Identification of novel flavin-dependent monooxygenase from Strobilanthes Cusia reveals molecular basis of indoles' biosynthetic logic.

BACKGROUND: Strobilanthes cusia (Nees) Kuntze is a traditional medical plant distributed widely in south China. The indole compounds that originated from the plant are responsible for its pharmacological activities. However, the reason why indole ingredients are accumulated in this herb and how it is biosynthesized has remained largely unknown. RESULTS: In this study, metabolic and transcriptional profiling measurement experiments of different S. cusia organs were carried out to understand the underlying molecular basis of indoles' biosynthetic logic. A metabolic investigation demonstrated that the indoles are primarily accumulated mainly in aerial parts, particularly in leaves. RNA-seq was employed to reveal the organ specific accumulation of indoles in different S. cusia organs. Meanwhile, a flavin-dependent monooxygenase gene (ScFMO1) was found in S. cusia, and it has capacity to produce indoxyl from indole by the fermentation assay. Finally, we assessed the outcomes of transient expression experiment in tobacco and confirmed that ScFMO1 localizes in cytoplasm. CONCLUSIONS: Our results suggest that ScFMO1 plays a key role in biosynthesis of indoles (Indigo, indirubin, indican, etc.), it will be useful for illuminating the molecular basis of the medicinal indoles' biosynthesis and developing strategies for improving their yields.

Combining the In Silico and In Vitro Assays to Identify Strobilanthes cusia Kuntze Bioactives against Penicillin-Resistant Streptococcus pneumoniae.

Leaves of Strobilanthes cusia Kuntze (S. cusia) are a widely used alexipharmic Traditional Chinese Medicine (TCM) in southern China for the prevention of cold and respiratory tract infectious diseases. One of the most common bacterial pathogens in the respiratory tract is the gram-positive bacterium Streptococcus pneumoniae. The antibiotic resistance of colonized S. pneumoniae makes it a more serious threat to public health. In this study, the leaves of S. cusia were found to perform antibacterial effects on the penicillin-resistant S. pneumoniae (PRSP). Confocal assay and Transmission Electron Microscopy (TEM) monitored the diminished cell wall integrity and capsule thickness of the PRSP with treatment. The following comparative proteomics analysis revealed that the glycometabolism-related pathways were enriched for the differentially expressed proteins between the samples with treatment and the control. To further delve into the specific single effective compound, the bio-active contents of leaves of S. cusia were analyzed by UPLC-UV-ESI-Q-TOF/MS, and 23 compounds were isolated for anti-PRSP screening. Among them, Tryptanthrin demonstrated the most promising effect, and it possibly inhibited the N-glycan degradation proteins, as suggested by reverse docking analysis in silico and further experimental verification by the surface plasmon resonance assay (SPR). Our study provided a research foundation for applications of the leaves of S. cusia as a TCM, and supplied a bio-active compound Tryptanthrin as a candidate drug skeleton for infectious diseases caused by the PRSP.

First Report of Corynespora cassiicola Causing Leaf spot of Strobilanthes cusia in China.

