Quantitative Determination and Toxicity Evaluation of Aristolochic Acid Analogues in Asarum heterotropoides F. Schmidt (Xixin) and Traditional Chinese Patent Medicines

Asarum (Xixin), which contains analogues of aristolochic acid (AA), is the only species of the genus Aristolochia included in the Chinese Pharmacopoeia 2020. However, the contents and nephrotoxic effects of AA analogs in Asarum (Xixin) and its formulations have not been clarified. An automatic, effective solid phase extraction process and UPLC-MS/MS method were established for the pretreatment and quantitative detection of AA analogues in commercially available traditional Chinese patent medicines. The cytotoxicity and DNA damage induced by five analogues of AA were evaluated by CCK8 using human kidney cells (HK-2) and comet assays. HPLC was used to detect the analogues of AA in Asarum heterotropoides F. Schmidt (Xixin). The results showed that the contents of AA I, AA II, and AA IIIa were below the detection limit, while AA IVa and AL I presented relatively high contents of Asarum heterotropoides F. Schmidt (Xixin), within the range of 66.50–121.03 μg/g and 19.73–43.75 μg/g, respectively. The levels of AA analogues were in the nanogram-per-gram level in the main traditional Chinese patent medicines. AA I and AL I exhibited relatively high cytotoxicity at 48 h in CCK8 assays, while AA II, AA IIIa, and AA IVa showed weak cytotoxicity even at 800–1,000 μM. AA I induced significant pathological alterations and direct DNA damage at 40 mg/kg and 20 mg/kg, respectively. No distinct nephrotoxicity or hepatotoxicity was observed in mice treated with AA II, AA IIIa, AA IVa, or AL I at 40 mg/kg in this study. Consumption of Asarum heterotropoides F. Schmidt (Xixin) with controlled doses and periods is relatively safe as the contents of AA analogues in Asarum heterotropoides F. Schmidt (Xixin) and its formulations were far below those causing acute toxicity in this study. But, the long-term toxicity of Asarum heterotropoides F. Schmidt (Xixin) still needs further study.

An Asarone Producing Aspergillus Niger From Asarum Heterotropoides With Whole Genome Sequencing

Asarone could decrease Aβ, APP, Beclin-1level and clearing Aβ accumulation, which makes it to be one potential therapeutic agent for AD treatment. This study investigated the endophytic fungus got from Asarum heterotropoides Fr.Schmidt var. mandshuricum (Maxim.) kitag. The phylogenetic tree based on single copy orthologues at protein level got from genome sequence analysis showed that the endophytic fungus we got was Aspergillus niger. The total length of nonredundant sequences was nearly 34.7 Mb, comprising approximately 61 contigs sequences. In total, 8,755 genes were predicted to encoding proteins, and 18,611,392 high-quality clean reads were generated using the Illumina NovaSeq platform with paired-end sequencing mode. In this study, the theoretical yield of cis-asarone was higher than the traditional ways extracted from essential oil, which was evaluated according to the reverse-phase high performance liquid chromatography (HPLC). The highest yield appeared from the slant cultivation instead of liquid fermentation, and the mature black spore were the final supporter of cis-asarone. This strain was capable of producing cis-asarone and could be a fascinating reservoir of cis-asarone for further industrial manufacture.

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.