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• Eleven new species are described. • New infraspecific combination created for I. pinnata. • All species illustrated in detailed photographic plates. • Conservation assessment and IUCN red list status is recommended for every species. Indigofera sect. Brachypodae DC. is a large group of approximately 40 species within the Cape Clade, mostly restricted to the Core Cape Region (CCR), with only two species extending eastwards to Grahamstown. This section has been subdivided into four subsections based primarily on morphological characters. Subsect. Brachypodae is the most diverse of the four subsections and is characterised by having sclerophyllous leaves, with a distinctly sunken midvein and (usually) revolute leaf margins. This group occurs almost exclusively within Fynbos, with a few species growing in coastal Strandveld vegetation. Revised here, nineteen species are recognized in this subsection, eleven of which are new to science. Additionally, a new infraspecific combination is created for I. pinnata following nomenclatural and identification confusion. All species are formally described here, with photographic plates of each and illustrations of corollas and calyces provided. Many of the new species were either unrecognised at the time of the last revision in Flora Capensis 2, or have only been discovered recently. Distribution maps and information regarding habitat preference, phenology, and ecology and recommended IUCN red list status of each species are also provided. [ABSTRACT FROM AUTHOR]

• Taxonomic description of new and little known Indigofera species from South Africa. • Four new Indigofera species, one new variety and two little known species are described. • One established taxon is raised to specific status and described with a new name. • An earlier name is found which has priority over an established species name. • All taxa are associated with the Pondoland Centre of Endemism, ranging to surrounding floristic areas. This second paper describes and clarifies new and poorly known Indigofera species, belonging to sect. Pinnatae (E.Mey.) Harv., from the Pondoland Centre of Endemism and associated neighbouring areas in the Eastern Cape and KwaZulu-Natal provinces, South Africa. Five new species (one with two infraspecific taxa) are recognized: Indigofera abbottii Schrire and K.W.Grieve , I. hillburttii Schrire and K.W.Grieve , I. tysonii Schrire and K.W.Grieve , I. herrstreyi Schrire and K.W.Grieve var. herrstreyi and I. herrstreyi var. helodes K.W.Grieve and Schrire, and I. ngwenyana Schrire and K.W.Grieve. Descriptions are provided for the poorly known I. corniculata E.Mey. and I. gerrardiana Harv., taxa rarely taken up in the literature and both having been mostly synonymised under I. dregeana E.Mey. that occurs outside the area of the present study. The name I. tenuissima E.Mey. was found to be the earliest, correct, name for the widespread species I. longipes N.E.Br. ex Baker f., leaving the coastal Zululand taxon – currently named I. tenuissima E.Mey. – requiring further evaluation. [ABSTRACT FROM AUTHOR]

• Four species new to science are described. • Detailed photographic plates and distribution maps are included. • Conservation assessments are provided for all four species. Four new Indigofera species are described from the Greater Cape Floristic Region (GCFR). Indigofera barkeri is a new annual from the Richtersveld, with small flowers, 3.5–4.0 mm long, pinnately trifoliolate leaves and laterally flattened seeds. Indigofera dodii is endemic to the area around Cape Town, growing on granite and shaly slopes. It has apically congested racemes, flowers 6.0–6.5 mm long, digitately trifoliolate leaves, fruit with a hispid indumentum and tuberculate seed testas. Indigofera gariepensis , endemic to dry riverbeds in the eastern Richtersveld, is recognized by its alternate, silky-velutinous leaflets and calyx lobes ± equal in length to its 5.5–6.5 mm long magenta flowers. Indigofera tanquana is known from only two collections and is endemic to the western Tanqua Karoo. It is easily recognized by its broad old grey stems, spinescent branches, simple grey-canescent leaves that aggregate on woody brachyblasts and reddish-magenta flowers. Detailed morphological descriptions are presented with composite photographic plates for each species. Notes on ecology, detailed diagnoses comparing with closely related species, a distribution map and an IUCN red list conservation assessment for each species is also provided. [ABSTRACT FROM AUTHOR]

• Indigofera heterantha is an important medicinal shrub. • Melatonin is an important growth promoter that elicite production of secondary metabolites. • Adventitious roots conserve the plants by producing pharmacologically important secondary metabolites. • Melatonin enhances the effectiveness of AgNPs synthesis in in-vitro culture. Indigofera heterantha is a medicinal shrub of the family Fabaceae that naturally produces pharmaceutically important polyphenolics and at the verge of extinction due to ruthless exploitation. The adventitious roots provide bases for the conservation of endangered plants by decreasing pressure on their wild populations and help in the large-scale production of pharmacologically important secondary metabolites. The main objective of the current study was to establish a feasible protocol for adventitious roots and to investigate the effect of melatonin on the biosynthesis of biomass, secondary cell products, and greener synthesis of silver nanoparticles (AgNPs) for various biological activities. Melatonin is an important growth promoter and plays a key role in plant defense that can be used as elicitors for large-scale production of secondary metabolites. Different concentrations of melatonin revealed that 1.5 mg l−1 melatonin was effective for the biosynthesis of maximum biomass during log phases of growth kinetics. The same melatonin concentration enhanced total phenolics content (TPC; 68.28 mg/g-DW), total flavonoids content (TFC; 24.34 mg/g-DW), total phenolics production (TPP; 153.63 g l−1), total flavonoids production (TFP; 54.80 g l−1), antioxidant activity and total secondary metabolites in adventitious roots. The melatonin-treated roots (300 mM) were able to produce maximum biogenic AgNPs, characterized by UV–vis spectroscopy (UV), scanning electron microscope (SEM), Fourier transform infrared (FTIR), and X-ray diffraction (XRD) and were used for various biological activities. These NPs displayed maximum antimicrobial activities and further revealed that AgNPs and aqueous root extract displayed higher cytotoxic activities. The protocol developed here provides an alternative strategy for germplasm conservation and melatonin proved to be one of the best elicitors for different activities and has the potential of scaling up by various pharmaceutical industrial applications. [ABSTRACT FROM AUTHOR]

• Taxonomic description of new and poorly known Indigofera species from South Africa. • Four new Indigofera species, two new varieties and one little known species are described. • One established species, but with an illegitimate name, is described with a new name. • All taxa are associated with the Pondoland centre of Endemism, ranging to surrounding floristic areas. • Indigofera is the third largest Fabaceae genus in South Africa, still with many undescribed species. New and poorly known Indigofera species from the Pondoland Centre of Endemism and associated surrounding areas in the Eastern Cape and southern KwaZulu-Natal provinces, South Africa are described and clarified. Four new species (one with two infraspecific taxa) are recognized: Indigofera huntleyi var. huntleyi Schrire and K.W.Grieve and I. huntleyi var. angustifolia Schrire and K.W.Grieve, I. pondoensis Baker f. ex Schrire and K.W.Grieve, I. hydra Schrire and K.W.Grieve and I. vanwykii Schrire and K.W.Grieve. A description is provided for the poorly known I. grata E.Mey. and a new name, I. sylvicola Schrire and K.W.Grieve published for the illegitimately named I. micrantha E.Mey. [ABSTRACT FROM AUTHOR]

• A taxonomic revision of section Oligophyllae is presented • Four new species to science are described • Indigofera nigromontana / spinescens species complex is resolved • Detailed photographic plates and distribution maps are included • Updated conservation assessments provided for all nine species Indigofera L. section Oligophyllae DC. comprises nine species, phylogenetically located within the Cape Clade, largely restricted to the Greater Cape Floristic Region (GCFR) of South Africa. The group is characterised by having mostly trifoliolate, glabrous or sparsely hairy leaves that emerge from woody brachyblasts, bracts that often remain persistent post anthesis, and fruits sparsely strigose to glabrous. In this study, nine species are recognized in section Oligophyllae and are described and photographically illustrated. Distribution maps and information regarding habitat preference and ecology and recommended IUCN red listings are also provided. Indigofera nigromontana was found to include three morphologically and geographically distinct species. The type is in the Garden Route District of the GCFR and retains the name I. nigromontana. A Namaqualand species, I. spinescens , was previously treated as a synonym of I. nigromontana, but is here recognized as a separate species, and a Drakensberg foothills species that has often been referred to as I. spinescens , is here also recognized as a new species, I. basotha. Indigofera ernstii is easily distinguished by its 5–9 foliolate leaves with a long petiole and rachis, found in the far northern reaches of the GCFR. Indigofera salteri has the largest flowers in this section, with 3–5 foliolate leaves and is known from a single location near Clanwilliam. Indigofera cogmaniana bears some resemblance to I. denudata , but differs in having orbicular leaflets, subsessile 2-flowered inflorescences with 3–6 mm long pedicels, a tiny calyx and is only found around the town of Montagu. [ABSTRACT FROM AUTHOR]

