Insight into tetrahydrofuran lignans from Isatis indigotica fortune with neuroprotective and acetylcholinesterase inhibitor activity.

Nine tetrahydrofuran lignans, including three undescribed spiro-lignans, were isolated from Isatis indigotica Fortune (Brassicaceae). Extensive spectroscopic analyses achieved the structure elucidation of these tetrahydrofuran lignans, and quantum chemical calculation combined with the MAEΔΔδ parameter. Notably, isatispironeols A-B have a unique spiro[dienone-tetrahydrofuran] molecular core. These spiro[dienone-tetrahydrofuran] lignans showed comparable neuroprotective effects as the positive control in the H2O2-induced SH-SY5Y cells model. In addition, (-)-(7R,8S,1'R,7'R,8'R)-isatispironeol A possessed more significant AChE inhibitory activity, further interact sites were also predicted by the in silico assay.

Sulfonated alkaloids from an aqueous extract of Isatis indigotica roots.

Eleven new sulfonated alkaloids (1 - 11) having diverse structures were isolated from an aqueous extract of the Isatis indigotica root (ban lan gen). Their structures were determined by spectroscopic data analysis, chemical method, and theoretical calculation, of which (-)-4 was proved by single crystal X-ray diffraction.

Computational Workflow to Study the Diversity of Secondary Metabolites in Fourteen Different Isatis Species.

The screening of real features among thousands of ions remains a great challenge in the study of metabolomics. In this research, a workflow designed based on the MetaboFR tool and "feature-rating" rule was developed to screen the real features in large-scale data analyses. Seventy-four reference standards were used to test the feasibility, with 83.21% of real features being obtained after MetaboFR processing. Moreover, the full workflow was applied for systematic characterization of 14 species of the genus Isatis, with the result that 87.72% of real features were retained and 69.19% of the in-source fragments were removed. To gain insights into metabolite diversity within this plant family, 1697 real features were tentatively identified, including lipids, phenylpropanoids, organic acids, indole derivatives, etc. Indole derivatives were demonstrated to be the best chemical markers with which to differentiate different species. The rare existence of indole derivatives in Isatis cappadocica (cap) and Isatis cappadocica subsp. Steveniana (capS) indicates that the biosynthesis of indole derivatives could play a key role in driving the chemical diversity and evolution of genus Isatis. Our workflow provides the foundations for the exploration of real features in metabolomics, and has the potential to reveal the chemical composition and marker metabolites of secondary metabolites in plant fields.

[Cloning and functional characterization of lignan glycosyltransferase gene IiUGT349 in Isatis indigotica].

Based on the transcriptome data of Isatis indigotica, a total of 110 putative glycosytransferases were identified. Through prokaryotic expression and enzymic activity assay in vitro, a novel lignan glycosyltransferase gene was screened out and named IiUGT349, which catalyzed lariciresinol into lariciresinol-4-O-ß-D-glucoside and lariciresinol-4'-O-ß-D-glucoside. Bioinformatics analysis suggested that IiUGT349 contained an open reading frame(ORF) of 1 401 bp encoding a protein of 467 amino acids. A protein analysis indicated that IiUGT349 have a predecited molecular weight of 52.77 kDa and pI of 5.96. Phylogenetic analysis showed that IiUGT349 belonging to UGT90 family shared low amino acid sequence identity with the reported lignan glycosyltransferases, which may represent a novel type of lignan glycosyltransferases. Quantitative real-time PCR(qRT-PCR) analysis showed that IiUGT349 was expressed in roots, stems, young leaves and leaves, with the highest expression level in stems. Further biochemical analysis showed that the optimal reaction time of IiUGT349 recombinant protein was 12 h and the optimal temperature was 45 ℃. Subcellular localization demonstrated that IiUGT349 was located in the cytoplasm and nucleus of plants. In this study, a new glucosyltransferase gene IiUGT349 from I. indigotica belonging to the UGT90 family was cloned, which laid a foundation to further investigate its' function and elucidate the lignan glycosides biosynthesis pathway and plays an important role for great significance for the synthetic biology of active lignan glycosides.

Depicting the inhibitory potential of polyphenols from Isatis indigotica root against the main protease of SARS CoV-2 using computational approaches.

