was the active ingredient with the most potential in the volatiles from ARR on P. operculella control and merit further study as botanic fumigant.

The Relative Content and Distribution of Absorbed Volatile Organic Compounds in Rats Administered Asari Radix et Rhizoma Are Different between Powder- and Decoction-Treated Groups.

Asari Radix et Rhizoma (ARR) is an important traditional Chinese medicine. Volatile organic compounds (VOCs) are the main active constituents of ARR. Research on the metabolite profile of VOCs and the difference of absorbed constituents in vivo after an administration of ARR decoction and powder will be helpful to understand the pharmacological activity and safety of ARR. In this study, headspace solid-phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS) was applied to profile the VOCs from ARR in rats in vivo. A total of 153 VOCs were tentatively identified; 101 were original constituents of ARR (98 in the powder-treated group and 43 in the decoction-treated group) and 15 were metabolites, and their metabolic reactions were mainly oxidation and reduction, with only two cases of methylation and esterification, and 37 unclassified compounds were identified only in the ARR-treated group. Of the 153 VOCs identified, 131 were reported in rats after oral administration of ARR for the first time, containing 79 original constituents, 15 metabolites, and 37 unclassified compounds. In the powder-treated group, methyleugenol, safrole, 3,5-dimethoxytoluene (3,5-DMT), 2,3,5-trimethoxytoluene (2,3,5-TMT), and 3,4,5-trimethoxytoluene (3,4,5-TMT) were the main absorbed constituents, the relative contents of which were significantly higher compared to the decoction-treated group, especially methyleugenol, safrole, and 3,5-DMT. In the decoction-treated group, 3,4,5-TMT, 2,3,5-TMT, kakuol, and eugenol were the main constituents with a higher content and wider distribution. The results of this study provide a reference for evaluating the efficacy and safety of ARR.

Asaroidoxazines from the Roots of Asarum asaroides Induce Apoptosis in Human Neuroblastoma Cells.

Plants in the family Aristolochiaceae contain phenanthrene skeleton-containing chemical constituents that exhibit nephrotoxic, carcinogenic, mutagenic, anti-inflammatory, and cytotoxic effects. Two new phenanthrene-containing 1,2-oxazin-6-ones, designated as asaroidoxazine A (1) and asaroidoxazine B (2), and a known aristolactam, 5-methoxyaristololactam I (3), were isolated from the roots of Asarum asaroides. The structures of compounds 1 and 2 were determined using spectroscopic methods and X-ray crystallography. Treatment of SH-SY5Y human neuroblastoma cells with 1 µM of asaroidoxazine A (1) induced nuclear condensation as well as caspase-3/7 activation, indicating that this compound is a strong apoptosis inducer in neuronal cells. This is the first report of apoptosis induction by phenanthrene-containing oxazines.

Protective effect of Asarum sieboldii essential oil on ovalbumin induced allergic rhinitis in rat.

BACKGROUND: The study was aimed to investigate the protective effect of Asarum sieboldii Miq. essential oil (AEO) on ovalbumin (OVA)-induced allergic rhinitis (AR) in rats. METHODS AND RESULTS: Sixty Sprague-Dawley male rats were randomly divided into six groups (n=10): control, model, cetirizine (Cet, 4.65 g/kg), and AEO (0.5, 1.5, 3 g/kg) groups. All animals except the control group received repeated intranasal instillation with 20 µl of 20% OVA in Al(OH)3 saline solvent for 15 days. The control group was intranasally instilled with 5 mg/ml of Al(OH)3 instead of the same procedure. In the 15 days, Cet and AEO were orally administrated for 28 days. At the end of the drug administration, 20 µl of 5% OVA was given to animals to stimulate allergic reaction, then the rat behavioral detection, assessment of the patho-morphological changes in nasal mucosa, and the serum biomarkers were determined. The result showed that AEO could significantly reduce the amount of nasal secretions, sneezing, and the degree of nasal scratching in AR rats with EC50 = 1.5 and 2.8 g/kg, respectively. The degree of nasal mucosal inflammation in AEO group improved, the levels of immunoglobulin E (IgE), histamine, IL-4, IL-5, IL-17 were decreased, and the level of IFN-γ was increased obviously with EC50 = 2 g/kg. CONCLUSION: The study suggested that the possible mechanism might be related with the inhibition of histamine release and regulation of the cytokine levels, which plays an important role in the treatment of AR.

[Identification based on HPLC and anti-inflammatory targets as well as related constituents analysis of Asarum heterotropoides var. mandshuricum and A. sieboldii].