The flowering plant Indigofera heterantha generally known as Indigo of Himalayan, was extracted with n-hexane, ethyl acetate, and methanol by using Soxhlet apparatus. The extracts were studied for their antioxidants and enzyme inhibition activities. Against DPPH free radical-scavenging activity methanol extract was found moderately active (IC 50 : 158.41 ± 1.1.66 µg/mL). The ethyl acetate and n-hexane extracts exhibited the best ABTS radical scavenging activity (IC 50 : 31.32 ± 2.50 and 52.0 ± 0.89 µg/mL respectively). Against the metal chelating activity, n-hexane extract was active IC 50 : 52.36 ± 0.95 µg/mL. The n-hexane and ethyl acetate extracts demonstrated the best β –carotene antioxidant activity (IC 50 : 22.32 ± 0.84 and 60.66 ± 0.35 μg/mL respectively). Against acetylcholinesterase (AChE), ethyl acetate and methanol extracts were moderate active (IC 50 : 133.58 ± 0.870, and 137.97 ± 0.97 µg/mL respectively). The ethyl acetate extract showed Butyrylcholinesterase (BChE) inhibitory activity with IC 50 value of 121.9 ± 1.13 µg/mL. Against tyrosinase activity, ethyl acetate and n-hexane extracts activity with an IC 50 value of 79.25 ± 0.15 and 131.48 ± 0.88 µg/mL respectively. The phenolic contents of the methanolic extract of I. heterantha were also determined by using LC-MS/MS. The major constituents were Quinic acid (57,333.04 μg/g), malic acid (1135.16 μg/g), gallic acid (201.13 μg/g), isoquercitrin (113.63 μg/g), Rutin (92.97 μg/g), and Salicylic acid (40.45 μg/g). Keeping in view the overall biological activities of I. heterantha, it might be used in food industries and pharmaceuticals as a potential functional food ingredient. [ABSTRACT FROM AUTHOR]

Indigo and indirubin are derived from indoxyl molecules, which generally occur as indoxyl glycosides in woad (Isatis tinctoria L.) and other indigo-producing plants. Indoxyl glycosides are biosynthesized from indole via 3-hydroxylation to form indoxyl, followed by one or more glycosylations. Enzymes that attach and remove sugars to and from indoxyl have already been isolated and characterized, while enzymes that convert indole into indoxyl in plants have remained elusive, until the identification of P450s and flavin-containing monooxygenases that hydroxylate indole. A P450 gene from woad (named CYP71B102) was heterologously expressed in E. coli , resulting in the formation of indigo and indirubin, as well as isatin and 2-oxindole, which along with indoxyl are putative precursors of indirubin. The addition of either isatin or 2-oxindole to the recombinant E. coli reduced the levels of indigo and increased the amount of indirubin, whereas coexpression of CYP71B102 with isatin hydroxylase (which degrades isatin) increased the levels of indigo and decreased the amount of indirubin, albeit slightly. The results suggest that CYP71B102 hydroxylates indole at both the 2- and 3- positions to produce 2-oxindole and indoxyl, respectively, and that the coupling of indoxyl with either 2-oxindole or isatin forms indirubin, while dimerization of indoxyl forms indigo. This P450 gene is thus likely involved in the biosynthesis of indirubin in woad, as well as the formation of indigo and its glycosidic precursors, even if other types of enzymes, such as flavin-containing monooxygenases, may be involved in indole hydroxylation in other indigo-producing plants. [Display omitted] • P450 enzyme from Isatis tinctoria produces indigo and indirubin when expressed in E. coli. • Indirubin production by CYP71B102 is increased by addition of either 2-oxindole or isatin. • Isatin hydroxylase, which degrades isatin, increases indigo production by CYP71B102. • P450s may be involved in the biosynthesis of indirubin and indigo precursors in plants. [ABSTRACT FROM AUTHOR]

Herbivory is one of the key processes shaping life history traits linked to plant fitness and the leaf-cutting ants are amongst the most voracious and polyphagous herbivores of the Neotropics. They extensively harvest aboveground plant parts to grow their symbiotic fungus, including vegetative and reproductive tissues. Although leaf-cutting ant herbivory is expected to reduce plant reproductive success, the potentially different outputs of damage to vegetative and reproductive parts are still poorly known. This study investigates the influence of Atta opaciceps herbivory on the reproductive success of three plant species native to the Caatinga dry forest: Croton argyrophylloides (Euphorbiaceae), Cenostigma pyramidale , and Indigofera suffruticosa (Leguminosae). We selected 10 adult and active nests of A. opaciceps based on the presence of individuals of the three focal plant species in their vicinity. For each colony, we marked four individuals of each plant species (totaling 12 individuals per colony), which were then subjected to one of four herbivory treatments: (1) ant herbivory on both reproductive and vegetative parts, (2) manually simulated herbivory on the reproductive parts alone, (3) ant herbivory exclusively on vegetative parts, and (4) no herbivory. From January to April 2020, we monitored all 120 individuals, recording the number of inflorescences, flower buds, flowers, fruits, and the proportion of flowers maturing into fruits (fruit set, as a proxy of reproductive success). Results revealed variations in reproductive structure production and divergent responses to leaf-cutting ant herbivory among our focal species. In terms of inflorescence, flower bud production, and fruit set C. argyrophylloides showed reduced numbers when subjected to higher levels of total herbivory (reproductive and vegetative parts), but increased inflorescence and flower bud production when plants suffered only leaf damage. Cenostigma pyramidale individuals under partial herbivory displayed lower flower bud numbers than protected ones. In contrast, I. suffruticosa displayed conflicting results, with fully exposed plants producing more inflorescences than partially exposed counterparts and more flowers than leaf-damaged and protected individuals. Despite the varied effects on different plant parts across all three study species, the overall reproductive success (i.e. fruit set) of only one species (C. argyrophylloides) was significantly reduced by leaf-cutting ant herbivory. The resilience of the two other plant species to leaf-cutting ant pressure may be attributed to compensatory mechanisms, shedding light on the intricate interplay between herbivores and plants in the Caatinga dry forest. As leaf-cutting ants proliferate in response to anthropogenic disturbances, the patterns described in this study may become more pronounced, potentially affecting the organization of plant communities in the Caatinga dry forest. [Display omitted] • Leaf-cutting ants' herbivory alters the production of inflorescences, flowers and fruits of plant species in the Caatinga dry forest. • Damage to reproductive or vegetative parts similarly affects the production of reproductive plant parts. • Fruit set remains unaffected by leaf-cutting ants in certain plant species but are reduced in others. • Caatinga dry forest plant species exhibit divergent reproductive responses to leaf-cutting ant herbivory. [ABSTRACT FROM AUTHOR]

• Biological and chemical profile of Indigofera argentea, a medicinal plant from Pakistan was assessed. • Flavonoids and lithocholic acid derivatives of steroids were major compounds. • All extracts showed promising results against α-glucosidase and carbonic anhydrase. • Atalanine showed higher inhibition affinity against the three enzymes in silico. The study aimed to assess the phytochemical composition and enzyme inhibitory potential of methanol, n -hexane, chloroform, and n -butanol extracts/fractions of Indigofera argentea Berm. f. (whole plant), a medicinal plant from Pakistan. Chemical profiling of I. argentea crude methanol extract was done via ultra-high performance liquid chromatography mass spectrometry (UHPLC-MS) in both the positive and negative ionization modes. Standard in vitro bioassays were used to evaluate the enzyme inhibition potential against carbonic anhydrase, xanthine oxidase, and α-glucosidase. Additionally, nine dominant compounds as tentatively identified by UHPLC-MS profiling were docked against the tested enzymes to provide additional insights into the binding affinities against the target enzymes. UHPLC-MS chemical profiling showed the tentative presence of seventy-six secondary metabolites. The identified compounds include flavonoids and lithocholic acid derivatives of steroids in major amounts along with terpenes. Lithocholic acid derivatives were first reported from this plant and consider being potential candidates for treating diabetes. All extracts showed promising results against α-glucosidase with percentage inhibition of 82.8–21.2%, while for the xanthine oxidase the percentage inhibition values varying from 67.8–47.3%. Furthermore, moderate inhibition was observed against carbonic anhydrase. The methanolic extracts showed good inhibition against B. subtilis, K. pneumonia, P. vulgaris and P. aeruginosa as compared to Ciprofloxacin. Docking studies showed that atalanine showed higher inhibition affinity against the three studied enzymes as compared to morphine. Indigofera argentea showed promising enzyme inhibitory activities and can be considered as a potential source of bioactive secondary metabolites. Isolation of the bioactive compounds and further mechanistic studies could provide further insights into the biopharmaceutical potential of this plant as a nutraceutical and/or dietary supplement. [ABSTRACT FROM AUTHOR]