The pandemic disease COVID-19, caused by SARS CoV-2, has created a global crisis. Presently, researchers across the globe are in a quest to identify/develop drugs or vaccines by targeting different non-structural proteins (Nsps) of SARS CoV-2. One such important drug target is Nsp5/main protease (Mpro) which plays a critical role in the viral replication. This cysteine protease/Mpro of SARS CoV-2 has high sequence similarity with the same protease from SARS CoV-1. Previously, it has been shown experimentally that eight polyphenols derived from the root of Isatis indigotica show inhibitory effect on the cleavage/catalytic activity of the SARS CoV-1 Mpro. But whether these polyphenols exhibit any inhibitory effect on SARS CoV-2 Mpro is unclear. To explore this possibility, here, we have adopted various computational approaches. Polyphenols that qualified the pharmacological parameters (indigo, sinigrin, hesperetin and daidzein) and two well-known Mpro inhibitors (N3 and lopinavir) were subjected to molecular docking studies. Two of them (sinigrin and hesperetin) were selected by comparing their binding affinities with N3 and lopinavir. Sinigrin and hesperetin interacted with the two most important catalytic residues of Mpro (His41 and Cys145). Molecular dynamics studies further revealed that these two Mpro-polyphenol complexes are more stable and experience less conformational fluctuations than Mpro-N3/lopinavir complex. The Mpro-hesperetin complex was more compact and less expanded than Mpro-sinigrin complex. These findings were additionally validated by MM-GBSA analysis. As a whole, our study revealed that these two polyphenols may be potent SARS CoV-2 Mpro inhibitors and may possibly be considered for COVID-19 treatment.

Polysaccharides derived from Chinese medicinal herbs: A promising choice of vaccine adjuvants.

Adjuvants have been used in vaccines for a long time to promote the body's immune response, reducing vaccine dosage and production costs. Although many vaccine adjuvants are developed, the use in human vaccines is limited because of either limited action or side effects. Therefore, the development of new vaccine adjuvants is required. Many studies have found that natural polysaccharides derived from Traditional Chinese medicine (TCM) possess good immune promoting effects and simultaneously improve humoral, cellular and mucosal immunity. Recently polysaccharide adjuvants have attracted much attention in vaccine preparation because of their intrinsic characteristics: immunomodulation, biocompatibility, biodegradability, low toxicity and safety. This review article systematically analysed the literature on polysaccharides possessing vaccine adjuvant activity from TCM plants, such as Astragalus polysaccharide (APS), Rehmannia glutinosa polysaccharide (RGP), Isatis indigotica root polysaccharides (IRPS), etc. and their derivatives. We believe that polysaccharide adjuvants can be used to prepare the vaccines for clinical use provided their mechanisms of action are studied in detail.

Isatidis Radix and Isatidis Folium: A systematic review on ethnopharmacology, phytochemistry and pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Isatidis Radix (called Banlangen, BLG in Chinese) and Isatidis Folium (called Daqingye, DQY in Chinese) are common traditional edible-medicinal herbs in detoxifying for thousands of years, have been traditionally applied in traditional Chinese medicine for centuries. Both of them are bitter in taste, coolness in nature, acting on the heart and stomach channels. They are often used to treat influenza and other viral infectious diseases in clinic, as well as could treat fever, dizziness, and cough and sore throat caused by lung heat. AIMS OF THE REVIEW: This review aimed at summarizing the latest and comprehensive information of BLG and DQY on the ethnopharmacology, phytochemistry, pharmacology, toxicity and clinical application to explore the therapeutic potential of them. In addition, outlooks and perspective for possible future researches that related are also discussed. MATERIALS AND METHODS: Related information concerning BLG and DQY were gathered from the internet database of Google Scholar, PubMed, Baidu Scholar, GeenMedical, CNKI and Web of Science, as well as other relevant textbooks, reviews, and documents (e.g., Chinese Pharmacopoeia, 2020 edition, Chinese herbal classic books and PhD and MSc thesis, etc.). Among of them with the keywords including "Isatis indigotica" "Isatidis Radix", "Isatidis Folium", "phytochemistry", "pharmacology", "toxicology", "clinical application" etc. and their combinations. RESULTS: To date, 39 Chinese patent medicines containing BLG and/or DQY have been developed on basis of the data of NMPA. Besides, 304 and 142 compounds have been found in BLG and DQY, respectively. The main chemical differences between BLG and DQY were concentrated on alkaloids and lignans, such as indican, indirubin, (R, S)-epigoitrin, 4(3H)-quinazolinone, clemastanin B and isatindigotindolines A-D. In 2020 Edition ChP, (R, S)-goitrin and indirubin are now used as the official marker to monitor the quality of BLG and DQY, respectively. Modern pharmacology has mainly studied some monomer components such as 4(3H)-quinazolinone, clemastanin B, erucic acid and adenosine, etc., all of which have shown good effects. These active compounds can resist various viruses, such as influenza virus, respiratory syncytial virus, herpes simplex virus, etc.. By regulating the level of immunity and a variety of inflammatory factors, inhibit the growth and reproduction of the virus. At the same time, it is worth noting that different components of BLG and DQY lead to BLG is more powerful in antiviral and immunomodulatory activity than DQY, while DQY possesses a higher intensity than BLG in anti-oxidant activity. CONCLUSION: By collecting and collating a large number of literature and various data websites, we concluded that the common compounds are mainly alkaloids. Recent findings regarding the phytochemical and pharmacological properties of BLG and DQY have confirmed their traditional uses in antiviral, antibacterial and treatment immune diseases. Without doubt, their significant differences on ethnopharmacology, phytochemistry and pharmacology can be used as evidence of separate list of BLG and DQY. For shortcomings, some comprehensive studies should be well designed for further utilization of BLG and DQY.