The present work is to establish an HPLC characteristic chromatograms of Asarum heterotropoides var. mandshuricum(AH) and A. sieboldii(AS), combined with cluster analysis for the identification of the two species, and predict their potential anti-inflammatory related targets by network pharmacological method. Eighty-nine samples(12 batches of AS and 77 batches of AH) were analyzed, and 11 characteristic peaks were identified by reference substances, UV spectrum and LC-MS. Cluster analysis showed that AS and AH were divided into two groups, and the ratio of characteristic peak areas can be used to distinguish them. When the ratio of characteristic peak sarisan to kakuol was greater than 5, it was AS, and when the ratio was less than 2, it was AH. The network pharmacological analysis of 119 constituents of Asari Radix et Rhizoma suggested that the anti-inflammatory effect of Asari Radix et Rhizoma might be related to COX-2, COX-1, iNOS, MAPK14, NR3 C1, PPARG and TNF. Among them, COX-2 is a relatively key target, which interacted with the characteristic constituents, asarinin, sesamin, safrole, methyleugenol and sarisan. The characteristic constituents asarinin and sesamin also interacted with the iNOS and MAPK14. Safrole and sarisan can also interact with iNOS, COX-1 and LAT4 H. Methyleugenol also showed interaction with COX-1 and LAT4 H. Since asarinin and sesamin interacted with three targets, COX-2, iNOS and MAPK14, it implied that they were the main active constituents for the anti-inflammatory activity of Asari Radix et Rhizoma. The COX-2 inhibitory activities of asarinin and sesamin were further studied by molecular docking and bioassay. The HPLC method established was simple, feasible and reliable, with predicted anti-inflammatory targets and anti-inflammatory constituents, which could provide a reference for improving the quality evaluation system of Asari Radix et Rhizoma.

Soft-templated mesoporous carbon-modified glassy carbon electrode for sensitive and selective detection of aristolochic acids.

In this study, ordered mesoporous carbon (OMC) was synthesized by applying a soft template method, and its mesoporous structure was characterized by scanning electron microscopy, transmission electron microscopy, and nitrogen adsorption-desorption techniques. X-ray diffraction and Raman spectroscopic analyses were conducted to demonstrate the high graphitization and topological defects at the sample surface. An electrochemical sensor based on an OMC-modified glassy carbon electrode (OMC/GCE) was constructed to detect aristolochic acids (AAs) using cyclic voltammetry and linear sweep voltammetry. The dependence of the experimental parameters including solution pH, scan rate, and accumulation time were examined and optimized. Under the optimal conditions, the response of OMC/GCE was linear over wide concentration ranges of AAs (0.6-10 µM and 10-50 µM), with sensitivities of -1.77 and -0.31 µA/µM, respectively. The limit of detection was calculated to be 0.186 µM (at S/N = 3). Furthermore, the proposed OMC/GCE was applied to detect AAs in Asarum sieboldini and the content of AAs was calculated to be 8.9 µg/g with high accuracy and precision. In addition, the modified electrode also exhibited good selectivity, reproducibility, and stability. Therefore, the OMC/GCE can be used as a platform for the determination of AAs.

Asatone and Isoasatone A Against Spodoptera litura Fab. by Acting on Cytochrome P450 Monoxygenases and Glutathione Transferases.

Asatone and isoasatone A from Asarum ichangense Cheng were determined to be defensive compounds to some insects in a previous investigation. However, the anti-insect activity mechanisms to caterpillar are still unclear. The compounds asatone and isoasatone A from A. ichangense were induced by Spodoptera litura. The anti-insect activity of asatone and isoasatone A to S. litura was further tested by weight growth rate of the insect through a diet experiment. Isoasatone A showed a more significant inhibitory effect on S. litura than asatone on the second day. The concentration of asatone was higher than isoasatone A in the second instar larvae of S. litura after 12 h on the feeding test diet. Both compounds caused mid-gut structural deformation and tissue decay as determined by mid-gut histopathology of S. litura. Furthermore, some detoxification enzyme activity were measured by relative expression levels of genes using a qPCR detecting system. Asatone inhibited the gene expression of the cytochrome P450 monooxygenases (P450s) CYP6AB14. Isoasatone A inhibited the relative expression levels of CYP321B1, CYP321A7, CYP6B47, CYP6AB14, and CYP9A39. Asatone increased the relative gene expression of the glutathione transferases (GSTs) SIGSTe1 and SIGSTo1, in contrast, isoasatone A decreased the relative gene expression of SIGSTe1 by about 33 fold. Neither compound showed an effect on acetylcholinesterase SIAce1 and SIAce2. The mechanism of anti-insect activity by both compounds could be explained by the inhibition of enzymes P450s and GSTs. The results provide new insights into the function of unique secondary metabolites asatone and isoasatone A in genus Asarum, and a new understanding of why A. ichangense is largely free of insect pests.