Insights into angiosperm evolution, floral development and chemical biosynthesis from the Aristolochia fimbriata genome.

Aristolochia, a genus in the magnoliid order Piperales, has been famous for centuries for its highly specialized flowers and wide medicinal applications. Here, we present a new, high-quality genome sequence of Aristolochia fimbriata, a species that, similar to Amborella trichopoda, lacks further whole-genome duplications since the origin of extant angiosperms. As such, the A. fimbriata genome is an excellent reference for inferences of angiosperm genome evolution, enabling detection of two novel whole-genome duplications in Piperales and dating of previously reported whole-genome duplications in other magnoliids. Genomic comparisons between A. fimbriata and other angiosperms facilitated the identification of ancient genomic rearrangements suggesting the placement of magnoliids as sister to monocots, whereas phylogenetic inferences based on sequence data we compiled yielded ambiguous relationships. By identifying associated homologues and investigating their evolutionary histories and expression patterns, we revealed highly conserved floral developmental genes and their distinct downstream regulatory network that may contribute to the complex flower morphology in A. fimbriata. Finally, we elucidated the genetic basis underlying the biosynthesis of terpenoids and aristolochic acids in A. fimbriata.

Chloroplast genome features of an important medicinal and edible plant: Houttuynia cordata (Saururaceae).

Houttuynia cordata (Saururaceae), an ancient and relic species, has been used as an important medicinal and edible plant in most parts of Asia. However, because of the lack of genome information and reliable molecular markers, studies on its population structure, or phylogenetic relationships with other related species are still rare. Here, we de novo assembled the complete chloroplast (cp) genome of H. cordata using the integration of the long PacBio and short Illumina reads. The cp genome of H. cordata showed a typical quadripartite cycle of 160,226 bp. This included a pair of inverted repeats (IRa and IRb) of 26,853 bp, separated by a large single-copy (LSC) region of 88,180 bp and a small single-copy (SSC) region of 18,340 bp. A total of 112 unique genes, including 79 protein-coding genes, 29 tRNA genes, and four rRNA genes, were identified in this cp genome. Eighty-one genes were located on the LSC region, 13 genes were located on the SSC region, and 17 two-copy genes were located on the IR region. Additionally, 48 repeat sequences and 86 SSR loci, which can be used as genomic markers for population structure analysis, were also detected. Phylogenetic analysis using 21 cp genomes of the Piperales family demonstrated that H. cordata had a close relationship with the species within the Aristolochia genus. Moreover, the results of mVISTA analysis and comparisons of IR regions demonstrated that the cp genome of H. cordata was conserved with that of the Aristolochia species. Our results provide valuable information for analyzing the genetic diversity and population structure of H. cordata, which can contribute to further its genetic improvement and breeding.

Complete Chloroplast Genomes and Comparative Analysis of Sequences Evolution among Seven Aristolochia (Aristolochiaceae) Medicinal Species.

Aristolochiaceae, comprising about 600 species, is a unique plant family containing aristolochic acids (AAs). In this study, we sequenced seven species of Aristolochia, and retrieved eleven chloroplast (cp) genomes published for comparative genomics analysis and phylogenetic constructions. The results show that the cp genomes had a typical quadripartite structure with conserved genome arrangement and moderate divergence. The cp genomes range from 159,308 bp to 160,520 bp in length and have a similar GC content of 38.5%⁻38.9%. A total number of 113 genes were identified, including 79 protein-coding genes, 30 tRNAs and four rRNAs. Although genomic structure and size were highly conserved, the IR-SC boundary regions were variable between these seven cp genomes. The trnH-GUG genes, are one of major differences between the plastomes of the two subgenera Siphisia and Aristolochia. We analyzed the features of nucleotide substitutions, distribution of repeat sequences and simple sequences repeats (SSRs), positive selections in the cp genomes, and identified 16 hotspot regions for genomes divergence that could be utilized as potential markers for phylogeny reconstruction. Phylogenetic relationships of the family Aristolochiaceae inferred from the 18 cp genome sequences were consistent and robust, using maximum parsimony (MP), maximum likelihood (ML), and Bayesian analysis (BI) methods.

DNA barcoding of Aristolochia plants and development of species-specific multiplex PCR to aid HPTLC in ascertainment of Aristolochia herbal materials.