Indigo is a fabric dye that requires reduction by microbial activity or chemical reagents to render it soluble in water. Sources of indigo for fermentation are primarily divided into composted indigo-containing plants and indigo extracted from plants. To elucidate the factors responsible for bacterial diversity, and for sustaining reduced state of indigo in different preparations, this study assessed fermentation-derived fluids using composted plant leaves, sukumo , and extracted indigo (Ryukyu-ai paste, and Indian indigo cake) prepared using different procedures. Regardless of the indigo source, obligate anaerobic bacteria, including the families Proteinivoraceae and Tissierellaceae , predominate (16.9–46.1%), suggesting their high affinity for this fermentation ecosystem (hyperalkaline and low redox potential). Moreover, bacterial communities in sukumo fermentations are more diverse than those from indigo extracts with the diversity tending to increase based on the fermentation period. Our results further suggest that the microbiota composition in sukumo fermentation is associated with the various bacterial nutrients derived from sukumo , including seed microorganisms. In addition, the debris derived from sukumo can reduce the pH stress experienced by the microorganisms. Further, regardless of 5.4 years difference in the fermentation age, the bacterial flora in two Ryukyu-ai batches exhibit similar features with low microbial diversities. The uniformity of the nutrient, along with the simple, yet strong, bacterial network in Ryukyu-ai fluids may be responsible for the stable bacterial flora composition. Taken together, these results indicate that the microbiota in indigo fermentation is highly influenced by the seed culture, the nutrient derived from raw materials, and the fermentation conditions. [Display omitted] • The complexity of the bacterial community in sukumo fluid depends on the complexity of its consituents and the fermentation environment. • The substrate along with the strong bacterial network in Ryukyu-ai fluids may be responsible for the strong sustainability. • Microbiota in indigo fermentation is influenced by various conditions such as the seed culture, the substrate, and fermentation conditions. [ABSTRACT FROM AUTHOR]

Two field cases of reddish-black pigmenturia occurred where cattle grazed on an established Cenchrus ciliaris (blue buffalo grass) pasture in South Africa. The pasture was noticeably invaded by Indigofera cryptantha , which was heavily grazed. Apart from the discolored urine, no other clinical abnormalities were detected. Urinalysis revealed hemoglobinuria, proteinuria and an alkaline pH. When the animals were immediately removed from the infested pasture, they made an uneventful recovery. However, a bull died when one of the herds could not be removed from the I. cryptantha -infested pasture. Macroscopically, the kidneys were dark red in color and the urinary bladder contained the dark pigmented urine. Microscopically, the renal tubules contained eosinophilic, granular pigment casts in the lumen. In addition, many renal tubular epithelial cells were attenuated with granular cytoplasm and were detached from the basement membranes. Chemical analysis was performed on dried, milled plant material and two urine samples collected during the field investigations. Qualitative UPLC-UV-qTOF/MS analysis revealed the presence of indican (indoxyl-β-glucoside) in the stems, leaves and pods of I. cryptantha and indoxyl sulfate was identified, and confirmed with an analytical standard, in the urine samples. It is proposed that following ingestion of I. cryptantha , indican will be hydrolysed in the liver to indoxyl and conjugated with sulfate. Indoxyl sulfate will then be excreted in relatively high concentrations in the urine. In the alkaline urine, two indoxyl molecules might dimerize to form leucoindigo with subsequent oxidation to indigo, thus, contributing to the dark pigmentation of the urine. It is also possible that indoxyl sulfate contributed to the renal failure and death of the bull. Although I. suffruticosa -induced hemoglobinuria has been described in Brazil, this is the first report of I. cryptantha -induced pigmenturia in cattle in South Africa. • Two outbreaks of reddish-black pigmenturia in cattle in South Africa. • Ingestion of the legume, Indigofera cryptantha. • Severe pigment nephrosis confirmed microscopically. • Qualitative analysis revealed indican in stems, leaves and pods. • Qualitative analysis confirmed indoxyl sulfate in urine samples. [ABSTRACT FROM AUTHOR]

Chemical investigation of ethyl acetate bark extracts of Indigofera ammoxylum red and white phenotypes led to the bio-guided isolation of four previously undescribed flavonoids, named (2 S ,3 R)-3′,7-dihydroxy-4′,6-dimethoxyflavanol (1), (2 S ,3 R)-6-methoxy-7-hydroxyflavanol (2), 2′,3′,7-trihydroxy-4′,6-dimethoxyisoflavone (7) and 2′,5' -dimethoxy-4′,5,7-trihydroxyisoflavanone (8), along with 14 known compounds (3 – 6 and 9 – 18). The previously undescribed structures were characterized based on NMR, HRESIMS, UV and IR data. Published spectroscopic data were used to deduce the structure of the known compounds. Eleven of the 18 isolated metabolites were evaluated for anti-inflammatory activity and cytotoxic activity against human liver carcinoma cells and human colon and colorectal adenocarcinoma cells. All tested compounds showed an anti-inflammatory activity (IC 50 NO < 25 μg/mL), and compounds 2 and 3 were more selective than the positive control dexamethasone. Afromorsin (6) showed promising cytotoxic properties against both cancer cell lines (IC 50 18.9 and 11.4 μg/mL). Feature-based molecular networking approach applied to bark and leaves extracts of the two phenotypes allowed to detect bioactive analogues, belonging to the families of flavones, isoflavones, flavanones, flavanols and flavonols, and to explore the chemodiversity of the species. The red and white phenotypes have a similar composition, whereas bark and leaves contain specific chemical entities. Finally, this approach highlighted a cluster of potentially bioactive and undescribed metabolites. [Display omitted] • The bark of the medicinal plant Indigofera ammoxylum possesses potent in vitro anti-inflammatory properties. • Six new phenolic compounds have been isolated from the ethyl acetate bark extract. • Two new flavonoids possess important and selective in vitro anti-inflammatory activity. • Feature-Based Molecular Networking allowed to explore the chemodiversity of the species. [ABSTRACT FROM AUTHOR]

• Tryptanthrin suppresses dsRNA-induced CXCL10 production in HUVEC. • TLR3-mediated induction of interferon-stimulated genes is inhibited by tryptanthrin independently of IFN-β. • Tryptanthrin suppresses the phosphorylation of STAT1 upon stimulation with both dsRNA and recombinant IFN-β. Tryptanthrin is a bioactive component of indigo plants such as Polygonum tinctrorium and known to have an anti-inflammatory activity. The aim of this study was to investigate the effects of tryptanthrin on Toll-like receptor 3 (TLR3)-mediated cytokine and chemokine expression in human umbilical vein endothelial cells (HUVEC). Herein, we found that tryptanthrin suppressed the expression of CXCL10 in HUVEC upon stimulation with a TLR3 ligand polyinosinic-polycytidylic acid (poly IC). Tryptanthrin did not inhibit poly IC-induced activation of interferon regulatory factor 3 (IRF3) or the mRNA expression of interferon (IFN)-β, while it significantly suppressed the expression of RIG-I, MDA5, and classical IFN-stimulated genes (ISGs). Tryptanthrin attenuated the phosphorylation and nuclear translocation of STAT1 in HUVEC stimulated with not only poly IC but also recombinant IFN-β. These results suggested that tryptanthrin inhibited poly IC-induced expression of CXCL10 and ISGs via suppressing the activation of STAT1 in HUVEC. Our findings indicate that tryptanthrin may be useful for regulating TLR3-mediated vascular inflammation. [ABSTRACT FROM AUTHOR]