Strobilanthes cusia (Nees) Kuntze is a vital medicinal and industrial herb, planted extensively in southern China (Hu, et al. 2011.). In July and August of 2021, leaf spot incidence on >60% plants and reduced yields >20% for fresh leaves were observed in S. cusia cultivar 'Malan No.1' across the Shufeng whole Township, Xianyou County, Fujian province. Initial symptoms on leaves were observed as small, dark-brown, spots surrounded by a yellow halo, expanding irregularly or into semicircular spots. As symptoms developed, the spots became dark brown, thin and fragile, forming small holes. In severe cases plants were defoliated. The pathogen was isolated from the margin of 60 symptomatic leaf lesions， surfacesterilized with 75% ethanol for 45 s, rinsed three times with sterile water, air dried, and cultured on PDA at 25°C in the dark. Pure cultures were obtained by single-spore isolation after subculture. Ten representative single-spore isolates (MY-1 to MY-10) from 154 pathogens in 10 sampling points were selected for morphological characterization and identification. After 7 days, mycelial colonies were gray to dark gray with few aerial hyphae. Conidia (32.3 to 132.8 × 5.8 to 8.4 µm, average 81.4 × 6.3 µm, n=50) were pale to brown, erect or curved, solitary or in chains, with 0 to 15 pseudosepta. Based on morphological characteristics, the isolates were preliminarily identified as Corynespora cassiicola. Genomic DNA of isolate MY-2 (randomly selected from 10 isolates as representative) was extracted from mycelia using the Ezup DNA extraction kit (Sangon Biotech Co., Ltd. Shanghai, China). The ITS (internal transcribed spacer) region of rDNA, TEF1-α (translation elongation factor 1 alpha) and TUB2 (beta-tubulin) genes were amplified and sequenced with primers ITS4/ITS5, EF1-728F/EF-986R (Wang et al. 2021) and Bt2a/Bt2b (Glass et al. 1995), respectively. BLASTN sequence analyses of ITS (538 bp), TEF1-α (302 bp) and TUB2 (436 bp) of isolate MY-2 (GenBank accessions OK355515, OM339443, OM339442) showed 100%, 97.6%, 100% identity with C. cassiicola in GenBank (Accession numbers JX908713, MW961421, AB539228). A neighbor-joining phylogenetic analysis based on ITS and TEF1-α sequences using MEGA7 showed that MY-2 clustered in the same clade with C. cassiicola. For pathogenicity tests, five S. cusia plants were inoculated onto the adaxial surface of leaves with mycelial plugs from ten isolates of 8-day-oldcultures on PDA. Five leaves per plant were inoculated, covered with wet cotton, and kept in a controlled greenhouse (26~33 °C, RH 80% ~ 90%). Leaves inoculated with sterile PDA plugs served as a negative control. At 3-5 days post inoculation, all 25 inoculated leaves of each isolate showed leaf spot lesions similar to those observed in the field, and control leaves were symptomless. C. cassiicola was successfully reisolated from the diseased leaves. The pathogenicity tests were repeated three times under the same conditions and similar results were observed. In view of morphology, pathogenicity and sequence results, the isolates were identified as C. cassiicola, a pathogen reported from many important crops (Lu et al. 2021). This is the first report of C. cassiicola as a pathogen in China which poses a potential threat to leaf production and S. cusia processing. References: Glass, N. L., et al. 1995. Appl. Environ. Microb. 61:1323 Hu, J.Q., et al. 2011. Flora of China. Science Press, Beijing, China. Volume 19: 407 Li, Q.L., et al. 2013. Plant Dis. 97 (5): 690 Lu, P. et al. 2021. Plant Dis. 105:3753 Wang S. H., et al. 2021.Forest Pathology, 51(2):1 Keywords: fungal disease, Strobilanthes cusia, medicinal plants, etiology, leaf spot.

Indigodole E from Strobilanthes cusia exhibits anti-IL-17A effect.

One new indazole alkaloid, indigodole E (1), was isolated from a traditional Chinese medicine Qing Dai prepared from the aerial parts of Strobilanthes cusia. The structure of 1 was elucidated by NMR, MS, UV, and IR spectra as well as optical rotation. Additionally, compound 1 could obviously inhibit not only IL-17A protein production at concentrations from 1.25 to 2.5 µg/mL, but also IL-17 gene expression at concentrations from 5.0 to 10.0 µg/mL without cytotoxicity toward Th17 and Jukat cells, respectively. Overall, indazole analogue 1 could be the anti-IL 17 A contributor of Qing Dai in this investigation.

High-Quality Genome of the Medicinal Plant Strobilanthes cusia Provides Insights Into the Biosynthesis of Indole Alkaloids.

