Plant P450 forms indigo and indirubin when expressed in Escherichia coli.

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.

Multi-analytical study of the medieval wall paintings from the rupestrian church Grotta del Crocifisso at Lentini (eastern Sicily): new evidence of the use of woad (Isatis tinctoria).

This study presents the results of the examination and characterisation of the wall paintings that decorate the rupestrian church named Grotta del Crocifisso, which is located in the territory of Lentini (eastern Sicily, few tens of kilometres from Catania and Syracuse). The earliest mural paintings in the church date back to the twelfth century AD. A multi-analytical approach was adopted for the characterisation of stone materials, secondary degradation products, and pigments. For this purpose, the following techniques were used: reflected light microscopy (RLM), polarised light microscopy on thin sections (PLM), X-ray powder diffraction (XRPD), mercury intrusion porosimetry (MIP), portable X-ray fluorescence (p-XRF), scanning electron microscopy (SEM-EDS), Fourier-transform infrared spectroscopy (FTIR), and Raman spectroscopy (RS). The lithic substrate and the plaster's coating layers were thoroughly characterised from compositional and textural points of view, and the use of locally available raw materials was established. Similarly, the newly formed crystalline phases produced by alteration processes of the original materials were recognised. The red, yellow, brown, and green pigments were easily identified by p-XRF and SEM-EDS. The use of "earth pigments" widely available in the surrounding area (various types of ochre) was thus highlighted. The recognition of the dark blue pigment created some additional issues in its identification, making further diagnostic methods necessary. In fact, the use of the most common mineral pigments was categorically excluded by both p-XRF and SEM-EDS, since no chromophore metallic elements were highlighted with the exception of trace amounts of iron. A combination of detailed microscopic observations together with the application of FTIR and RS supported the use of an organic pigment obtained from the maceration of woad (Isatis tinctoria). The green pigment is the result of a mixture between woad and yellow ochre. Woad is even today easily available in Sicily, and some additional experimental tests were carried out on Isatis tinctoria that had been freshly collected in the area (treated with traditional procedures). Over the past centuries, woad was widely used for dyeing fabrics, but its practice for wall paintings has only been sporadically proven. The identification reported in this case study could be considered a novelty at least in the Sicilian panorama. Supplementary Information: The online version contains supplementary material available at 10.1007/s12520-022-01656-6.

A Systemic Review for Ethnopharmacological Studies on Isatis indigotica Fortune: Bioactive Compounds and their Therapeutic Insights.

Isatis indigotica Fortune is a biennial Chinese woad of the Cruciferae family. It is primarily cultivated in China, where it was a staple in indigo dye manufacture till the end of the 17th century. Today, I. indigotica is used primarily as a therapeutic herb in traditional Chinese medicine (TCM). The medicinal use of the plant is separated into its leaves (Da-Qing-Ye) and roots (Ban-Lan-Gen), whereas its aerial components can be processed into a dried bluish-spruce powder (Qing-Dai), following dehydration for long-term preservation. Over the past several decades, I. indigotica has been generally utilized for its heat-clearing effects and bodily detoxification in TCM, attributed to the presence of several classes of bioactive compounds, including organic acids, alkaloids, terpenoids, and flavonoids, as well as lignans, anthraquinones, glucosides, glucosinolates, sphingolipids, tetrapyrroles, and polysaccharides. This paper aims to delineate I. indigotica from its closely-related species (Isatis tinctoria and Isatis glauca) while highlighting the ethnomedicinal uses of I. indigotica from the perspectives of modern and traditional medicine. A systematic search of PubMed, Embase, PMC, Web of Science, and Google Scholar databases was done for articles on all aspects of the plant, emphasizing those analyzing the bioactivity of constituents of the plant. The various key bioactive compounds of I. indigotica that have been found to exhibit anti-inflammatory, antimicrobial, anticancer, and anti-allergic properties, along with the protective effects against neuronal injury and bone fracture, will be discussed. Collectively, the review hopes to draw attention to the therapeutic potential of I. indigotica not only as a TCM, but also as a potential source of bioactive compounds for disease management and treatment.