Abstract Indospicine is a natural toxin occurring only in Indigofera plant species, including the Australian native species I. linnaei. These perennial legumes are resistant to drought and palatable to grazing livestock including cattle. Indospicine accumulates in the tissues (including muscle) of animals grazing Indigofera and these residues persist for several months after exposure. Dogs are particularly sensitive to indospicine with reports in past decades of hepatotoxicosis and mortalities in dogs after dietary exposure to indospicine-contaminated horse and camel meat. The risk for human consumption is not known, and the current study was undertaken to assess indospicine levels in cattle going to slaughter from divergent regions of Western Australia, and to predict the likelihood of significant residues being present. Muscle and corresponding liver samples from 776 cattle originating from the Kimberley and Pilbara Regions in the tropical north of the state, where I. linnaei is prevalent, and 640 cattle from the South West and South Coast Regions in the temperate south west of the state, where the plant is not known to occur, were collected at abattoirs over four seasons in 2015–2017. Indospicine levels were measured by LC-MS/MS and ranged from below detection to 3.63 mg/kg. No indospicine residues were detected in any of the animals originating from the South West and South Coast Regions. Prevalence of indospicine residues in cattle from the Kimberley Region was as high as 33% in spring and 91% in autumn, with positive animals being present in most consignments and on most properties. The average prevalence of indospicine residues from the Kimberley and Pilbara Regions throughout the survey period was 63%. @Risk best fit probability distributions showed ninety-fifth percentile (P95) indospicine concentrations of 0.54 mg/kg for muscle and 0.77 mg/kg for liver in cattle originating from the Kimberley and Pilbara Regions during the survey period. When considered with average Australian meat consumption data, the estimated consumer exposure from this P95 muscle was 0.32 μg indospicine/kg bw/day, which compared favourably with our calculated provisional tolerable daily intake (PTDI) of 1.3 μg indospicine/kg bw/day. However canine exposure is of potential concern, with active working dog exposure calculated to exceed this PTDI by a factor of 25, based on a P95 indospicine concentration of 0.54 mg/kg in muscle. Highlights • The hepatotoxin indospicine accumulates in tissues of animals grazing Indigofera. • Indospicine detected in meat of cattle from Indigofera regions collected at abattoir. • @Risk modelling employed to determine risk to human and canine consumers. • Measured indospicine levels were higher in autumn than in spring abattoir collections. [ABSTRACT FROM AUTHOR]

Highlights • Indigofera caerulea fruits from Djibouti were investigated. • Indigofera caerulea fruits have shown antibacterial and antioxidant activities. • Thin-layer chromatography-mass spectrometry coupling is used for analysis. • Rutin, methyl gallate and gallic acid were the major phenolic compounds. • Methyl gallate showed antibacterial activity against Staphylococcus aureus. Abstract Leaves and fruits of Indigofera caerulea , an endemic plant from Djibouti, are used in the traditional medicine to treat infectious diseases. However, there are no documented scientific reports on phytochemistry and antimicrobial properties of the fruit of this species. For the first time, the chemical compounds and the evaluation of the antimicrobial and antioxidant properties of Indigofera caerulea Rox fruits were investigated. The hexane and methanolic extracts showed a significant activity against both Staphylococcus aureus with a Minimal Inhibitory Concentration (MIC) of 256 and 128 μg/mL, respectively, and Enterococcus faecalis with a MIC of 128 μg/mL for the two extracts. The methanolic and acetone extracts showed effective free radical scavenging activities in 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays, while only the methanolic extract revealed the best ferric-reducing/antioxidant power (FRAP). The highest amount of both total polyphenolic content and total flavonoid content were detected in the methanolic extract (818.14 mg gallic acid equivalents (GAE)/g and 102.10 mg quercetin equivalents (QE)/g, respectively). About the phytochemistry of this extract, two phenolic compounds and three flavonoids were identified rapidly by HPTLC-MS, confirmed by NMR and quantified. Also, chlorogenic acid, caffeic acid and benzoic acid were identified by HPLC. Methyl gallate, the major compound isolated from the methanolic extract, showed antibacterial activity against S. aureus (MIC: 64 μg/mL). Twelve compounds were identified in hexane extract where fatty acids comprised 95.1% and steroids 3.5%. Thus, I. caerulea fruits could serve as a bio-based material for antioxidants and antibacterial compounds. [ABSTRACT FROM AUTHOR]

Indospicine, a non-proteinogenic analogue of arginine, occurs only in Indigofera plant species and accumulates in the tissues of animals grazing on Indigofera . Canine deaths have resulted from the consumption of indospicine-contaminated meat but only limited information is available regarding indospicine toxicity in humans. In this study three human cell lines, Caco-2 (colorectal adenocarcinoma), HT29-MTX-E12 (colorectal adenocarcinoma) and HepG2 (hepatocellular carcinoma), were used to investigate the cytotoxicity of indospicine and its metabolite 2-aminopimelic acid in comparison to arginine. Indospicine and 2-aminopimelic acid were more cytotoxic than arginine, displaying the highest toxicity in HepG2 liver cells. Intestinal transport in vitro also revealed a 2-fold higher transport rate of indospicine compared to arginine. The sensitivity of HepG2 cells to indospicine is consistent with observed canine hepatotoxicity, and considering the higher in vitro transport of indospicine across an intestinal barrier, it is possible that similar ill effects could be seen in humans consuming contaminated meat. [ABSTRACT FROM AUTHOR]

Indican is a secondary metabolite in Indigofera tinctoria ; its synthesis from indoxyl and UDP-glucose is catalyzed by a UDP-glucosyltransferase (UGT). In this study, we partially purified UGT extracted from I. tinctoria leaves and analyzed the protein by peptide mass fingerprinting. We identified two fragments that were homologous to UGT after comparison with the transcriptomic data of I. tinctoria leaves. The fragments were named itUgt1 and itUgt2 and were amplified using rapid amplification of cDNA ends polymerase chain reaction to obtain full-length cDNAs. The resultant nucleotide sequences of itUgt1 and itUgt2 encoded peptides of 477 and 475 amino acids, respectively. The primary structure of it UGT1 was 89% identical to that of it UGT2 and contained an important plant secondary product glycosyltransferase (PSPG) box sequence and a UGT motif. The recombinant proteins expressed in Escherichia coli were found to possess high indican synthesis activity. Although the properties of the two proteins it UGT1 and it UGT2 were very similar, it UGT2 was more stable at high temperatures than it UGT1. Expression levels of it UGT mRNA and protein in plant tissues were examined by UGT assay, immunoblotting, and semi-quantitative reverse transcription polymerase chain reaction. So far, we presume that it UGT1, but not it UGT2, primarily catalyzes indican synthesis in I. tinctoria leaves. [ABSTRACT FROM AUTHOR]

Organelles (chloroplast and mitochondria) are involved in some essential processes, including energy production and photosynthesis, while proteins coded by genes in organelle genomes regulate gene transcription. Research on Indigofera species has been limited by lack of organelle information on population genetics, species identification, and breeding researches. Here, we sequenced and assembled the organelle genomes of two ecologically and medicinally important Indigofera species, namely Indigofera amblyantha and I. pseudotinctoria. Comparative genome analysis among the chloroplast genomes of both species, and a further three Indigofera species revealed some intergenic 'hotspot' regions with potential as markers for species identification. mVISTA analysis revealed significant variations in two genes, ribosomal protein L32 (rpl32) and hypothetical chloroplast reading frame 1 (ycf1), evidenced by high nucleic acid diversity values (0.0222 for rpl32 and 0.0342 for ycf1). Furthermore, both genes exhibited nonsynonymous/synonymous substitution values greater than one between some pairwise chloroplast genomes, suggesting that they play an important role in evolution. We detected abundant repeated sequences in both mitogenomes. Notably, two large repeats with length of more than 1000 bp were identified in the I. amblyantha mitogenome, which might lead to the complex structure of I. amblyantha and I. pseudotinctoria mitogenomes. Our results further indicated that some homologous sequences in the mitogenomes migrated from chloroplast genes via intracellular gene transfer. Collectively, these findings provide valuable insights to guide future species identification and molecular evolution of Indigofera species. • The organelle genomes of Indigofera amblyantha and I. pseudotinctoria were sequenced for the first time. • Several 'hotspot' regions were identified among Indigofera species that could be considered as the potential markers. • The complex structure of I. amblyantha and I. pseudotinctoria mitogenomes may be attributed to repeat sequences. [ABSTRACT FROM AUTHOR]

Herbs with medicinal properties contribute to atheroprotective effects through their preventive and therapeutic interactions with macrophages. Scoparia dulcis and Indigofera tinctoria are examples of such medicinal owing to their antiinflammatory, antioxidant, antibacterial and anticancer properties. The aim of this study was to investigate the antiatherogenic potential of hot- and cold-water extracts of Scoparia dulcis (HwSd and CwSd) and Indigofera tinctoria (HwIt and CwIt). S. dulcis and I. tinctoria extracts were primarily analyzed for phytochemical constituents. In vitro model of atherosclerosis was established using IC-21 M2 phenotypic macrophages, which were stimulated using 7-ketocholesterol (7KCh) and lipopolysaccharide (LPS). In addition, their antiinflammatory and antioxidant potential against 7KCh and LPS-induced macrophage polarization mediators like intracellular ROS/ RNS and gene expression of pro-inflammatory mediators were studied. HwSd, CwSd, HwIt and CwIt contained alkaloids, amino acids, flavonoids, glycosides, reducing sugar, saponins, tannins and terpenoids. In addition, they improved oxidative stress caused by nitric oxide and reactive oxygen species, as well as exhibited anti-inflammatory activity. Furthermore, they modulated gene expression of pro-inflammatory mediators (iNOS, IL-1β, COX-2, TLR-4, MMP-9 and IL-6). Based on these results, we concluded that Scoparia dulcis and Indigofera tinctoria possess antiatherogenic potential and can be beneficial in treating several illnesses associated with chronic inflammation. [Display omitted] [ABSTRACT FROM AUTHOR]