Strobilanthes cusia (Nees) Kuntze is an important plant used to process the traditional Chinese herbal medicines "Qingdai" and "Nanbanlangen". The key active ingredients are indole alkaloids (IAs) that exert antibacterial, antiviral, and antitumor pharmacological activities and serve as natural dyes. We assembled the S. cusia genome at the chromosome level through combined PacBio circular consensus sequencing (CCS) and Hi-C sequencing data. Hi-C data revealed a draft genome size of 913.74 Mb, with 904.18 Mb contigs anchored into 16 pseudo-chromosomes. Contig N50 and scaffold N50 were 35.59 and 68.44 Mb, respectively. Of the 32,974 predicted protein-coding genes, 96.52% were functionally annotated in public databases. We predicted 675.66 Mb repetitive sequences, 47.08% of sequences were long terminal repeat (LTR) retrotransposons. Moreover, 983 Strobilanthes-specific genes (SSGs) were identified for the first time, accounting for ~2.98% of all protein-coding genes. Further, 245 putative centromeric and 29 putative telomeric fragments were identified. The transcriptome analysis identified 2,975 differentially expressed genes (DEGs) enriched in phenylpropanoid, flavonoid, and triterpenoid biosynthesis. This systematic characterization of key enzyme-coding genes associated with the IA pathway and basic helix-loop-helix (bHLH) transcription factor family formed a network from the shikimate pathway to the indole alkaloid synthesis pathway in S. cusia. The high-quality S. cusia genome presented herein is an essential resource for the traditional Chinese medicine genomics studies and understanding the genetic underpinning of IA biosynthesis.

Blue genome: chromosome-scale genome reveals the evolutionary and molecular basis of indigo biosynthesis in Strobilanthes cusia.

Natural plant dyes have been developed and used across many traditional societies worldwide. The blue pigment indigo has seen widespread usage across South America, Egypt, Europe, India and China for thousands of years, mainly extracted from indigo-rich plants. The utilization and genetic engineering of indigo in industries and ethnobotanical studies on the effects of cultural selection on plant domestication are limited due to lack of relevant genetic and genomic information of dye plants. Strobilanthes cusia (Acanthaceae) is a typical indigo-rich plant important to diverse ethnic cultures in many regions of Asia. Here we present a chromosome-scale genome for S. cusia with a genome size of approximately 865 Mb. About 79% of the sequences were identified as repetitive sequences and 32 148 protein-coding genes were annotated. Metabolic analysis showed that the main indigoid pigments (indican, indigo and indirubin) were mainly synthesized in the leaves and stems of S. cusia. Transcriptomic analysis revealed that the expression level of genes encoding metabolic enzymes such as monooxygenase, uridine diphosphate-glycosyltransferase and ß-glucosidase were significantly changed in leaves and stems compared with root tissues, implying their participation in indigo biosynthesis. We found that several gene families involved in indigo biosynthesis had undergone an expansion in number, with functional differentiation likely facilitating indigo biosynthesis in S. cusia. This study provides insight into the physiological and molecular bases of indigo biosynthesis, as well as providing genomic data that provide the basis for further study of S. cusia cultivation by Asia's traditional textile producers.

Antiviral Action of Tryptanthrin Isolated from Strobilanthes cusia Leaf against Human Coronavirus NL63.

Strobilanthes cusia (Nees) Kuntze is a Chinese herbal medicine used in the treatment of respiratory virus infections. The methanol extract of S. cusia leaf contains chemical components such as ß-sitosterol, indirubin, tryptanthrin, betulin, indigodole A, and indigodole B that have diverse biological activities. However, the antiviral action of S. cusia leaf and its components against human coronavirus remains to be elucidated. Human coronavirus NL63 infection is frequent among immunocompromised individuals, young children, and in the elderly. This study investigated the anti-Human coronavirus NL63 (HCoV-NL63) activity of the methanol extract of S. cusia leaf and its major components. The methanol extract of S. cusia leaf effectively inhibited the cytopathic effect (CPE) and virus yield (IC50 = 0.64 µg/mL) in HCoV-NL63-infected cells. Moreover, this extract potently inhibited the HCoV-NL63 infection in a concentration-dependent manner. Among the six components identified in the methanol extract of S. cusia leaf, tryptanthrin and indigodole B (5aR-ethyltryptanthrin) exhibited potent antiviral activity in reducing the CPE and progeny virus production. The IC50 values against virus yield were 1.52 µM and 2.60 µM for tryptanthrin and indigodole B, respectively. Different modes of time-of-addition/removal assay indicated that tryptanthrin prevented the early and late stages of HCoV-NL63 replication, particularly by blocking viral RNA genome synthesis and papain-like protease 2 activity. Notably, tryptanthrin (IC50 = 0.06 µM) and indigodole B (IC50 = 2.09 µM) exhibited strong virucidal activity as well. This study identified tryptanthrin as the key active component of S. cusia leaf methanol extract that acted against HCoV-NL63 in a cell-type independent manner. The results specify that tryptanthrin possesses antiviral potential against HCoV-NL63 infection.