Anti-microtubule activity of the traditional Chinese medicine herb Northern Ban Lan (Isatis tinctoria) leads to glucobrassicin.

Traditional Chinese medicine (TCM) belongs to the most elaborate and extensive systems of plant-based healing. The herb Northern Ban Lan (Isatis tinctoria) is famous for its antiviral and anti-inflammatory activity. Although numerous components isolated from I. tinctoria have been characterized so far, their modes of action have remained unclear. Here, we show that extracts from I. tinctoria exert anti-microtubular activity. Using time-lapse microscopy in living tobacco BY-2 (Nicotiana tabacum L. cv Bright Yellow 2) cells expressing green fluorescent protein-tubulin, we use activity-guided fractionation to screen out the biologically active compounds of I. tinctoria. Among 54 fractions obtained from either leaves or roots of I. tinctoria by methanol (MeOH/H2 O 8:2), or ethyl acetate extraction, one specific methanolic root fraction was selected, because it efficiently and rapidly eliminated microtubules. By combination of further purification with ultra-high-performance liquid chromatography and high-resolution tandem mass spectrometry most of the bioactivity could be assigned to the glucosinolate compound glucobrassicin. Glucobrassicin can also affect microtubules and induce apoptosis in HeLa cells. In the light of these findings, the antiviral activity of Northern Ban Lan is discussed in the context of microtubules being hijacked by many viral pathogens for cell-to-cell spread.

A Network Pharmacology-Based Investigation to the Pharmacodynamic Material Basis and Mechanisms of the Anti-Inflammatory and Anti-Viral Effect of Isatis indigotica.

PURPOSE: Isatis indigotica (Ii) is a cruciferous herb that is widely distributed in China, and its roots and leaves have been used in two renowned antipyretic detoxicate crude drugs in Chinese Pharmacopoeia, Radix (R) and Folium (F) Isatidis. However, the pharmacodynamic material basis and underlying mechanisms of the herbal efficacy remained to be elucidated. METHODS: Ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was adopted for the chemical profiling of R and F Isatidis. The active ingredients were screened out through the prediction of gastrointestinal absorption and druglikeness analysis using SwissADME. A herb-ingredient-target network was constructed through target prediction of the herbal active ingredients and anti-inflammation or anti-viral properties, followed by protein-protein interaction analysis. Then, the potential relevant signaling pathways were predicted by pathway enrichment. Finally, for verification, RAW 264.7 cell line was adopted to examine the anti-inflammatory and anti-viral activities of 6 representative ingredients in Ii. RESULTS: Seventy-three compounds have been identified from Ii through UPLC-Q-TOF-MS. A total of 17 potential active ingredients were screened through pharmacokinetics and drug-likeness evaluation using SwissADME. It was shown that key targets might include TNF, AKT1, SRC, IL2, CASP9, and CASP3 in our herb-ingredient-target network, and isovitexin, a flavonoid, tended to participate in the inflammatory response, indoles were more likely to affect the cell proliferation processes, and lignans might have a broader affinity to key targets than the other active ingredients, such as regulating immune system (targeting IL-2) and PI3K-Akt signaling pathway. In vitro, indigo and secoisolariciresinol diglucoside markedly reduced TNF-α expression in Poly (I: C)-incubated cells. Isovitexin significantly inhibited TNF-α expression, and isatin treatment markedly reduced IL-1ß expression in LPS-incubated cells. CONCLUSION: As the pharmacodynamics material basis of Ii, indoles, lignans, and flavonoids are believed to confer beneficial properties through various cellular aspects with multiple signaling pathways involved.