The anecdotal evidence is outstanding on the uses of Aristolochia plants as traditional medicines and dietary supplements in many regions of the world. However, herbal materials derived from Aristolochia species have been identified as potent human carcinogens since the first case of severe renal disease after ingesting these herbal preparations. Any products containing Aristolochia species have thus been banned on many continents, including Europe, America and Asia. Therefore, the development of a method to identify these herbs is critically needed for customer safety. The present study evaluated DNA barcoding of the rbcL, matK, ITS2 and trnH-psbA regions among eleven Aristolochia species collected in Thailand. Polymorphic sites were observed in all four DNA loci. Among those eleven Aristolochia species, three species (A. pierrei, A. tagala and A. pothieri) are used as herbal materials in Thai folk medicine, namely, in Thai "Krai-Krue". "Krai-Krue" herbs are interchangeably used as an admixture in Thai traditional remedies without specific knowledge of their identities. A species-specific multiplex PCR based on nucleotide polymorphisms in the ITS2 region was developed as an identification tool to differentiate these three Aristolochia species and to supplement the HPTLC pattern in clarifying the origins of herbal materials. The combination of multiplex PCR and HPTLC profiling achieves accurate herbal identification with the goal of protecting consumers from the health risks associated with product substitution and contamination.

Molecular Structure and Phylogenetic Analyses of Complete Chloroplast Genomes of Two Aristolochia Medicinal Species.

The family Aristolochiaceae, comprising about 600 species of eight genera, is a unique plant family containing aristolochic acids (AAs). The complete chloroplast genome sequences of Aristolochia debilis and Aristolochia contorta are reported here. The results show that the complete chloroplast genomes of A. debilis and A. contorta comprise circular 159,793 and 160,576 bp-long molecules, respectively and have typical quadripartite structures. The GC contents of both species were 38.3% each. A total of 131 genes were identified in each genome including 85 protein-coding genes, 37 tRNA genes, eight rRNA genes and one pseudogene (ycf1). The simple-sequence repeat sequences mainly comprise A/T mononucletide repeats. Phylogenetic analyses using maximum parsimony (MP) revealed that A. debilis and A. contorta had a close phylogenetic relationship with species of the family Piperaceae, as well as Laurales and Magnoliales. The data obtained in this study will be beneficial for further investigations on A. debilis and A. contorta from the aspect of evolution, and chloroplast genetic engineering.

LC-MS- and (1)H NMR-Based Metabolomic Analysis and in Vitro Toxicological Assessment of 43 Aristolochia Species.

Species of Aristolochia are used as herbal medicines worldwide. They cause aristolochic acid nephropathy (AAN), a devastating disease associated with kidney failure and renal cancer. Aristolochic acids I and II (1 and 2) are considered to be responsible for these nephrotoxic and carcinogenic effects. A wide range of other aristolochic acid analogues (AAAs) exist, and their implication in AAN may have been overlooked. An LC-MS- and (1)H NMR-based metabolomic analysis was carried out on 43 medicinally used Aristolochia species. The cytotoxicity and genotoxicity of 28 Aristolochia extracts were measured in human kidney (HK-2) cells. Compounds 1 and 2 were found to be the most common AAAs. However, AA IV (3), aristolactam I (4), and aristolactam BI (5) were also widespread. No correlation was found between the amounts of 1 or 2 and extract cytotoxicity against HK-2 cells. The genotoxicity and cytotoxicity of the extracts could be linked to their contents of 5, AA D (8), and AA IIIa (10). These results undermine the assumption that 1 and 2 are exclusively responsible for the toxicity of Aristolochia species. Other analogues are likely to contribute to their toxicity and need to be considered as nephrotoxic agents. These findings facilitate understanding of the nephrotoxic mechanisms of Aristolochia and have significance for the regulation of herbal medicines.

[Molecular identification of aucklandiae radix, vladimiriae radix, inulae radix, aristolochiae radix and kadsurae radix using ITS2 barcode].

