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Madder color (MC), a natural dye isolated from Rubia tinctorum, is a potent carcinogen that targets the outer stripe of outer medulla (OSOM) in the kidneys of rats. To clarify the role of MC components in renal carcinogenesis, we examined distributions of MC components and metabolites in the kidneys of rats treated with MC using desorption electrospray ionization-mass spectrometry imaging (DESI-MSI). Alizarin, lucidin, munjistin, nordamnacanthal, purpurin, pseudopurpurin, rubiadin, and some other metabolites detected and identified by liquid chromatography time-of-flight MS analysis of rat serum 1 h after MC administration were subjected to DESI-MSI. This analysis enabled visualization of the distribution of anthraquinones in the kidney, and the ion images showed a characteristic distribution according to their chemical structure. Among the components, lucidin and rubiadin specifically localized in the OSOM, suggesting that their genotoxicity was a direct cause of MC carcinogenesis. Alizarin showed greater distribution in the OSOM than the cortex and may therefore participate in renal carcinogenicity owing to its tumor-promoting activity. Overall, our data suggested that the distribution of carcinogenic components to the OSOM was responsible for the site-specific renal carcinogenicity of MC and that DESI-MSI analysis may be a powerful tool for exploring the mechanisms of chemical carcinogenesis.

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Three undescribed meroterpenoids, named bipolacochlioquinones A-C, together with seven known compounds, were isolated from the plant endophytic fungus Bipolaris victoriae S27 derived from the fresh stems of Rubia podantha Diels. Their structures were mainly determined by extensive spectroscopic analysis. The relative configurations of bipolacochlioquinones A-C were assigned using the ROESY spectrum, comparison of their spectral data with that reported in the literatures, and NMR calculations. Moreover, their complete absolute configurations were further established by electronic circular dichroism calculations using density functional theory. Among them, bipolacochlioquinone A is found to represent the first example of previously undescribed 6/6/6/6/6 pentacyclic dioxane-containing cochlioquinones, and bipolacochlioquinone B possesses a rare 6/6/6/6/5 pentacyclic system bearing a tetrahydrofuran ring fused to a polyketide and a sesquiterpenoid subunit. All compounds were evaluated for their inhibitory effects on tumor growth, metastasis, and the NF-κB signaling pathway. Among them, bipolacochlioquinone C and cochlioquinone A show the most potent cytotoxicities and NF-κB inhibitory activities. The effects of bipolacochlioquinone C and cochlioquinone A on the expression of NF-κB-associated proteins were also evaluated using western blotting. These results indicate that bipolacochlioquinone C and cochlioquinone A can inhibit the growth and metastasis of HCT116 and MDA-MB-231 cells by suppressing the NF-κB signaling pathway.

Alizarin (1,2-dihydroxyanthraquinone) and purpurin (1,2,4-trihydroxyanthraquinone), natural anthraquinone compounds from

Simple Summary A total of 84% of the autochthonous livestock breeds in Spain are in danger of extinction. This decline is mainly produced because they are not very productive and the farmers have been oriented to the most productive breeds, neglecting the rest. There are many factors that push us to conserve autochthonous breeds with little census. These animals are very adapted to their environment, so they are the ones that best exploit the geographical characteristics of their area; besides, their products have a high quality and these breeds have very beneficial genetic components: they are a source of very useful and not sufficiently known gene variants that make them a genetic heritage of undoubted value. If they become extinct, we would be losing an irreplaceable genetic quality. Rubia de El Molar sheep, an autochthonous breed of Madrid, Spain, is currently in serious danger of disappearing. In this work, the meat quality of lambs from Rubia de El Molar breed was studied to test both weight and sex effects on meat quality, assessed by means of instrumental methods. This is the first work that studies the instrumental quality of Rubia de El Molar suckling lamb meat. Abstract The effects of sex and weight on instrumental meat quality characteristics of Rubia de El Molar autochthonous ovine breed were studied. Four weight groups (10, 15, 20 and 25 kg, each of the groups with seven males and seven females) were assessed. A decrease of longissimus thoracis muscle (LT) lightness from 43.39 for 10 kg lambs to 38.84 for 25 kg group was observed. LT redness and yellowness indices and chromaticity values increased as weight increased. The percentage of juice expelled increased from 11.52 (10 kg) to 17.15 (15 kg). The percentage of intramuscular fat in biceps femoris (BF) and supraespinatus (SE) muscles increased as weight increased. Warner–Bratzler shear force (WBSF) in raw meat and hardness in cooked meat increased as liveweight increased. However, a decrease in the amount of soluble collagen in quadriceps femoris (QF), biceps femoris (BF) and infraespinatus (IE) muscles was observed as weight increased. Sex effect was only observed for intramuscular fat content in QF and BF muscles (2.4% in males and 2.9 in females) and WBSF in raw meat (26.12 N for males and 21.25 N for females). Weight had a greater effect than sex on Rubia de El Molar meat quality characteristics.