Indigofera , the third largest genus of Fabaceae, comprises approximately 750 species worldwide with a pantropical distribution. Eight Indigofera species, namely I. calcicola , I. delavayi , I. franchetii , I. hancockii , I. lenticellata , I. pendula , I. rigioclada , and I. szechuensis , are considered a species complex because of their morphological similarities and the phylogenetic analysis based on Internal Transcribed Spacer (ITS). Small populations of these species are allopatrically distributed in Hengduan Mountains in China. Although considerable EST-SSR markers have been developed from the transcriptome of I. szechuensis , no codominant markers have been applied to study population genetic structure of the complex. In this study, we selected 66 EST-SSR markers which were transferable in Indigofera szechuensis complex for estimating polymorphism, of which 44 EST-SSRs (66.67%) were polymorphic. Amplification with selected 23 polymorphic EST-SSRs revealed a moderately high genetic diversity in this complex. The mean value of Observed number of alleles, Expected heterozygosity, Polymorphism information content and Shannon's information index was 10.4783, 0.4335, 0.6228 and 1.4369 respectively. A total of 758 genotypes were detected, with an average of 32.9565 genotypes per locus. The Mantel test showed a significant correlation between genetic and geographic distance ( r = 0.0748, P = 0.0100). High differentiation and limited gene flow were detected among all populations ( F st = 0.3589, Nm = 0.5168). The PCoA and structure analysis grouped 31 populations of Indigofera szechuensis complex into five main species. I. delavayi was obviously separated from other species, and the result was in accordance with that of morphology and phylogeny. I. pendula was a separate species, and had two distinct phenotypes. Four I. szechuensis populations (Pop23, Pop24, Pop27 and Pop30) were the same species. I. calcicola , I. hancockii , I. rigioclada , I. franchetii , I. lenticellata , and I. szechuensis , distributed in southwest of Jinshajiang, could be considered as the same species; the species of I. franchetii , I. lenticellata , and I. szechuensis distributed in northeast of Jinshajiang could be considered as another separate species. Given the above information, the morphological classification of I. calcicola , I. franchetii , I. hancockii , I. lenticellata , I. rigioclada , and I. szechuensis was incredible. [ABSTRACT FROM AUTHOR]

Multidrug resistant (MDR) gram negative bacterial infections are most prevalent in the hospitals which are truly untreatable and remedial verdict are very deprived. In the current investigation, two compounds (Quercetin and Pinitol) and fractions have been isolated and characterized from Indigofera barberi using chromatographic and NMR studies. Antimicrobial assays reveal IB-Me, IB-D and IB-E fractions and quercetin have shown eloquent antimicrobial activity on clinically isolated bacteria such as Pseudomonas aeruginosa, E. coli, Klebsiella pneumonia and Citrobacter sp. Molecular docking studies demonstrated quercetin has exhibited highest binding affinity with DNA gyrase B and subsequently eleven pharmacophore features viz . five HBD/HBA, two hydrophobic, one HBA and three aromatic (Pi) rings were identified using MOE. Pharmacophore based virtual screening, docking, binding energies and binding affinity with Born-volume (GB/VI) studies demonstrated thirteen lead molecules which were specifically interacted with Asn46, Asp73, Arg76, Thr163, Lys103, Arg136 residues and conserved water molecules within ATP pocket of DNA gyrase B. These scaffolds comply with Lipinski rule of five, toxicity and drug likeness properties deliberated as drug compounds. Hence, these compounds could be endorsed as persuasive DNA gyrase B inhibitors which can be considered as novel compounds in the clinical management of MDR Gram negative bacterial infections. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A novel pterocarpan derivative, stachyodin A, was isolated from Indigofera stachyodes. • The absolute configurations were determined by single crystal X-ray and ECD data. • Structurally, stachyodin A features a 6/5/5/6 tetracyclic ring system with an unusual spirotetrahydrofuran ring. • The isolates showed inhibition of NO production in LPS-activated BV-2 cells. Abstract Stachyodin A (1), a rearrangement product of pterocarpan featuring a 6/5/5/6 tetracyclic ring system with an unusual spirotetrahydrofuran ring, and stachyodin B (2), a new dihydrochalcone, along with five known analogues (3 – 7) were isolated from the roots of Indigofera stachyodes. The structures of 1 and 2 were elucidated on the basis of their HRESIMS and NMR spectroscopic data, and their absolute configurations were determined by X-ray crystallographic analysis and electronic circular dichroism (ECD) data, respectively. Compounds 2 – 4 showed inhibition of nitric oxide production in lipopolysaccharide-activated BV-2 microglial cells. [ABSTRACT FROM AUTHOR]

Indigofera szechuensis Craib, a species endemic to Hengduan Mountains in China, is widely distributed across a range of habitats, including slopes, trailsides, and riverbanks. This species serves as a good model to examine flexible adaptation of plants. In this study, we reported the transcriptome sequence of this species and developed a set of SSR markers for such evolutionary studies. Approximately 19.4 million clean reads from the leaves of I . szechuensis were generated through transcriptome sequencing, and 43,896 unigenes with an average length of 798.54 bp were obtained. A total of 27,320 (62.2%) unigenes were annotated for their functions, and 17,422 and 8017 unigenes could be aligned to the GO and COG database, respectively. By searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG), 5682 unigenes were assigned to 118 KEGG pathways that belong to five main categories, i.e ., organismal systems, cellular processes, environmental information processing, metabolism, and genetic information processing. We further identified 4052 potential SSR sites. Using 102 randomly selected EST-SSRs, 73 markers were successfully amplified with the expected size in I . szechuensis and 67-71 markers of these 73 SSRs in eight other Indigofera species. We firstly established a public trascriptome platform for the species-rich genus Indigofera . The SSR sites identified here provide additional resources for development of molecular markers for further evolutionary studies for this species and congeners. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate the free radical scavenging potential and high performance thin layer chromatography (HPTLC) fingerprinting of Indigofera tinctoria ( I. tinctoria ). Phytochemical analysis was carried out using standard methods, and free radical scavenging activity of the plant was determined using 2,2-diphenyl-1-picrylhydrazy (DPPH), nitric oxide (NO) and superoxide anion ( O 2 − ) radical scavenging capacities. HPTLC plate was kept in CAMAG TLC Scanner 3 and the Rf values at fingerprint data were recorded by WINCATS software. Aqueous extract of I. tinctoria reliably showed the total phenolics (267.2±2.42 mg/g), flavonoids (75.43±3.36 mg/g) and antioxidants (349.11±8.04 mg/g). The extract was found to have DPPH (52.08%), NO (23.12%) and O 2 − (26.79%) scavenging activities at the concentration of 250 μg/mL and the results were statistically significant compared with ascorbic acid standard ( p <0.05). HPTLC results confirmed that the extract contained several potential active components such as phenols, flavonoids, saponins and terpenoids as the slides revealed multi-colored bands of varying intensities. This study confirmed that the plant had multipotential antioxidant and free radicals scavenging activities. [ABSTRACT FROM AUTHOR]

A new isoflavone 7-hydroxy 3′-methoxyisoflavone ( 1 ) is isolated from the seeds of Indigofera heterantha . The structure of this new compound was established using spectroscopic techniques such as ID, 2D NMR, and mass spectrometry. Density functional theory calculations are carried out for the first time for geometric, electronic and spectroscopic properties of 1 (isoflavone). DFT calculations have been performed at B3LYP/6-311G(d,p) level of theory for obtaining geometric and spectroscopic properties of compound 1 . The simulated vibrational spectrum of compound 1 at B3LYP/6-311G(d,p) shows nice correlation with the experimental IR spectrum after a scaling factor of 0.973. 1 H and 13 C NMR chemical shifts were calculated using Cramer’s re-parameterized function WP04 at 6-311G(d,p) basis set, and show nice correlation with the experimental data. Four conformers were considered for NMR chemical shift calculations. Electronic properties such as band gap, Ionization potential and electron affinities were also simulated for the first time; however, no comparison could be made with the experiment. [ABSTRACT FROM AUTHOR]