IL-17A inhibitions of indole alkaloids from traditional Chinese medicine Qing Dai.

ETHNOPHARMACOLOGICAL RELEVANCE: Qing Dai, a famous traditional Chinese medicine (TCM), is prepared by a traditional fermentation process with the aerial part of Strobilanthes cusia. Currently, this TCM could treat various clinical inflammatory diseases, such as ulcerative colitis and psoriasis, however, the bioactive components of Qing Dai are unknown clearly. AIM OF THE STUDY: To isolate and identify the anti-IL-17A components of Qing Dai. MATERIALS AND METHODS: Silica, RP-18 gels, and size exclusion resin were used for column chromatography to isolate the pure compounds. The structures of isolates were elucidated by NMR, MS, UV, IR spectra, and optical rotation. IL-17A protein and gene expressions were also evaluated in the Th17 cell model and luciferase reporter assay, respectively. RESULTS: Two indole alkaloids, including one new indigodole D and cephalandole B, were isolated from Qing Dai. Indigodole D could inhibit IL-17A protein production during the Th17 polarization (EC50: 2.16 µg/mL) or after the polarization (EC50: 5.99 µg/mL) without cytotoxicity toward Th17 cells. Cephalandole B did not inhibit the IL-17A protein secretion. Nevertheless, both isolates notably inhibited IL-17A gene expression, especially cephalandole B, in a dose-dependent manner in Jukat cells with IL-17A luciferase reporter. CONCLUSIONS: Indole alkaloids, indigodoles A, C, D, tryptanthrin, and indirubin could contribute to anti-IL 17A properties of Qing Dai. The possible biogenetic mechanisms of above-mentioned indoles were also speculated in this investigation for further promising anti-IL-17 lead drugs development.

Indole alkaloids indigodoles A-C from aerial parts of Strobilanthes cusia in the traditional Chinese medicine Qing Dai have anti-IL-17 properties.

Qing Dai (Naturalis Indigo) is a traditional Chinese medicine (TCM) used as a topical agent in moderate psoriasis, targeting interleukin-17 (IL-17). In this study, it was prepared from the aerial parts of Strobilanthes cusia. Three undescribed indole alkaloid derivatives, indigodoles A-C, along with seven known compounds were isolated from this preparation of Qing Dai and their structures were elucidated from spectroscopic data, including NMR, MS, UV, IR, optical rotation, and CD. As well, most compounds were tested against IL-17. Indigodole C and tryptanthrin could significantly inhibit IL-17 production of Th17 cells. In addition, indigodole A and indirubin showed notably anti-IL-17 gene expression in dose-dependent effects without cytotoxicities toward Th17 and Jurkat cells, respectively. Overall, our studies indicate that the aforementioned indole alkaloids could contribute to anti-IL 17 properties of Qing Dai.

A novel isocoumarin with anti-influenza virus activity from Strobilanthes cusia.

Strobilanthes A (1), a novel isocoumarin with an unusual tetrahydro-4H-pyran-4-one moiety fused isocoumarin core skeleton, together with a known compound (2) was isolated from Strobilanthes cusia. Its chemical structures were elucidated by 2D NMR spectroscopy, mass spectrometry and single-crystal X-ray diffraction analysis. The biosynthetic pathway of 1 could be supposed to be originally derived from 3-methylisocoumarin, a product of AA-MA pathway. Both of two compounds displayed anti-influenza virus activity in vitro.