A broad palette of natural dyes is often mentioned with reference to dyed textiles from ancient China. However, few scientific works address the problem of correctly identifying these dyes, often referring simply to unidentified sources. The aim of this work was the creation of a database of mass spectra of molecules related to Chinese natural dyes, intended to be used for their identification in archaeological textiles. High pressure liquid chromatography coupled with electrospray ionisation and quadrupole time-of-flight detection (HPLC-ESI-Q-ToF) was used to analyse twenty-eight reference specimens of Asian dyes, including relatively common dyes, such as sappanwood (Biancaea sappan), gromwell (Lithospermum erythrorhyzon), safflower (Carthamus tinctorius), madder (Rubia tinctorum, Rubia cordifolia and Rubia akane), and unusual dyes, such as rosewood (Dalbergia sp.), rhubarb (Rheum emodi), dragon's blood (Daemonorops sp.), gamboge (Garcinia hanburyi), violet (Viola yedoensis), etc. All analyses were repeated after artificial ageing, thus showing which molecules are more likely to be considered as markers for the identification of these dyes in ancient textiles. Because of the dye sources chosen, most molecular classes of natural organic colourants were included in the mass spectra database, i.e. anthraquinones, naphthoquinones, flavonoids, neoflavonoids, alkaloids, chalcones, carotenoids, xanthenes, polyphenols, etc. The information obtained from the reference specimens was then used to identify the dyes in thirty-one textiles from Dunhuang (northwestern Gansu, China, 7th-10th century AD) in the British Museum's collection. The sampling was guided by the data previously gathered from multispectral imaging and fibre optic reflectance spectroscopy (FORS), presented in a separate paper. Mixtures of dyes were commonly found, leading to hypothesise the re-dyeing or recycling of some of the textiles. A very broad palette of dyes suggested possible different origins for these textiles and a tendency to experiment with dyeing. These data, integrated with the ones obtained non-invasively, represent a significant source of information for researchers investigating textiles from the Silk Road.

In this study, silk fabrics were dyed with madder (Rubia tinctorium L.) and gall oak (Quercus infectoria Olivier). One fabric was dyed only with madder and the other four fabrics were dyed with bot...

The archaeological complex of Dunhuang (northwestern Gansu, China) is considered a pearl on the Silk Road and the content of its caves revolutionised oriental studies. The British Museum hosts a significant number of textiles and textile fragments from the site. Although mostly catalogued and studied from the point of view of textile production, weaving techniques and iconography, they have never undergone dye analysis, which is here presented for the first time. Due to the value, preciousness and fragility of these textiles, sampling is not always an option. Therefore, a non-invasive approach combining multispectral imaging (MSI) and fibre optic reflectance spectroscopy (FORS) was tested. The final aims of this research were to explore the advantages and limitations of such an approach with application to Asian dyes and ultimately to provide new insights into the technological skills related to dyestuff production and use in ancient China. To achieve these aims, twenty-nine reference specimens of Asian dyes were sourced, including sappanwood (Biancaea sappan), sandalwood (Pterocarpus santalinus), different species of madder (Rubia spp.), rosewood (Dalbergia sp.), rhubarb (Rheum emodi), dragon's blood (Daemonorops sp.), Indian lac (Kerria lacca), gromwell (Lithospermum erythrorhyzon), safflower (Carthamus tinctorius), amur-cork tree (Phellodendron amurense), gamboge (Garcinia hanburyi), violet (Viola yedoensis), pagoda tree (Sophora japonica). Silk was dyed using these raw materials and the reference samples were analysed by colorimetry before and after ageing. These measurements highlighted the most light-sensitive dyes to be turmeric, safflower, sappanwood and sandalwood. The reference samples were also used to create a database of reflectance and apparent absorption spectra obtained by FORS, as well as to record the reflectance, luminescence and absorption properties of specific dyes by MSI. The combined information obtained from MSI and FORS brought to the fore the signature behaviour of certain dyes (protoberberine-based dyes, safflower, gromwell, tannins, madder, sappanwood, Indian lac and indigo), which enabled their unequivocal identification and the mapping of their distribution in thirty-one Dunhuang textiles from the British Museum's collection. The data presented in this work will be beneficial to other researchers and conservators investigating textiles from the Silk Road using a non-invasive approach and for the assessment of textiles and their suitability for display.

Cancer is one of the leading causes of human death. Nanotechnology could offer new and optimised anticancer agents in order to fight cancer. It was shown that metal nanoparticles, in particular silver nanoparticles (AgNPs) were effective in cancer therapy. In this study, AgNPs were synthesised using Rubia tinctorum L. extract (Ru-AgNPs). Then, cytotoxicity effects of the Ru-AgNPs against MDA-MB-231 carcinoma cell line and human dermal fibroblast as normal cell line were performed. Furthermore, anti-apoptotic effects of Ru-AgNPs on these cancer and normal cell lines were compared using acridine orange/propidium iodide staining, flow cytometry analysis and real-time qPCR in apoptosis gene markers. Results of UV-vis spectroscopy showed that Ru-AgNPs have a peak at 430 nm, which indicated synthesis of AgNPs. Ru-AgNPs had spherical shape and average size of 12 nm. Ru-AgNPs have cytotoxicity on MDA-MB-231 cells and decrease cancerous cell viability (IC50 = 4 µg/ml/48 h). Ru-AgNPs could induce apoptosis in MDA-MB-231 cells through upregulation of Bax and downregulation of Bcl-2 gene expression. The results opened up new avenues to develop Rubia based metal complexes as an anticancer agent.

Two RA-series bicyclic hexapeptides, RA-XXV (4) and RA-XXVI (5), which have no N-methyl group at Tyr-5, were isolated from the roots of Rubia cordifolia L. Their amino acid compositions and sequences were determined by interpretation of MS, and 1D and 2D NMR data and their relative structures were elucidated by XRD analysis of 4 and RA-XXVI acetate (6). The absolute stereochemistry of 4 was established by the total synthesis of 4, and that of 5, by the chemical correlation with 4. Peptides 4 and 5 exhibited cytotoxicity toward human promyelocytic leukemia HL-60 (IC50 =0.062 and 0.066 μm, respectively) and human colonic carcinoma HCT-116 (IC50 =0.028 and 0.051 μm, respectively) cell lines. Analysis of the conformational structures of 4 and 6 in the crystalline state and those of 4 and 5 in solution revealed that the N-methyl group at Tyr-5 functions to make this series of peptides preferentially adopt the active conformation.