The extractions of indigo (Indigofera tinctoria L.) and safflower (Carthamus tinctorius L.) red and yellow from wool fibres are investigated using high performance liquid chromatography with photo-diode-array detection (HPLC–PDA) which is frequently coupled to Mass Spectrometry (HPLC–PDA–MS) for identification purposes. The efficiencies of dimethyl sulfoxide (DMSO) and dimethyl formamide (DMF) to extract indigo and safflower from wool, are compared for a wide range of temperature (from 40 to 120°C) and treatment time (from 1 to 30min). Extraction procedures are investigated by measuring integrated HPLC peak areas of selected dyestuff components, evaluating simultaneously the effects of solvent (DMSO and DMF), treatment temperature (T) and time (t). Thus, cross-influence of the different extraction parameters (solvent, T and t) is taken into account. Indigotin, isatin and indirubin are monitored to evaluate the extraction of indigo. Carthamin, a decomposition product of carthamin, apigenin, safflomin A, 6-hydroxykaempferol-3-O-β-D-glucoside, anhydrosafflor yellow Ct1 and Ct4 are monitored to investigate the extractions of safflower red and yellow. Overall, it is reported that DMSO gives better extraction yields than DMF. The best conditions for the extraction of the marker compounds of the three dyes are summarized as follows. For indigotin, treatments of the wool with DMSO at 80°C for t>5min or at 120°C min for t=1min give the best yield. Longer treatment (t>1min) at 120°C results to decomposition of indigotin. For carthamin, treatments of the wool with DMSO at 80°C for t>5min or at 120°C min for short t (<5min) give the best yield, considering that longer (>5min) treatment at 120°C results to decomposition of the marker compound. Finally, for safflomin A treatments of the wool with DMSO at 100°C for t>10min or at 120°C min for t=5min give the best result. [ABSTRACT FROM AUTHOR]

Highlights: [•] We examined parts of indigo plant for cytotoxic and antioxidant effects. [•] The highest amount of antioxidants was in Indigo methanol extract. [•] Indigo extracts effective in suppressing proliferation of cancer cells. [•] The results varied on the different parts of plant and types of cancers. [Copyright &y& Elsevier]

Highlights: [•] To investigate I. suffruticosa extracts on expression of GSTP and NQO1 in hepatocytes. [•] Suffruticosa extracts increased GSTP and NQO1 levels and GSTP promoter activity in hepatocyte. [•] Suffruticosa extracts up-regulated binding affinity of Nrf2 with GPEI and ARE activation. [•] ERK inhibitor abolished I. suffruticosa extracts induced GSTP and NQO-1 expression. [•] Suffruticosa extracts increased GSH levels in mouse liver, RBC and hepatocytes. [ABSTRACT FROM AUTHOR]

Abstract: Poisoning by Indigofera lespedezioides is reported in horses in the state of Roraima, northern Brazil. The main clinical signs are anorexia, sleepiness, unsteady gait, severe ataxia, weakness, stumbling, and progressive weight loss. To induce the disease experimentally, a 7-year-old horse was introduced in a small paddock invaded by the plant. The first nervous signs were observed 44 days from the start of grazing. The animal was euthanized on day 59. No significant gross lesions were observed upon necropsies of the experimental horse as well as one spontaneously affected horse. Upon histologic examination neuronal lipofuscinosis was observed in the brain, cerebellum, and spinal cord. Wallerian-type degeneration was observed on some mesencephalic tracts. Neuronal and axonal degeneration and lipofuscinosis were observed on electron microscopy examination. Indospicine was detected in four samples of I. lespedezioides with concentrations ranging from 63 to 1178 μg/g whereas nitro toxins could be detected in only one of the samples at a concentration of 2.5 mg/g. In conclusion, poisoning by I. lespedezioides is very similar to those poisonings by Indigofera linnaei and Indigofera hendecaphylla. Based on the preponderance of indospince and lack of nitro toxins in the samples it is proposed that indospicine is the toxic compound responsible for the poisoning. [Copyright &y& Elsevier]

Abstract: Background: Topical indigo naturalis ointment is clinically proved to be an effective therapy for plaque-type psoriasis. Indirubin, as the active component of indigo naturalis, inhibits cell proliferation of epidermal keratinocytes. However, the detailed underlying mechanism is not fully understood. Objective: To further investigate the anti-proliferating effects of indigo naturalis and indirubin on epidermal keratinocytes. Methods: The decreased expression of CDC25B in indigo naturalis- or indirubin-treated epidermal keratinocytes, as revealed by cDNA microarray analysis, was studied. The CDC25B expression was examined under different serum concentrations and compared between primary and immortalized keratinocytes. The activation of EGFR and the effect of EGF on the cell proliferation and CDC25B expression were also investigated in epidermal keratinocytes. RT/real-time PCR and western blot method were used to analyze the CDC25B expression at the mRNA and protein levels, respectively. Results: Indigo naturalis and indirubin were confirmed to down-regulate CDC25B expression significantly at both the mRNA and protein levels. The growth-dependent expression of CDC25B was demonstrated by the increased expression in serum-stimulated and immortalized keratinocytes. The activation of EGF receptor, known to be highly expressed in psoriatic lesions, was inhibited by indigo naturalis or indirubin. The cell proliferation and CDC25B expression of epidermal keratinocytes were induced by EGF alone and confirmed to be inhibited by indigo naturalis or indirubin. Conclusion: Except being a common therapeutic target in various cancers, CDC25B also plays an important role in the hyper-proliferation of epidermal keratinocytes which can be suppressed by anti-psoriatic drug indigo naturalis and its component, indirubin. [Copyright &y& Elsevier]

Abstract: Ethnopharmacological relevance: Indigo naturalis is used by traditional Chinese medicine to treat various inflammatory diseases. Aim of the study: Topical indigo naturalis ointment showed efficacy in treating psoriasis in our previous clinical studies. In this study, we investigated the anti-inflammatory effects of the extract of indigo naturalis (QD) and its main components indirubin, indigo, and tryptanthrin in human neutrophils. Materials and methods: Superoxide anion (O2 −) generation and elastase release were measured by spectrophotometry. Some important signals including mitogen-activated protein kinase (MAPK), cAMP, and calcium were studied by Western blot analysis, an enzyme immunoassay, and spectrofluorometry. Results: QD significantly inhibited O2 − generation and elastase release in formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP)-activated human neutrophils in a concentration-dependent fashion, while neither indirubin, indigo, nor tryptanthrin produced a comparable result. QD attenuated the FMLP-induced phosphorylation of extracellular regulated kinase, p38 MAPK, and c-Jun N-terminal kinase. Furthermore, QD inhibited calcium mobilization caused by FMLP. However, QD did not affect cellular cAMP levels. On the other hand, neither indirubin, indigo, nor tryptanthrin produced similar changes in human neutrophils. Conclusions: Taken collectively, these findings indicate that QD, but not indirubin, indigo, or tryptanthrin, inhibited O2 − generation and elastase release in FMLP-induced human neutrophils, which was at least partially mediated by the inhibition of MAPK activation and regulation of calcium mobilization. [Copyright &y& Elsevier]

Abstract: By considering controversial discussions in the literature with regard to gland denomination in Indigofera species, as well as the taxonomic value of secretory structures in Leguminosae, we aim to morphologically detail glands that had been previously observed in I. microcarpa and I. sabulicola, and to investigate the occurrence of glands in vegetative and reproductive organs of other six Neotropical species that belong to the genus. Glands analyzed through scanning electronic microscopy (SEM) in combination with anatomic analyses correspond to secretory trichomes that are classified into seven types. Main variations in relation to types occurred with regard to head shape and peduncle size. Trichome heads were multicellular, with a thin cuticle. Hollow heads with conspicuous inner space characterized only one type (type 1); the other trichome types had massive heads. Peduncles, which varied from biseriate to multiseriate, had thick, pecto-cellulosic cell walls. Trichomes were found on stems, stipules, petioles, rachis, petiolules, leaflets, bracteoles, sepals, standards and fruits, more commonly along the margins. Each of the eight Indigofera species analyzed had at least two different trichome types out of the seven types that occurred in reproductive and vegetative organs of these taxa. Various types of secretory trichomes were found in I. campestris, I. lespedezioides, I. microcarpa, I. spicata, I. suffruticosa and I. truxillensis. Stems and rachis were the vegetative organs in which a greater variety of trichomes occurred, and sepals were parts of reproductive organs with the same status. Five out of the seven secretory trichome types occurred on both vegetative and reproductive organs. Distribution and gland types differed between species and these gland distribution patterns can be used as diagnostic characters. Reports of glands in Indigofera campestris, I. hirsuta, I. lepedezioides, I. suffruticosa, I. spicata and I. truxillensis, their recognition as secretory trichomes, and the morphological variety of types found for such trichomes are novel data for Indigofera. [Copyright &y& Elsevier]