Physiological and biochemical responses of Isatis indigotica to deficit irrigation in a cold and arid environment.

Water shortage and wastage are critical challenges to sustainable agricultural development, especially in arid and semiarid regions worldwide. Isatis indigotica (woad), as a traditional Chinese herb, was planted in a large area in a cold and arid environment of Hexi. Regulated deficit irrigation can reduce the growth of some vegetative organs by changing the accumulation and distribution of photosynthetic products in crops, thus increasing the economic yield of crops. In agricultural production, crop productivity may be improved by mulched drip irrigation and deficit irrigation. Hence, a field experiment was conducted to investigate the responses of photosynthesis, malondialdehyde, osmotic regulators, antioxidant enzyme activities, and the yield of woad to water deficit at different growth stages. The growth stage of woad was divided in four stages: seedling, vegetative growth, fleshy root growth, and fleshy root maturity. During vegetative growth, fleshy root growth, and fleshy root maturity, three water gradients were set for plants with mild (65-75% in field water capacity, FC), moderate (55-65% in FC), and severe (45-55% in FC) deficits, respectively. In contrast, an adequate water supply (75-85% in FC) during the growth period was designed as the control (CK). The net photosynthetic rate (Pn), transpiration rate, and stomatal conductance of woad significantly decreased (P< 0.05) by moderate and severe water deficits. Still, rehydration after the water deficit could produce a noticeable compensation effect. In contrast, malondialdehyde and proline accumulation significantly increased under moderate and severe water deficits. At the same time, the superoxide dismutase, peroxidase, and catalase all had high activities (increased significantly by 19.87-39.28%, 19.91-34.26%, and 10.63-16.13% compared with CK, respectively), but yields were substantially lower, compared to CK. Additionally, the net photosynthetic rate was negatively correlated with antioxidant enzyme activity. The economic yield of plants subjected to continuous mild water deficit during both vegetative and fleshy root growth was not significantly different from that in CK. Still, the water use efficiency improved significantly. Therefore, the continuous mild water deficit during vegetative and fleshy root growth could improve the physiological and biochemical mechanisms of the plant, representing an optimal irrigation strategy for woad in cold and arid areas.

Isatis tinctoria L. (Woad): A Review of its Botany, Ethnobotanical Uses, Phytochemistry, Biological Activities, and Biotechnological Studies.

Isatis tinctoria L. (Brassicaceae), which is commonly known as woad, is a species with an ancient and well-documented history as an indigo dye and medicinal plant. Currently, I. tinctoria is utilized more often as medicinal remedy and also as a cosmetic ingredient. In 2011, I. tinctoria root was accepted in the official European phytotherapy by introducing its monograph in the European Pharmacopoeia. The biological properties of raw material have been known from Traditional Chinese Medicine (TCM). Over recent decades, I. tinctoria has been investigated both from a phytochemical and a biological point of view. The modern in vitro and in vivo scientific studies proved anti-inflammatory, anti-tumour, antimicrobial, antiviral, analgesic, and antioxidant activities. The phytochemical composition of I. tinctoria has been thoroughly investigated and the plant was proven to contain many valuable biologically active compounds, including several alkaloids, among which tryptanthrin, indirubin, indolinone, phenolic compounds, and polysaccharides as well as glucosinolates, carotenoids, volatile constituents, and fatty acids. This article provides a general botanical and ethnobotanical overview that summarizes the up-to-date knowledge on the phytochemistry and biological properties of this valuable plant in order to support its therapeutic potential. Moreover, the biotechnological studies on I. tinctoria, which mainly focused on hairy root cultures for the enhanced production of flavonoids and alkaloids as well as on the establishment of shoot cultures and micropropagation protocols, were reviewed. They provide input for future research prospects.