Rubiaceae-type cyclopeptides (RAs) are one of characteristic constituents isolated from Rubia species, which are candidates of innovative anti-tumor drugs due to their significant bioactivity. However, approaches on the systematic characterization and quantification of RAs are still not available because of low contents and complicated purification. In this study, an ultra performance liquid chromatography coupled triple quadrupole tandem mass spectrometry (UPLC-QqQ-MS/MS) method was established and validated for qualitative and quantitative analysis of 14 RAs (1-14) in 20 Rubia plants from China. The separation was achieved on a Waters ACQUITY UPLC® BEH C18 column (50 × 2.1 mm, 1.7 μm) using gradient elution with a mobile phase consisting of 0.1% formic acid in acetonitrile and water. Multiple reaction monitoring (MRM) in positive mode was used to enable the selective detection of RAs from the Rubia root and rhizome extracts within 10 min. This method was proved to be specific, sensitive, precise, and accurate with the limits of detection and quantification at 0.6–11.4 ng/mL and 1.9–34.2 ng/mL, respectively, and the relative standard deviation (RSD) of the overall intra-day and inter-day precision less than 5.24%. Satisfactory recovery was obtained from 83.80% to 111.77%, with the RSD less than 5.32%. Totally, 67 samples were then qualitatively and quantitatively analyzed by this method and 51 of them were proved to contain RAs. Thereinto, R. podantha and R. yunnanensis from Yunnan were the two most abundant species. Additionally, RAs were detected in 8 Rubia species for the first time. Then chemometric approaches were revealed to explain the relationship between samples based on their contents of RAs. This study demonstrated that the method was not only useful for RAs source discovery and chemotaxonomy of Rubia species, but also could be extended to standardization of RAs medical materials and their new drug research and development.

Rubia yunnanensis is an important medicinal plant with various bioactive secondary metabolites. In order to reduce the dependence on wild populations of the species, we aim to establish in vitro culture system that can produce Rubiaceae-type cyclopeptides (RAs) and quinones. Agrobacterium rhizogenes-mediated transformation of stem segments of in vitro grown R. yunnanensis plants using four A. rhizogenes strains was studied and transformation conditions were optimized. Hairy roots appeared with the highest frequency (68.89%) when stem segments (with leaves) without pre-culture were immersed in A. rhizogenes A4 strain bacterial suspension for 30 min, co-cultured on Murashige and Skoog (MS) solid medium in the dark for three days, and afterwards incubated in darkness. PCR analysis of rolB and rolC genes confirmed transformed nature of six hairy root clones. The hairy roots grew rapidly, especially showing the highest accumulation of biomass in MS liquid medium compared to in vitro grown plants and calli. Histological observation of hairy root revealed anatomical difference in vascular cylinder, where the cells exhibited high mitotic activity characterized by vigorous growth. The UPLC-MS/MS analysis revealed that the amount of RAs in the hairy roots grown in ½MS liquid medium (4.611 μg g−1 DW) was higher than that in in vitro grown plants (0.331 and 4.096 μg g−1 DW for shoots and roots respectively) and calli (1.082 μg g−1 DW), but still far lower than that in the roots of seed-borne plants (80.296 μg g−1 DW). However, the hairy roots accumulated high level of quinones (2320.923 and 5067.801 μg g−1 DW for MS and ½MS liquid media respectively), of the same order of magnitude as the roots of seed-borne plants (7409.973 μg g−1 DW). Hairy root culture of R. yunnanensis, with high accumulation of biomass and production of quinones, may offer an attractive perspective for the production of the RAs and quinones that could be further optimized for pharmaceutical use.

Simple Summary Rubia de El Molar is in danger of extinction, and this work is part of a research project funded by the Regional Government of Madrid that aims to know its productive characteristics to prevent its disappearance. The aim of this work is to study the effect of weight and sex on suckling lamb meat sensory characteristics of Rubia de El Molar breed. According to sensory characteristics, two weight groups of animals were detected: light carcasses (10 and 15 kg) and heavy carcasses (20 and 25 kg). Heavy carcasses’ meat was tougher, more elastic, and received lower pleasantness scores than light animals. Sex had a lower effect on meat sensory parameters than weight, and meat from male lambs showed higher flavour intensity and also higher pleasantness scores than female meat. Abstract Fifty-six Rubia de El Molar ovine breed lamb carcasses were divided into 8 groups (n = 7 per group) according to weight (10, 15, 20 and 25 kg) and sex (male and female) to study the effect of these factors on meat sensory characteristics, assessed by means of a trained panel. Heavier animals showed a more-springy meat and also received lower scores for pleasantness. Assessors gave lower scores for flavour and pleasantness in female meat. A high correlation was detected between hardness and springiness, number of chews, and pleasantness. Juiciness, greasiness, and flavour were also sensory variates highly correlated. Pleasantness was only correlated to hardness. Changes from 10 to 25 kg did not affect juiciness, greasiness, flavour, and number of chews. There are no significant differences in the sensory quality of lambs slaughtered at 10 and 15 kg live weight, and also between 20 and 25 kg groups. Heavy carcasses (20 and 25 kg) showed a more hard and springy meat than light carcasses (10 and 15 kg). Besides, the 10 and 15 kg animals group received higher scores for pleasantness. This work showed differences in suckling lambs’ meat sensory parameters between Rubia de El Molar and other ovine breeds.