Abstract: Two experiments were completed to determine the influence of season/year and species on forage quality of Indigofera accessions. In Experiment 1, leaf material was collected from five Indigofera species (i.e., I. amorphoides, I. arrecta, I. brevicalyx, I. costata and I. cryptantha) by harvesting six middle plants of a plot in the autumn of 2003 and the autumn of 2004. In Experiment 2, edible forage (leaves+<3mm stems) material were collected by harvesting six middle plants in the autumn of 2004 and the spring of 2004. Both leaf meal and edible forage material were analyzed for dry matter (DM), ash, crude protein (CP), neutral detergent fibre (NDF), in vitro digestible organic matter (IVDOM), as well as Ca, P, Mg, Cu, Zn and Mn concentrations. In Experiment 1, the interaction effects occurred between species and year of cutting (P<0.05) in studied parameters except Cu concentration. In Experiment 2, interactions (P<0.05) occurred between species and season effect for ash content as well as Ca and Mg concentrations in the edible material. The CP content of the leaf meal ranged between 223 and 311g/kgDM and that of edible forage material between 81 and 287g/kgDM. Spring growth had a higher (P<0.05) CP content than autumn growth in all species. The NDF content of leaves ranged between 189g/kgDM in I. amorphoides and 504g/kgDM in I. costata, and that in edible material ranged between 328g/kgDM in I. arrecta and 654g/kgDM in I. cryptantha. The in vitro OM digestibility of leaves ranged between 568g/kgDM in I. cryptantha and 717g/kgDM in I. amorphoides, while that of edible material ranged between 507 and 722g/kgDM in I. cryptantha. In contrast to the trend with CP and NDF, in vitro OM digestibility of dry material tended to decrease from the spring of 2004 to the autumn of 2004 harvest. Higher levels of Ca, P, Mg, Zn and Cu occurred in the leaf meal of the first harvest than in the re-growth harvest. All of the species had Ca, Mg, Zn and Mn levels that could meet the requirements of ruminants, but P and Cu were slightly deficient for some of the species in the autumn harvest, making it essential to supplement P and Cu from other sources during this period to meet animal requirements. [Copyright &y& Elsevier]

Abstract: Indigofera mysorens is a shrub used for its antidiabetic activity in rural India. Here, we elucidate the antidiabetic potential of Indigofera mysorensis extract. Ethanolic extract of the whole shrub of Indigofera (EEI) at 300mg/kg for 10 days, produced a 63% reduction in plasma glucose, 41% reduction in plasma triglyceride and 77% reduction in plasma insulin levels in insulin resistant db/db mice, which is better than insulin sensitizer, troglitazone (400mg/kg). EEI unlike sulphonylureas failed to show any acute hypoglycemic effect in normoglycemic Swiss albino mice (SAM). Even in a chronic study (10 days) in SAM, EEI (300mg/kg) like insulin sensitizers showed no effect on plasma glucose, but an 81% reduction in plasma insulin levels. When challenged with 3gm/kg sucrose, SAM treated with EEI (300mg/kg, 7 days) failed to show any effect on the absorption of sugar, whereas standard drug, acarbose (10mg/kg) showed 52% reduction in the area under the plasma glucose curve. EEI failed to show any significant transactivation of PPARγ, a proposed target of synthetic insulin sensitizers. Taken together, our data indicate that the antidiabetic effect of the ethanolic extract of Indigofera is due to its insulin sensitizing property and is clearly different from that of sulfonylurea or acarbose. [Copyright &y& Elsevier]

Abstract: An investigation of aliphatic nitro-compounds in Indigofera carlesii resulted in the identification of two blended novel compounds: 2-O-acryl-3,6-di-O-(3-nitropropanoyl)-α-d-glucopyranose and 6-O-acryl-2,3-di-O-(3-nitropropanoyl)-α-d-glucopyranose. The structures of the new compounds were elucidated on basis of spectral analysis. [Copyright &y& Elsevier]

Abstract: Andean hillsides dominate the landscape of a considerable proportion of Cauca Department in Colombia. The typical cropping cycle in the region includes monocrops or intercrops of maize (Zea mays L.), beans (Phaseolus vulgaris L.) and/or cassava (Manihot esculenta Crantz). Cassava is usually the last crop before local farmers leave plots to natural fallow until soil fertility is recovered and a new cropping phase can be initiated. Previous studies on land use in the Río Cabuyal watershed (6500ha) show that a considerable proportion of land (about 25–30%) remains under natural fallow every year. The focus of our studies is on systems of accelerated regeneration of soil fertility, or improved fallow systems, as an alternative to the natural regeneration by the native flora. Fallow improvement studies were conducted on plots following cassava cultivation. The potential for soil fertility recovery after 12 and 28 months was evaluated with two fast growing trees, Calliandra calothyrsus Meissn (CAL) and Indigofera constricta L.(IND), and one shrub, Tithonia diversifolia (Hemsl.) Gray (TTH), as slash/mulch fallow systems compared to the natural fallow (NAT). All planted slash/mulch fallow systems produced greater biomass than the natural fallow. Greatest dry biomass (16.4Mgha−1 year−1) was produced by TTH. Other planted fallows (CAL and IND) produced about 40% less biomass than TTH and the control (NAT) about 75% less. Nutrient levels in the biomass were especially high for TTH, followed by IND, CAL, and NAT. The impact of fallow management on soil chemical, physical and biological parameters related to residual soil fertility during the cropping phase was evaluated. Soil parameters most affected by slash/mulch fallow systems included soil total N, available N (ammonium and nitrate), exchangeable cations (K, Ca, Mg and Al), amount of P in light fraction, soil bulk density and air permeability, and soil macrofauna diversity. Results from field studies suggest that the Tithonia slash/mulch fallow system could be the best option to regenerate soil fertility of degraded volcanic-ash soils of the Andean hillsides. [Copyright &y& Elsevier]

Aqueous extract of leaves of Indigofera suffruticosa (AELIs) were studied for adverse effects in preimplantation mouse embryos. Two-cell mouse embryos were cultured for 94 h in human tubal fluid medium (HTF), and AELIs at a concentration of 5 or 10 mg/ml. On Day 4 of culture, morulae and blastocysts were collected for morphological analysis of blastomeres. We found that embryos exposed to the higher concentration of AELIs (10 mg/ml) did not develop and all embryos persisted at the two-cell stage. Those embryos exposed to the lower concentration (5 mg/ml) showed development until morula, blastocyst and hatched blastocyst stages that were similar to the controls. These results suggest that use of AELIs may be hazardous to humans who make use of it in folk medicine. [Copyright &y& Elsevier]

The effect of intraperitoneal injection of 400 mg/kg of ethanolic extract of Indigofera aspalathoides (EEIA) against Dalton''s ascitic lymphoma has been evaluated in Swiss albino mice. A significant increase in the life span, a decrease in the cancer cell count and body weight were noted in the tumour-induced mice after treatment with EEIA. These observations indicate that the plant is having protective effect in Dalton''s ascitic lymphoma. [Copyright &y& Elsevier]

The chemical investigation of the whole plant of Indigofera spicata Forsk (Fabaceae), a medicinal plant from Cameroon, resulted in the isolation of a new benzofuran, named spibenzofuran (1a), together with ten known secondary metabolites including one benzofuran (2), one flavonoid (3), one saponin (6), two triterpenes (4 and 5), two steroids (8 and 11), one phthalate (7) and two fatty acids (9 and 10). All these compounds have been isolated for the first time from this plant. The structures of the isolated compounds were established by spectroscopic analysis including HR-ESI-MS, 1D and 2D NMR and by comparison of our data with the reported data. The isolated compounds might be considered as the chemophenetic markers of this species, and antibacterial and urease inhibitory activities of some isolated compounds were assessed. Image 1 • A new benzofuran from Indigofera spicata Forsk. • Structure elucidation with NMR and MS analyses. • Antibacterial and urease activities of isolated compounds. • Chemophenetic significance of the isolated compounds. [ABSTRACT FROM AUTHOR]

In this work, we extract the natural dye ingredient found in Indigofera Tinctoria leaves, and investigate its nonlinear optical absorption by employing the Z-scan technique. The dye is extracted from Indigo powder which is commercially available for hair coloring applications. The powder, which is made by grinding Indigofera Tinctoria leaves into fine particles, is mixed with water, sonicated, centrifuged and filtered to give an optically clear extract. Linear absorption of the extract is measured in the visible spectral region using a spectrophotometer. Nonlinear absorption in the green spectral region is measured using 5 ns (5 × 10−9 s), 532 nm laser pulses obtained from a frequency-doubled Nd:YAG laser. The Indigofera Tinctoria dye is found to show excellent nonlinear absorption, which arises from strong reverse saturable absorption occurring in the sample at this wavelength. The third order nonlinear absorption coefficient, which is obtained by numerically fitting the Z-scan data to the nonlinear transmission equation, has a high value in the order of 10−10 m/W. This high nonlinear absorption, which is in the same range as that of materials such as graphene oxide, leads to an excellent optical limiting behavior, making Indigofera Tinctoria a potential candidate for fabricating optical limiter devices for eye and sensor protection. • Natural dye present in Indigofera Tinctoria leaf powder (hair colorant) is extracted by ultrasonication and centrifugation. • Absorption, excitation and emission spectra are measured. Open aperture Z-scans are done using 532nm, 5ns laser pulses. • Strong reverse saturable absorption is observed (β eff ≈ 10-10 m/W) which results in excellent optical limiting action. • Optical limiters made from this dye can potentially protect human eyes and light detectors from harmful laser radiation. • To the best of our knowledge, this is the first such study carried out in Indigofera Tinctoria natural dye extract. [ABSTRACT FROM AUTHOR]