The indole alkaloids from the roots of Isatidis Radix.

The root of Isatis indigotica is used as a traditional Chinese medicine (termed Isatidis Radix) due to its antiviral effects. We examined compounds isolated from Isatidis Radix and elucidated the structures of three new natural alkaloids, and we examined the possible mechanisms or active targets of indole alkaloids occurring in blood of rats treated by gavage. Three new natural products were isolated from Radix Isatidis for the first time, including 1-methoxy-2-indoleacetonitrile, 1-hydroxy-3-indoleacetonitrile, 8-Methoxy-1, 2-dihydroquinoline, and 4 compounds isolated from this medicinal material for the first time. Their structures were elucidated using nuclear magnetic resonance. The components of Isatidis Radix were analysed using liquid chromatography tandem mass spectrometry, and 33 compounds were detected in plasma of treated rats; 24 of these compounds were indole alkaloids, and they included the newly identified compounds. Molecular docking and in vitro antithrombin activity tests showed HA inhibition activity of indoleacetonitriles.

Indole alkaloid glycosides from Isatis tinctoria roots.

Isatindigoside A and B (1 - 2), two new indole alkaloid glycosides along with five known ones (3 - 7) were obtained from the roots of I. tinctoria. Their structures were determined as isatindigoside A (1), isatindigoside B (2), isatindosulfonicacid A 3-O-ß-D-glucopyranoside (3), indole-3-acetonitrile 6-O-ß-D-glucopyranoside (4), isatindigobisindoloside A (5), isatindigobisindoloside B (6) isatindigobisindoloside F (7), by physicochemical properties and spectroscopic methods including 1 D, 2 D NMR, IR, HR-ESI-MS data. Nitric oxide (NO) inhibitory activities of all of the isolated compounds (1 - 7) were also evaluated. Compounds 2 and 7 showed inhibitory effects against LPS-stimulated RAW 264.7 cells with IC50 values of 27.6 µM and 18.8 µM, respectively.

Isatisindigoticanine A, a novel indole alkaloid with an unpresented carbon skeleton from the roots of Isatis tinctoria.

Isatisindigoticanine A (1), a new indole alkaloid with an unusual carbon skeleton of a benzofuran-3-one unit connected with a 1H-indole-3-yl unit and a 4-(1,2-dihydroxyethyl)-6-oxa-3-azabicyclo[3.1.0]hexan-2-one unit via a C-3－C-8'' bond and a C-4'－C-8'' bond, was obtained from the roots of Isatis tinctoria. Its structure was determined by physicochemical properties and spectroscopic methods including 1 D, 2 D NMR, IR, HRESIMS data. The absolutely configurations were deduced by comparison of its experimental CD and calculated ECD spectra. Nitric oxide (NO) inhibitory activities of isatisindigoticanine A was also evaluated in the LPS-stimulated RAW 264.7 cells, however, no inhibitory effect was presented.

Development of a thin-layer chromatography bioautographic assay for neuraminidase inhibitors hyphenated with electrostatic field induced spray ionisation-mass spectrometry for identification of active Isatis indigotica root compounds.