Counter-current chromatography (CCC) is a unique liquid–liquid partition chromatography and largely relies on the partition interactions of solutes and solvents in two-phase solvents. Usually, the two-phase solvents used in CCC include a lipophilic organic phase and a hydrophilic aqueous phase. Although a large number of partition interactions have been found and used in the CCC separations, there are few studies that address the role of water on solvents and solutes in the two-phase partition. In this study, we presented a new insight that H2O (water) might be an efficient and sensible hydrophobic agent in the n-hexane-methanol-based two-phase partition and CCC separation of lipophilic compounds, i.e., anti-cancer component mollugin from Rubia cordifolia. Although the n-hexane-methanol-based four components solvent systems of n-hexane-ethyl acetate-methanol-water (HEMWat) is one of the most popular CCC solvent systems and widely used for natural products isolation, this is an interesting trial to investigate the water roles in the two-phase solutions. In addition, as an example, the bioactive component mollugin was targeted, separated, and purified by MS-guided CCC with hexane-methanol and minor water as a hydrophobic agent. It might be useful for isolation and purification of lipophilic mollugin and other bioactive compounds complex natural products and traditional Chinese medicines.

Treatment of kidney stones is based on symptomatic medications which are associated with side effects such as gastrointestinal symptoms (e.g., nausea, vomiting) and hepatotoxicity. The search for effective plant extracts without the above side effects has demonstrated the involvement of antioxidants in the treatment of kidney stones. A local survey in Morocco has previously revealed the frequent use of Rubia tinctorum L. (RT) for the treatment of kidney stones. In this study, we first explored whether RT ethanolic (E-RT) and ethyl acetate (EA-RT) extracts of Rubia tinctorum L. could prevent the occurrence of urolithiasis in an experimental 0.75% ethylene glycol (EG) and 2% ammonium chloride (AC)-induced rat model. Secondly, we determined the potential antioxidant potency as well as the polyphenol composition of these extracts. An EG/AC regimen for 10 days induced the formation of bipyramid-shaped calcium oxalate crystals in the urine. Concomitantly, serum and urinary creatinine, urea, uric acid, phosphorus, calcium, sodium, potassium, and chloride were altered. The co-administration of both RT extracts prevented alterations in all these parameters. In the EG/AC-induced rat model, the antioxidants- and polyphenols-rich E-RT and EA-RT extracts significantly reduced the presence of calcium oxalate in the urine, and prevented serum and urinary biochemical alterations together with kidney tissue damage associated with urolithiasis. Moreover, we demonstrated that the beneficial preventive effects of E-RT co-administration were more pronounced than those obtained with EA-RT. The superiority of E-RT was associated with its more potent antioxidant effect, due to its high content in polyphenols.

Synthetic dyes release huge amount of waste and uniform colourants lead to health hazard. It also disturbing the ecobalance of the nature. Natural dyes are mostly derived from plants, invertebrates, or minerals. The most of the natural dyes are vegetable dyes from plant sources-roots, berries, bark, leaves, and wood—and other organic sources such as fungi and lichens. Natural dyes exhibit good biodegradability and are more compatible with the environment. In spite of their inferior fastness, natural dyes are more acceptable to environmentally conscious people around the world. The present study deals with the natural dyes extracted from Rubia Cordifloria. The extracted dye used to dye selected silk fabric and myrobalan mordant used for dye ability, fastness properties, absorbency test. Two shades with different concentration have been developed. Absorbency properties of the dyes extracted from madder and sinking test was determined. The colour fastness through washing and rubbing (Wet and Dry) was an excellent satisfaction in both different concentrations.

The anticancer activities of Rubia cordifolia and its constituents have been reported earlier, but their influence on the crosstalk of complex cancer-related signaling metabolic pathways (i.e., transcription factors; TF) has not yet been fully investigated. In this study, R. cordifolia root extract was subjected to the cancer signaling assay based bioactivity-guided fractionation, which yielded the following compounds viz., three anthraquinones, namely alizarin (1), purpurin (2), and emodin (3); two lignans, namely eudesmin (4) and compound 5; and two cyclic hexapeptides, namely deoxybouvardin RA-V (6), and a mixture of 6+9 (RA-XXI). The structures of the isolated compounds were determined by NMR spectroscopy and HRESIMS. The isolated compounds 1, 2, 3, 6, and a mixture of 6+9 were tested against a panel of luciferase reporter genes that assesses the activity of a wide-range of cancer-related signaling pathways. In addition, reference anthraquinones viz., chrysophanol (11), danthron (12), quinizarin (13), aloe-emodin (14), and α-lapachone (15) were also tested. Among the tested compounds, the cyclic hexapeptide 6 was found to be very active against several signaling pathways, notably Wnt, Myc, and Notch with IC50 values of 50, 75, and 93 ng/mL, respectively. Whereas, the anthraquinones exhibited very mild or no inhibition against these signaling pathways. Compound 6 being the most active, we tested it for stability in simulated intestinal (SIF) and gastric fluids (SGF), since the stability in biological fluid is a key short-coming of cyclic hexapeptides. The anticancer activity of 6 was found to remain unchanged before and after the treatment of simulated gastric/intestinal fluids, indicating that RA-V was stable. As a result, it could be bioavailable when orally used in therapeutics and possibly a drug candidate for cancer treatment. The mechanism for the preferential inhibition of these pathways and the possible crosstalk effect with other previously reported signaling pathways has been discussed.

Objective Nanoharvesting from intact plants, organs, and cultured cells is a method in which nanoparticles are co-incubated with the target tissue, which leads to the internalization of nanoparticles. Internalized nanoparticles are coated in situ with specific metabolites that form a dynamic surface layer called a bio-corona. Our previous study showed that metabolites that form the bio-corona around anatase TiO2 nanoparticles incubated with leaves of the model plant Arabidopsis thaliana are enriched for flavonoids and lipids. The present study focused on the identification of metabolites isolated by nanoharvesting from two medicinal plants, Ocimum sanctum (Tulsi) and Rubia tinctorum (common madder). Results To identify metabolites that form the bio-corona, Tulsi leaves and madder roots were incubated with ultra-small anatase TiO2 nanoparticles, the coated nanoparticles were collected, and the adsorbed molecules were released from the nanoparticle surface and analyzed using an untargeted metabolomics approach. Similar to the results in which Arabidopsis tissue was used as a source of metabolites, TiO2 nanoparticle bio-coronas from Tulsi and madder were enriched for flavonoids and lipids, suggesting that nanoharvesting has a wide-range application potential. The third group of metabolites enriched in bio-coronas isolated from both plants were small peptides with C-terminal arginine and lysine residues.