• We prepared TMF- O -glycosides and free TMF from indigo leaves. • Free TMF suppressed the gene expression of pro-inflammatory cytokines. • Both TMF- O -glycosides and free TMF protected against ulcerative colitis in mice. • Free TMF was generated by digestion and absorption of TMF- O -glycosides. • TMF- O -glycosides exert in vivo anti-inflammatory effects through free TMF. Persicaria tinctoria (Aiton) Spach, also called Polygonum tinctorium Lour., (family Polygonaceae) for indigo plant has been traditionally useful as a medicinal or edible plant with a variety of biological activities. Of these, much attention has been paid to their anti-inflammatory activities. We have recently demonstrated that indigo leaves contain high levels of flavonol O -glycosides with 3,5,4'-trihydroxy-6,7-methylenedioxyflavone (TMF) as an aglycone. In this study, we attempted to evaluate anti-inflammatory activities of TMF- O -glycosides and free TMF prepared from indigo leaves after extraction with hot water. Free TMF was found to appreciably down-regulate the gene expression of pro-inflammatory cytokines including interleukin (IL)-1β, IL-6, inducible nitric oxide, and tumor necrosis factor-α in cultured macrophage cells stimulated with lipopolysaccharide while up-regulating the expression of anti-inflammatory IL-10. However, no study has been conducted regarding in vivo anti-inflammatory activities of TMF- O -glycosides and free TMF until now. Here, we assessed in vivo anti-inflammatory effects of these dietary compounds on ulcerative colitis in a murine model of inflammatory bowel disease by the induction with dextran sulfate sodium (DSS). Histological evaluation revealed that both TMF- O -glycosides and free TMF effectively protected against DSS-induced ulcerative colitis. The analysis of digested products by liquid chromatography and mass spectrometry led us to detect free TMF as a predominant metabolite in the feces of mice fed with TMF- O -glycosides. Moreover, free TMF was later detected as glucuronyl conjugates of TMF in the liver of mice fed with both fractions. These results indicate the effective digestion of TMF- O -glycosides and the subsequent absorption of free TMF in the gut of mice for exerting anti-inflammatory effects. Taken together, our findings suggest that dietary TMF- O -glycosides could be promising natural sources for the utilization as herbal medicine and nutraceuticals to expect in vivo anti-inflammatory activities. [ABSTRACT FROM AUTHOR]

Abstract: A review is given of the legume tribe Indigofereae for the region of southern Africa including Namibia. The overall distribution, taxon statistics, phylogenetic relationships, uses, bibliography and a checklist of taxa in the tribe are provided covering the c. 340 taxa of Indigofereae within the region. [Copyright &y& Elsevier]

Summary: Indigo, also known in Morocco as Nila, is a dye widely used in the coloring of Moroccan handicrafts. It is obtained from fermentation reactions on the leaves and branches of true indigo, Indigofera tinctoria, which is a widespread plant in tropical Africa and Asia. We report a case of fatal poisoning in a 3-year-old child after administration of indigo for therapeutic purposes. Death resulted from multiple organ failure. The toxicity of this compound is little known in the literature and deserves to be explored through toxicokinetic and toxicodynamic studies, in order to better determine the toxic constituents of the dye. [ABSTRACT FROM AUTHOR]

Abstract: Although smoke treatments have successfully been used for promoting the germination of many seeds, there are no reports on the effect of smoke on secondary metabolite production in plants. This study highlights the effects of smoke-water on shoot growth and accumulation of indigo in shoots of Isatis indigotica Fort., a Chinese medicinal plant. Results showed that seedlings treated with smoke-water at a dilution of 1:1000 achieved the highest fresh and dry shoot mass, which was significantly (P <0.05) different from the control and other smoke-water dilutions tested. A higher concentration of smoke-water (1:500) significantly increased (P <0.05) the indigo concentration in shoots compared with untreated seedlings. The effect of 1:1000 and 1:2000 dilutions gave non-significant increases in the concentration of indigo. These findings suggest the possible use of smoke-water for promoting the growth and accumulation of secondary metabolites in plants. [Copyright &y& Elsevier]

• Hydroponic cultivation of Polygonum tinctorium was demonstrated in plant factory. • Flowering was drastically enhanced by blue light under 12-h photoperiod. • Indican content of leaves was greatly increased by blue light under 24-h photoperiod. • Blue light involves in increase of Pt'IGS and Pt'BGL expression levels in leaves. The photoperiod is a day-length-dependent seasonal change of physiological or developmental activities, such as flowering, in many plant species. Polygonum tinctorium (P. tinctorium) is an important industrial crop producing indigo blue dyes, and indican is an important substance as a precursor of indigo. Here, we report the day-length-dependent responses of growth, flowering, and indican synthesis in P. tinctorium. Indigo plants were grown in a hydroponic system under artificial light conditions in a completely-controlled plant factory. The growth parameters and indican content of leaves were measured and compared. Certain growth parameters (such as fresh weight and number of leaves) under 24-h continuous irradiation were significantly higher than those under other day-length conditions. Under 12-h photoperiod conditions, the flowering rate of plants with blue LED treatment increased six-fold compared with fluorescent white light treatment, while none of the plants flowered with red LED treatment. In the leaves, the relative expression levels of Pt'IGS and Pt'BGL were significantly higher in newer leaves compared to older ones. Indican content was greatly enhanced by blue light under 24-h continuous irradiation, which was reflected by increased expression levels of Pt'IGS. These findings demonstrate that there is a new regulatory mechanism for the indican synthesis pathway through blue light signalling. Blue light provides feasible strategy for artificially regulating indican synthesis and flowering in P. tinctorium. [ABSTRACT FROM AUTHOR]

Indospicine is a hepatotoxic non-proteinogenic amino acid that has only been found in Indigofera plants including a number of species abundant in central Australia. These legumes are palatable to feral camels and indospicine residues derived from these plants accumulates in camel meat tissue. Indospicine-contaminated camel meat has caused liver disease and mortalities in dogs after a short period of dietary exposure. Since camel meat is also exported for human consumption, this raises concern about the human health risks of consuming indospicine-contaminated camel meat harvested from central Australia. The objective of this study was to assess the indospicine levels in camel meat and its associated potential health risk to the Australian population and the vulnerable sub-population of indigenous Australians from the consumption of such feral camel meat. Two main quantitative probabilistic exposure assessments were conducted through the use of Monte Carlo simulations. The first exposure assessment considered the indospicine concentrations in real-field camel meat samples. The second assessment was based on a simulated probabilistic distribution of indospicine residues in camel meat, involving eight (8) predictive factors that were derived from our published research data and other secondary sources. The first assessment predicted that the estimated risk of developing indospicine₋induced hepatotoxicosis from contaminated camel meat consumption was 800 per 10 000 of the average Australian population (8.0%), and higher for the indigenous Australians (10.0%). The second exposure assessment demonstrated there was a higher risk (>31%) for the whole Australian population and for the indigenous Australian population. In conclusion, this risk assessment study shows that, while there is a low risk amongst Australians of hepatotoxicosis being associated with the consumption of indospicine-contaminated camel meat, such consumption is still a cause for concern, since the assessment based on the simulated probabilistic distribution of indospicine concentrations in camel meat demonstrated a substantially higher risk. It is therefore advisable that an indospicine residue monitoring program should be implemented to ensure food safety of Australian camel meat. Additionally, the risk of dietary indospicine exposure from other livestock grazing Indigofera in all global regions should also be considered. Image 1 • Heptatotoxic indospicine forms residues in meat tissue of camels grazing Indigofera. • Dogs have died from eating contaminated meat and human health risk is of concern. • Quantitative probabilistic exposure assessments characterised potential health risk. • Assessments based on real-field camel meat samples suggest low health risk. • Residue monitoring program warranted to ensure food safety of Australian camel meat. [ABSTRACT FROM AUTHOR]