Woad extract containing cream improves significantly dry, irritated, and pruritic skin.

Objective of this open, noninterventional, noncontrolled study was to investigate the tolerability and performance of a woad extract containing cream in subjects with dry, pruritic skin after twice daily application over 2 weeks. Assessments included sensorial characteristics, skin condition, pruritus (numeric rating scale [NRS], dynamic pruritus score [DPS]), skin dryness (itch-controlled days [ItchCD], overall dry skin [ODS]), transepidermal water loss (TEWL), skin hydration and quality of life (5PLQ, DLQI). All sensorial characteristics were well perceived (>4), with tolerability (4.77 ± 0.47) and no negative skin feelings (4.73 ± 0.74) achieving the highest scores (0-5 scale). Dry skin and symptoms improved significantly (p < .001) for all variables: NRS (from 4.8 ± 1.74 to 1.83 ± 2.18), ItchCD (from 8.23 ± 4.40 to 4.81 ± 4.07), ODS (from 1.80 ± 0.85 to 0.65 ± 0.65), hydration (23.33 ± 1.33 to 40.70 ± 1.86), and quality of life (5PLQ: from 7.12 ± 4.25 to 4.24 ± 3.67; DLQI: from 5.29 ± 4.79 to 3.00 ± 3.98). The DPS revealed a moderate antipruritic effect. The TEWL decreased slightly (p = .511). No side effects were reported. The study suggests that, the cream is effective, and well tolerated in treating dry, irritated, and pruritic skin.

Geographic population structure in an outcrossing plant invasion after centuries of cultivation and recent founding events.

Population structure and genetic diversity of invasions are the result of evolutionary processes such as natural selection, drift and founding events. Some invasions are also molded by specific human activities such as selection for cultivars and intentional introduction of desired phenotypes, which can lead to low genetic diversity in the resulting invasion. We investigated the population structure, diversity and origins of a species with both accidental and intentional introduction histories, as well as long-term selection as a cultivar. Dyer's woad (Isatis tinctoria; Brassicaceae) has been used as a dye source for at least eight centuries in Eurasia, was introduced to eastern USA in the 1600s, and is now considered invasive in the western USA. Our analyses of amplified fragment length polymorphisms (AFLPs) from 645 plants from the USA and Eurasia did not find significantly lower gene diversity (Hj) in the invaded compared to the native range. This suggests that even though the species was under cultivation for many centuries, human selection of plants may not have had a strong influence on diversity in the invasion. We did find significantly lower genetic differentiation (Fst) in the invasive range but our results still suggested that there are two distinct invasions in the western USA. Our data suggest that these invasions most likely originated from Switzerland, Ukraine and Germany, which correlates with initial biological control agent survey findings. Genetic information on population structure, diversity and origins assists in efforts to control invasive species, and continued combination of ecological and molecular analyses will help bring us closer to sustainable management of plant invasions.

Extraction of Indigo from Some Isatis species and Dyeing Standardization Using Low-technology Methods

Fresh leaves of four Isatis species culture form of I. tinctoria L and wild forms of I. buschiana Schischkin, I. candolleana Boiss. (endemic) and I. tinctoria L. subsp. corymbosa. (Boiss.) were used for indigo production. Dyes were extracted by fermentation and hot water application. The extracted dyes were optimized with different pH and reducing agents. Results showed that the dye from hot water application produced the desired dying quality at pH 11. Reducing agent concentrations had no significant effect on color quality. Dark blue and blue colors were obtained from I. tinctoria and I. candolleana extracts although I. tinctoria subsp. corymbosa and I. buschiana produced mostly yellow-gray colors. Light, dry and wet rubbing fastness values varied between 3 and 3/4 while washing fastness was between 2 and 4/5. The highest indigo amounts were determined spectrophotometrically as 4.19 mg/g and 2.53 mg/g in I. tinctoria and I. candolleana, respectively. Results also showed that harvesting season was important for indigo production and the highest indigo amount was observed in mid-June.