Calcium-dependent protein kinases (CDPKs) are essential regulators of plant growth and development, biotic and abiotic stress responses. Inactivation of the auto-inhibitory domain (AID) of CDPKs provides the constitutive activity. This study investigated the effect of overexpressed native and constitutive active (AtCPK1-Ca) forms of the AtCPK1 gene on abiotic stress tolerance and the ROS/redox system in Rubia cordifolia transgenic callus lines. Overexpression of the native AtCPK1 increased tolerance to salinity and cold almost in two times, when AtCPK1-Ca - in three times compare to control culture. A more interesting effect of overexpression of the AtCPK1 and AtCPK1-Ca was observed for heat resistance. The native form of AtCPK1 increased resistance to heating by 45%, while the AtCPK1-Ca increased by 80%. At the same time, another type of mutation of the AID (AtCPK1-Na, not active) did not affect the tolerance of the cell culture to stresses. We suppose, in this process, the ROS/redox system might be involved. Levels of intracellular ROS, ROS-generating enzymes expression and activities (Rbohs, Prx) and ROS-detoxifying enzymes (SOD, Cat, Apx and Prx) changed in a coordinated manner and in strict interconnection, depending of the callus growth phase and correlated with improved stress tolerance caused by AtCPK1. Because overexpression of both the AtCPK1 and AtCPK1-Ca did not significantly change callus growth, we propose that inactivation of AID of the AtCPK1 or its ortholog, might be an interesting instrument for improvement of plant cells resistance to abiotic stress.

Ethnopharmacological relevance The roots of Rubia yunnanensis Diels (Chinese name ‘Xiao-Hong-Shen’), a traditional Chinese medicine native to Yunnan province (China), have a long history of use for treating several diseases, such as tuberculosis, rheumatism and cancers. A bicyclic hexapeptidic glucoside named RA-XII was isolated from R. yunnanensis, which has been reported to exert anti-inflammatory and antitumor activities. Aim of the study This study was designed to investigate the antitumor activity and potential mechanism of RA-XII on colorectal cancer (CRC) cell lines. Materials and methods Sulforhodamine B assay, clonogenic assay and cell cycle analysis were conducted to assess the anti-proliferative activity of RA-XII on CRC cells. GFP-LC3B plasmid transfection, MDC and AO staining assays, cathepsin activity assay, and siRNAs against several genes were used to investigate the effect of RA-XII on autophagy. Western blotting was used to examine the expression levels of proteins associated with cell cycle arrest, apoptosis and autophagy. Human CRC xenograft-bearing BALB/c nude mice were used to evaluate the antitumor effect of RA-XII in vivo. Results RA-XII showed favorable antineoplastic activity in SW620 and HT29 cells in vitro and in vivo. RA-XII did not induce apoptosis indicated by no obvious changes on mitochondrial membrane potential and apoptosis-related marker proteins in SW620 or HT29 cells. Treatment of RA-XII inhibited the formation of autophagosomes, which is implied by the GFP-LC3 fluorescent dots, MDC-stained autophagic vesicles and LC3 protein expression. It was indicated that RA-XII suppressed autophagy by regulating several signaling pathways including mTOR and NF-κB pathways. Pharmacological or genetic inhibition of autophagy could enhance the cytotoxicity of RA-XII while autophagy inducer could rescue RA-XII-induced cell death. Besides, RA-XII could increase the susceptibility of CRC cells to bortezomib. Conclusion Our study demonstrated that RA-XII exerted antitumor activity independent of apoptosis, and suppressed protective autophagy by regulating mTOR and NF-κB pathways in SW620 and HT29 cell lines, which suggested that RA-XII is a key active ingredient for the cancer treatment of Rubia yunnanensis and possesses a promising prospect as an autophagy inhibitor for CRC therapy.

Background: Plant extracts have long been regarded as useful medicines in the treatment of human diseases. Rubia cordifolia Nakai has been used as a traditional medicine, as it has pharmacological properties such as antioxidant and anti-inflammatory activity. However, the biological functions of TMARg, isolated from the roots of R. cordifolia, in osteoblast differentiation remain unknown. This study was performed to investigate the pharmacological effects and intracellular signaling of TMARg in the osteoblast differentiation of pre-osteoblast MC3T3-E1 cells and mesenchymal precursor C2C12 cells. Methods: Cell viability was evaluated using an MTT assay. Early and late osteoblast differentiation was examined by analyzing the activity of alkaline phosphatase (ALP), and by staining it with Alizarin red S (ARS). Cell migration was determined by using migration assays. Western blot analysis and immunocytochemical analysis were used to examine the intracellular signaling pathways and differentiation proteins. Results: In the present study, TMARg showed no cytotoxicity and increased the osteoblast differentiation in pre-osteoblasts, as assessed from the alkaline phosphate (ALP) staining and activity and ARS staining. TMARg also induced BMP2 expression and increased the p-smad1/5/8-RUNX2 and β-catenin pathways in both MC3T3-E1 and C2C12 cells. Furthermore, TMARg activated mitogen-activated protein kinases (MAPKs) and increased the cell migration rate. In addition, the TMARg-mediated osteoblast differentiation was suppressed by BMP and Wnt inhibitors with the downregulation of BMP2 expression. Conclusion: These findings demonstrate that TMARg exerts pharmacological and biological effects on osteoblast differentiation through the activation of BMP2 and β-catenin signaling pathways, and suggest that TMARg might be a potential phytomedicine for the treatment of bone diseases.