The identification of neuraminidase inhibitors from natural products is a promising strategy in the field of anti-influenza research. In this study, a new thin-layer chromatography (TLC) bioautographic assay for the screening of neuraminidase inhibitors from natural products was developed. This TLC bioassay is based on the one-step reaction of neuraminidase with the sodium salt of 5­bromo­4­chloro­3-indolyl-α-d-N-acetylneuraminic acid (substrate) and the subsequent formation of blue coloured products. Neuraminidase inhibitory activity was shown by the development of white spots against the blue TLC background. The key factors affecting the assay (such as enzyme concentration, substrate concentration, incubation time, reaction time, and pH) were investigated and optimised by a combination of a one-factor-at-a-time design and a Box-Behnken design/response surface method. The developed TLC bioautographic method was applied to identify neuraminidase inhibitory compounds in the roots of Isatis indigotica. Eleven active compounds including six alkaloids, three lignans, one sterol, and one fatty acid were identified in situ by direct coupling with an electrostatic field induced spray ionisation-mass spectrometry approach through analysis of their MSn (n = 4) data or comparison with reference substances. The developed TLC bioautographic assay is simple, rapid, and efficient for screening potential neuraminidase inhibitors from natural products.

Bisindole alkaloids with nitric oxide inhibitory activities from an alcohol extract of the Isatis indigotica roots.

Eight bisindole alkaloids including six undescribed ones (1a/1b-5) were isolated from an alcohol extract of the Isatis indigotica roots. Their structures and absolute configurations were supported by extensive spectroscopic data analysis, including 1D, 2D NMR, HRESIMS data, specific rotation data, and comparison of the experimental and calculated ECD data. Compounds 1a and 1b were determined to be a pair of enantiomers with a ratio of approximately 1:1 by chiral-phase chromatography analysis while compound 4 was elucidated as a new type of bisindole alkaloid with the aglycone categorized as bis(indole-1'/3″-yl)methane. All the isolated compounds were tested for their nitric oxide (NO) inhibitory effects and 1-4 and 6 exhibited inhibitory effects with IC50 values ranging from 11.0 to 37.6 µM.

Combination of N, N'-dicyclohexyl-N-arachidonic acylurea and tacrolimus prolongs cardiac allograft survival in mice.

Current immunosuppressive agents for organ transplantation are not ideal because of their strong toxicity and adverse effects. Hence, there is an urgent need to develop novel immunosuppressive agents. The compound N, N'-dicyclohexyl-N-arachidonic acylurea (DCAAA) is a novel highly unsaturated fatty acid from the traditional Chinese medicinal plant Radix Isatidis. In this study, we systematically investigated the toxicity, immunosuppressive effect and mechanisms underlying the activity of DCAAA. The toxicity tests showed that DCAAA treatment did not lead to red blood cell hemolysis and did not affect the liver and kidney functions in mice. The lymphocyte transformation test showed that DCAAA treatment inhibited lymphocyte proliferation in a dose-dependent manner. An in vivo cardiac allotransplantation experiment showed that DCAAA treatment could suppress the immune rejection and significantly prolong the survival of cardiac allografts in recipient mice by reducing the proportion of CD4+ T cells in the spleen and grafts, concentration of interferon-γ in the supernatant and serum and infiltration of inflammatory cells into the grafts. Moreover, a combination treatment with DCAAA and tacrolimus had a synergistic effect in preventing acute rejection of heart transplants. In vitro molecular biology experiments showed that DCAAA treatment inhibited activation of the T-cell receptor-mediated phosphoinostide 3-kinase-protein kinase B pathway, thereby arresting cell cycle transition from the G1 to the S phase, and inhibiting lymphocyte proliferation. Overall, our study reveals a novel, low-toxicity immunosuppressive agent that has the potential to reduce the toxic side effects of existing immunosuppressive agents when used in combination with them.

Discovery, synthesis, and optimization of an N-alkoxy indolylacetamide against HIV-1 carrying NNRTI-resistant mutations from the Isatis indigotica root.

From an aqueous decoction of the traditional Chinese medicine "ban lan gen" (the Isatis indigotica root), an antiviral natural product CI - 39 was isolated as an NNRTI (non-nucleoside reverse transcriptase inhibitor) (EC50 = 3.40 µM). Its novel structure was determined as methyl (1-methoxy-1H-indol-3-yl)acetamidobenzoate by spectroscopic data and confirmed by single crystal X-ray diffraction. Through synthesis and structure-activity relationship (SAR) investigation of CI - 39 and 57 new derivatives (24 with EC50 values of 0.06-8.55 µM), two optimized derivatives 10f and 10i (EC50: 0.06 µM and 0.06 µM) having activity comparable to that of NVP (EC50 = 0.03 µM) were obtained. Further evaluation verified that 10f and 10i were RT DNA polymerase inhibitors and exhibited better activities and drug resistance folds compared to NVP against seven NNRTI-resistant strains carrying different mutations. Especially, 10i (EC50 = 0.43 µM) was more active to the L100I/K103N double-mutant strain as compared to both NVP (EC50 = 0.76 µM) and EFV (EC50 = 1.08 µM). The molecular docking demonstrated a possible binding pattern between 10i and RT and revealed activity mechanism of 10i against the NNRTI-resistant strains.