In order to identify Aucklandiae Radix, Vladimiriae Radix, Inulae Radix, Aristolochiae Radix and Kadsurae Radix using ITS2 barcodes, genomic DNA from sixty samples was extracted and the ITS2 (internal transcribed spacer) regions were amplified and sequenced. The genetic distances were computed using MEGA 5.0 in accordance with the kimura 2-parameter (K2P) model and the neighbor-joining (NJ) phylogenetic tree was constructed. The results indicated that for Aucklandiae Radix (Aucklandia lappa), Vladimiriae Radix (Vladimiria souliei and V. souliei var. cinerea), Inulae Radix (Inula helenium), Aristolochiae Radix (Aristolochia debilis) and Kadsurae Radix (Kadsura longipedunculata), the intra-specific variation was smaller than inter-specific one. There are 162 variable sites among 272 bp after alignment of all ITS2 sequence haplotypes. For each species, the intra-specific genetic distances were also smaller than inter-specific one. Furthermore, the NJ tree strongly supported that Aucklandiae Radix, Vladimiriae Radix, Inulae Radix, Aristolochiae Radix and Kadsurae Radix can be differentiated. At the same time, V. souliei (Dolomiaea souliei) and V. souliei var. cinerea( D. souliei var. cinerea) belonging to Vladimiriae Radix were clearly identified. In conclusion, ITS2 barcode could be used to identify Aucklandiae Radix, Vladimiriae Radix, Inulae Radix, Aristolochiae Radix and Kadsurae Radix. Our study may provide a scientific foundation for clinical safe use of the traditional Chinese medicines.

Molecular identification and cytotoxicity study of herbal medicinal materials that are confused by Aristolochia herbs.

Herbal materials derived from Aristolochia species contain carcinogenic aristolochic acids (AAs) and have been used as traditional Chinese medicines (TCMs) or adulterants of other TCMs. The purpose of this study is to identify the TCMs Stephaniae Tetrandrae Radix, Akebiae Caulis, Aucklandia Radix and Aristolochiae Fructus by sequencing the matK, rbcL, trnH-psbA and trnL-trnF DNA regions. The cytotoxicities of AAs and these TCMs were also studied in COS-7 and HEK-293 cells. Diagnostic polymorphic sites were identified in all the four DNA loci for the differentiation of genuine herbs from their adulterants/substitute. The 48 h IC50 of AAI were 78 µM (COS-7) and 70 µM (HEK-293) while the IC50 of AAII were higher than 100 µM in both cell lines. Except Aucklandia Radix, cytotoxicity study also showed that AA-containing herbs were more toxic than their corresponding genuine herbs and substitute.

Identification of Baiying (Herba Solani Lyrati) commodity and its toxic substitute Xungufeng (Herba Aristolochiae Mollissimae) using DNA barcoding and chemical profiling techniques.

Baiying derived from Solanum lyratum Hance is a commonly consumed natural product for ethnomedical treatment of cancer. One of the substitutes present in the market is a carcinogenic aristolochic acids-containing herb Xungufeng derived from Aristolochia mollissima Thunb. The purpose of this study is to establish DNA barcodes, thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and cytotoxicity assay to differentiate Baiying from Xungufeng. A total of 30 DNA sequences from five DNA barcodes (ITS, matK, rbcL, trnH-psbA and trnL-trnF) were generated to differentiate S. lyratum from A. mollissima and authenticate ten samples of Baiying and Xungufeng commodities. Using aristolochic acids as standard markers, TLC and HPLC analyses also successfully authenticated these commodities. In vitro cytotoxicity assay using HEK-293 and Vero cells demonstrated that Xungufeng was significantly more toxic than Baiying. This is the first study applying an integrated molecular, chemical and biological approach to differentiate traditional Chinese medicine from Aristolochia adulterant.

Cardiocrinum seeds as a replacement for Aristolochia fruits in treating cough.

AIM OF THE STUDY: The antitussive Chinese herb Madouling derived from Aristolochia species is banned due to aristolochic acid-induced nephropathy. A substitute is found dispensed as Madouling in Taiwan. This study aims to determine the source plant and verify the antitussive properties of the Madouling substitute used in Taiwan. MATERIALS AND METHODS: Forensically informative nucleotide sequencing (FINS) approach based on the trnL-trnF and psbA-trnH regions was applied to facilitate identification of the genuine species and substitute. The antitussive effect of both genuine Madouling and the substitute were evaluated in guinea pigs. RESULTS: FINS approach based on the trnL-trnF and psbA-trnH regions readily identified the sample of Madouling in Taiwan to the seeds of Cardiocrinum giganteum var. yunnanense. Ethanol extracts of the substitute showed significant antitussive properties in guinea pigs. CONCLUSION: Cardiocrinum seeds may have potential as a replacement of Aristolochia fruits.