A rapid and sensitive method was developed and validated for the quantitative determination of xanthopurpurin (XPP) in rat plasma using ultra-performance liquid chromatography-electrospray ionization-Orbitrap mass spectrometry. XPP inhibits IgE production and prevents peanut-induced anaphylaxis. The XPP and emodin (internal standard) were determined in negative ion mode with m/z 239.0350 → 211.0400 and 269.0455 → 241.0507, respectively. The separation process was achieved using an ACQUITY UPLC HSS T3 column with acetonitrile and 0.1% formic acid in water (85:15). The linear range was 0.5-100 ng/mL, and the correlation coefficient (r2 ) was > 0.993. The inter-day and intra-day precision was within an acceptable range of 15%. The extraction recovery and matrix effect were 78.9-87.2% and 94.3-98.5%, respectively. Under different conditions, the XPP was stable in the range of 5.6-10.6%. This method was successfully applied to study the pharmacokinetics of XPP with an oral dose of 10.0 mg/kg and intravenous dose of 2.0 mg/kg in rats. The absolute oral bioavailability of XPP was 4.6%.

The roots of Rubia cordifolia L. (RCL) have become an important medicine for abnormal uterine bleeding (AUB) and hemorrhage syndrome in Traditional Asian medicine. However, the underlying mechanism and the material basis of RCL for treating AUB has not been fully elucidated. In this study, quantitative evaluation of quinones, systematic pharmacology and experimental verification were adopted. Firstly, the Disease-Ingredient-Target network was established by Cytoscape, which was consistent with 23 compounds and 47 target genes. The hub targets were discovered by Maximal Clique Centrality (MCC) method with Cytohubba plugins of Cytoscape, and top 20 nodes were ranked by MCC. It was assumed that mollugin is the main ingredient of RCL for treating AUB. Pathways on which RCL acted were obtained from observation of its biological functions, KEGG pathways and Reactome pathway enrichment analysis. The possible mechanism of RCL for treating AUB was revealed for improvment of the blood clotting system, blood circulation, arachidonic acid metabolism and inflammation. Then, a novel method for evaluating the quality of RCL was established, and the content of mollugin in RCL was the higher than others. Finally, pharmacologic experiments confirmed that RCL could improve the inflammation by inhibiting the activity of COX-2 and cPLA2 enzyme, ameliorate blood hypercoagulability by affecting coagulation cascade and fibrinolytic system. It was found that RCL inhibited the expression COX-2 and PAI-1 by reducing HIF-1α expression. The trend of each index of mollugin was consistent with that of RCL, indicating that it played an important role in RCL for treating AUB. The above results could provide a novel method for the quality evaluation of RCL and was expected to give us more important information regarding the use of RCL as a promising drug candidate for AUB, offering a fertility preserving medical, non-hormonal treatment choose for women with AUB.

The genus RubiaL. includes valuable species in terms of important agricultural,industrial, and pharmacological characteristics. Red dye obtained from theroots of Rubia tinctorum L.,naturally found in the flora of Turkey and known as common madder, has beenused as the source of a natural dye since ancient times. In this work, apreliminary report is given on anatomical and palynological traits of R. tinctorum distributed in the AegeanRegion of Turkey examined by light and scanning electron microscopy. In theroot anatomy, the cortex composed of multilayered parenchyma cells, thevascular tissue organized in collateral vascular bundles, 1(-2)-rowed raycells, and the pith with a cavity at the center are observed. In the leafanatomy, the bifacial and amphistomatic leaf, the dorsiventral mesophyll withone layer of columnar palisade parenchyma cells and a few layers of irregularlyorganized spongy parenchyma cells, and the midrib with a large collateralvascular bundle surrounded by parenchymatous bundle sheath cells are recognized.Pollen grains are shed as monads, small, mostly spheroidal in equatorial view,generally hexacolpate and have a microechinate-perforate exine ornamentation., Rubia L.cinsi tarımsal, endüstriyel ve farmakolojik açıdan önemli türleri içermektedir.Türkiye florasında doğal olarak bulunan ve kızılboya olarak bilinen Rubia tinctorum L. eski tarihlerden beriköklerinden elde edilen kökboyası ile doğal boya kaynağı olarakkullanılmaktadır. Bu çalışmada, Türkiye’nin Ege Bölgesi’nde yayılış gösteren R. tinctorum’un ışık ve taramalıelektron mikroskobu ile incelenen anatomik and palinolojik özellikleri üzerinebir ön değerlendirme raporu verilir. Kök anatomisinde, çok sıralı parankimahücrelerinden meydana gelen korteks, kollateral iletim demetlerinden oluşaniletim doku, 1(-2) sıralı ışın hücreleri ve merkezinde bir boşluk bulunan özgözlemlenmektedir. Yaprak ayası anatomisi incelendiğinde, bifasiyal veamfistomatik tipte yaprak, 1 sıralı silindir şeklinde palizat parankiması ilebirkaç sıralı düzensiz dizilmiş sünger parankimasından oluşan dosiventral tiptemezofil ve parankimatik demet kını hücreleri tarafından çevrelenen büyükkollateral bir iletim demetine sahip orta damar bulunmaktadır. Polentanecikleri tek tek dökülür, küçük boyutlu, ekvatoral görünümde sıklıklasferoidal şekilli, genellikle hekzakolpat ve mikroekinat-perforat ekzin süsünesahiptir.