Antiviral activities of Radix isatidis polysaccharide against pseudorabies virus in swine testicle cells.

BACKGROUND: Radix isatidis has been used in China and other Asian countries for its antiviral and anti-inflammatory effects for thousands of years. However, the antiviral effect of Radix isatidis polysaccharide against pseudorabies virus (PRV) is still unknown. METHODS: The polysaccharide were isolated from extract of the roots of Radix isatidis. MTT assays were used to determine the preventive effect, inhibitory effect and antiviral effect of Radix isatidis polysaccharide on PRV in vitro. RESULTS: This study found that different concentrations of polysaccharides from this plant can inhibit PRV replication by 14.674-30.840%, prevent infection at rates of 6.668-14.923%, and kill this virus at rates of 32.214-67.422%. CONCLUSION: These results broaden the understanding of this traditional Chinese herb and provide a theoretical basis for further research. Moreover, Radix isatidis polysaccharide could be used for antiviral therapy.

Four pairs of alkaloid enantiomers from Isatis indigotica Fortune Ex Land with neuroprotective effects against H2O2-induced SH-SY5Y cell injury.

In the current study, four pairs of new enantiomeric alkaloids (1a/1b-4a/4b) were obtained from the leaves of Isatis indigotica Fortune Ex Land. Their structures were elucidated through spectroscopic methods and quantum mechanical calculations. Biologically, all isolates were evaluated for their neuroprotective effects against H2O2-induced SH-SY5Y cell injury. As a result, 1a and 1b exhibited enantioselective neuroprotective effects, further Annexin V-FITC/PI analysis showed that apoptosis ratios of 1a and 1b were reduced to 20.93% and 17.87%, respectively.

Performance of polyvinyl pyrrolidone-isatis root antibacterial wound dressings produced in situ by handheld electrospinner.

Antibacterial dressings are an increasingly important tool for the prevention and management of wound infections, particularly in light of concerns surrounding conventional drug-resistant antibiotics. Handheld electrospinning devices provide opportunities for the rapid application of antibacterial dressing materials to wounds, but spinning formulations need to be compatible with live biological surfaces. We report the development of a new antibacterial formulation compatible with handheld electrospinning, and its manufacture directly on a wound site. Nanofibrous dressing mats were produced from polyvinyl pyrrolidone (PVP) containing isatis root (Indigowoad root or Ban-Lan-Gen), a traditional Chinese medicine, commonly used for the treatment of infectious disease. The resulting wound dressing mats of PVP/isatis root exhibited well-defined fibrous structures and excellent surface wetting, and permeability characteristics. The presence of isatis root conferred antibacterial activity against gram negative and gram positive strains. Moreover, in a Kunming mouse skin injury model, direct electrospinning of PVP/isatis root formulations on to wound sites produced near complete wound closure after 11 days and epidermal repair in histological studies.

Alkaloid Enantiomers from Isatis tinctoria with Neuroprotective Effects against H2O2-Induced SH-SY5Y Cell Injury.

Six pairs of alkaloid enantiomers including 11 new alkaloids (1A: /1B: -5A: /5B, 6A: ) were isolated from the leaves of Isatis tinctoria. Their structures were established by extensive spectroscopic analyses. Enantiomers were separated successfully by chiral chromatographic column and the absolute configurations of all isolates were determined by comparison of experimental and calculated electronic circular dichroism spectra. The isolated alkaloids were evaluated for their neuroprotective activities against H2O2-induced cell injury in human neuroblastoma SH-SY5Y cells. The results showed that 5A/5B: and 6A/6B: exhibited potent neuroprotective activities at 50 µM compared with the H2O2-treated group.