Real-time quantitative polymerase chain reaction (RT-qPCR) has been widely applied in gene expression and transcription abundance analysis because of its high sensitivity, good repeatability, and strong specificity. Selection of relatively stable reference genes is a precondition in order to obtain the reliable analysis results. However, little is known about evaluation of a set of reference genes through scientific experiments in Rubia plants. Here, 15 candidate reference genes were selected from R. yunnanensis transcriptome database and analyzed under abiotic stresses, hormone treatments, and different tissues. Among these 15 candidate reference genes, heterogeneous nuclear ribonucleoprotein (hnRNP), TATA binding protein (TBP), ribosomal protein L5 (RPL5), malate dehydrogenase (MDH), and elongation factor 1-alpha (EF-1α) were indicated as the five most stable reference genes by four statistical programs (geNorm, NormFinder, BestKeeper, and RefFinder). Ultimately, the validity of reference genes was confirmed by normalizing the expression of o-succinylbenzoate-CoA ligase (OSBL) and isochorismate synthase (ICS) involved in the anthraquinone biosynthesis pathway in different tissues and hormone treatments. Meanwhile, four other putative genes involved in the anthraquinone biosynthesis pathway were also normalized with the selected reference genes, which showed similar expression levels with those given by transcriptome data. This work is the first research that aims at a systematic validation on the stability of reference genes selected from R. yunnanensis transcriptome data and will be conducive to analyze gene expression in R. yunnanensis or other Rubia species.

Hydrogen bonding is a vital feature of a large ensemble of chemical structures. Soluble epoxide hydrolase (sEH) has been targeted for development of the treatment for inflammation-associated diseases. Compounds 1 and 2 were purified from Rubia philippinensis, and their structures were established via physical data analysis. Compound 1 possesses intramolecular hydrogen bonding, sufficiently robust to transfer heteronuclear magnetization via a nonbonded interaction. The bonding strength was assessed using the 1H NMR chemical shift temperature coefficients (−1.8 ppb/K), and the heteronuclear coupling constants were measured. The stereochemical details were investigated using interproton distance analysis and ECD. Purified compounds displayed moderate sEH-inhibitory activity.

Rubia cordifolia L., is an industrially viable medicinal crop and is widely exploited for the therapeutic potential of its bioactive metabolite, purpurin. The present investigation aimed to explore the chemotypic and molecular variability in seven wild populations of R. cordifolia from South Eastern Ghats region of India. Thirty-eight individuals were assessed for molecular fingerprinting (ISSR markers) and densitometric quantification of purpurin and alizarin. The populations of Yelagiri Hills and Shervaroy Hills contained the highest levels of alizarin (0.115 and 0.093%, respectively) while Pachamalai and Kolli Hills revealed the highest purpurin content (0.284 and 0.280%, respectively). Genetic diversity was generally higher in the same populations that produced higher metabolite content, with the exception of Pachamalai, suggesting a highly prioritized conservation concern. The study revealed a Nei's total gene diversity at species level of 0.266 and of 0.187 at population level, with an average population genetic differentiation of 0.28. No clear genetic or chemical structure was retrieved between the studied populations, with individuals from different locations clustering together, and no significant correlation was obtained between metabolites and genetic diversity or between these and the populations' geographic distances.

The objective of the current work was to evaluate the spectrum-effect relationships between high-performance liquid chromatography fingerprints and analgesic and anti-inflammatory effects of Rubia cordifolia L. extract (RCE), and to identify active components of RCE. Chemical fingerprints of ten batches of RC from various sources were obtained by HPLC, and similarity and hierarchical clustering analyses were carried out. Pharmacodynamic assays were performed in adjuvant-induced arthritis rat model to assess the analgesic and anti-inflammatory properties of RCE. The spectrum-effect relationships between chemical fingerprints and the analgesic and anti-inflammatory effects of RCE were established by gray correlation analysis. UPLC-ESI-MS was used to identify the structures of potential active components, by reference standards comparison. The results showed that a close correlation existed between chemical fingerprints with analgesic and anti-inflammatory activities, and alizarin, 6-hydroxyrubiadin, purpurin and rubiadin might be the active constituents of RCE. In addition, RCE attenuated pathological changes in adjuvant-induced arthritis. The current findings provide a strong basis for combining chemical fingerprints with analgesic and anti-inflammatory activities in assessing the spectrum-effect relationships of RCE.

Rubia cordifolia is a common traditional Chinese medicine that promotes blood circulation and eliminates blood stasis, and has been used to cure diseases related to blood stasis syndrome (BSS) clinically for many years. It has been previously demonstrated that anti-thrombosis and pro-angiogenesis can improve BSS. However, the anti-thrombotic and pro-angiogenic activities of Rubia cordifolia have not been well investigated.To determine the potential anti-thrombotic and pro-angiogenic activities of Rubia cordifolia and to elucidate the underlying mechanisms. In addition, the major chemical constituents of Rubia cordifolia extract (QC) were qualitatively analysed by UPLC-Q-TOF/MS to explore the association between pharmacological activity and chemical constituents.The QC samples were composed of a 95% ethanol extract and an aqueous extract following extraction using 95% ethanol. UPLC-Q-TOF/MS was used to analyse the major chemical constituents of QC. For the anti-thrombotic experiment of QC, a phenylhydrazine (PHZ)-induced AB strain zebrafish thrombosis model was used. The zebrafish larvae were stained using O-dianisidine, and the heart and caudal vein of the zebrafish were observed and imaged with a fluorescence microscope. The staining intensity of erythrocytes in the heart (SI) of each group and the morphology of thrombus in the caudal vein were used to assess the anti-thrombotic effect of QC. For the pro-angiogenic assay of QC, the intersegmental blood vessel (ISV) insufficiency model of Tg(fli-1: EGFP)y1 transgenic zebrafish (Flik zebrafish), which was induced by the VEGF receptor tyrosine kinase inhibitor II (VRI), was used. The morphology of the intact ISVs and defective ISVs was observed to evaluate the pro-angiogenic activity of QC. The mechanism involved in promoting angiogenesis was studied with real-time PCR.A total of 12 components in QC were identified based on standard compounds and references, including nine anthraquinones and three naphthoquinones. After treatment with QC, the PHZ-induced thrombosis in AB strain zebrafish larvae decreased to a certain degree, which we believe was related to its dosages, and the therapeutic effect within the 50-200 µg/mL QC treatment groups was especially prominent (P0.01, P0.001) compared to that in the PHZ model group. Similarly, QC also recovered the loss of the ISVs, which was induced by VRI in Flik zebrafish larvae, which have a certain dose-effect relationship. The pro-angiogenic activity of QC was also conspicuous (P0.01, P0.001) compared to that of the VRI model group. The following real-time PCR assay proved that QC significantly restored the VRI-induced downregulation of vWF, VEGF-A, kdrl, and flt-1 in Flik zebrafish (P0.05, P0.01, P0.001).A total of 12 compounds from QC were analysed by UPLC-Q-TOF/MS. The data of the pharmacological experiments demonstrated that QC presented anti-thrombotic and pro-angiogenic activities in zebrafish, and the principal active components were likely anthraquinones and naphthoquinones. Thus, the current study provided a theoretical basis for the clinical use of Rubia cordifolia as a traditional Chinese medicine in promoting blood circulation and eliminating stasis.

Irrigation with brackish water is common in the semiarid region of the Brazilian Northeast, potentiating the process of salinization of the soil in these areas. The hydroponic system stands out as an important strategy for crop management in semiarid regions, since crops show greater tolerance to the salt stress under hydroponic conditions, which minimize the effects of soil salinization. Thus, the objective of this work was to evaluate the effects of salt stress on the growth, fruit yield and mineral nutrition of sweet pepper in hydroponic system, using brackish water and coconut fiber substrate. The experiment was conducted in Recife, State of Pernambuco, Brazil (8°3'15''S, 34°52'53''W). A randomized block experimental design in a 5x2 factorial arrangement with five replications was used, consisting of five water salinity levels of the nutrient solution (2.0; 3.5; 4.5; 5.5 and 6.5 dS m-1) and two sweet pepper cultivars (Bruno and Rubia). The increasing salinity level of the nutrient solution decreased the fruit yield of the cultivars evaluated, reduced the potassium, phosphorus and sulfur, and increased the sodium and chlorine contents in the leaf tissues. The Ca contents of the cultivar Bruno decreased with increasing salinity, and increased in the cultivar Rubia. The cultivar Rubia stood out with the highest fruit yield and ionic selectivity, accumulating high amounts of potassium and calcium and maintaining low Na and Cl contents. The fruit yield of the cultivar Rubia (39.9 t ha-1) grown under solution with electrical conductivity of up to 3.5 dS m-1, denotes the potential viability of growing this cultivar under hydroponic conditions. A irrigação com águas salobra, frequente na região semiárida do nordeste brasileiro, potencializa o processo de salinização do solo nessas áreas. O cultivo hidropônico destaca-se como uma importante estratégia de convivência com o semiárido, uma vez que as culturas apresentam maior tolerância ao estresse salino nestas condições de cultivo, as quais minimizam os efeitos da salinização dos solos. O objetivo deste trabalho foi avaliar os efeitos da salinidade sobre o crescimento, produção e nutrição mineral de pimentão em cultivo hidropônico utilizando água salina em substrato de fibra de coco. O experimento foi conduzido em Recife, Pernambuco, Brasil (8°3'15'' S, 34°52'53'' W). Foi utilizado o delineamento experimental em blocos casualizados com fatorial 5x2, com cinco repetições, consistindo em cinco níveis de salinidade da solução nutritiva (2,0; 3,5; 4,5; 5,5 e 6,5 dS m-1) e duas cultivares de pimentão (Bruno e Rúbia). O incremento dos níveis de salinidade da solução nutritiva diminuiu a produção de frutos das cultivares avaliadas, reduziu os teores de potássio, fósforo e enxofre e aumentou os teores de sódio e cloro no tecido foliar. O teor de Ca da cv. Bruno decresceu em função do aumento da salinidade, e a apresentou aumento na cv. Rúbia. A cv. Rúbia destacou-se pela maior produtividade e maior eficiência na seletividade iônica, sendo capaz de acumular mais potássio e cálcio, e manter menores teores de Na e Cl. A produtividade da cv. Rúbia (39,9 t ha-1) quando cultivada em solução com condutividade elétrica de até 3,5 dS m-1, indica a viabilidade potencial de seu cultivo em condições hidropônicas.

Five new quinone derivatives, rubiasins D-F (1-3), rubialatones A and B (4 and 5), have been isolated from the roots and rhizomes of three Rubia species, Rubia alata, R. wallichiana and R. schumanniana, together with 26 known quinones (6-31). Their structures have been elucidated on the basis of NMR, MS spectra and computational methods. The compounds have been evaluated for their toxicity to the saprophytic nematode Caenorhabditis elegans and the root-knot nematode Meloidogyne incognita. Compounds 1, 6 and 7 showed toxicity to C. elegans with the LC50 values at 8.50, 9.44 and 44.82 mu g/mL, respectively; and 6 showed toxicity to M. incognita with the LC50 value at 35.22 mu g/mL. Meanwhile, compounds 1 and 6 showed inhibitory effect on egg hatch of C. elegans with the LC50 values at 5.60 and 48.95 mu g/ml. (C) 2018 Elsevier Ltd. All rights reserved.

An important example of a mixture of red colourants from plant and insect sources has been found during a recent investigation of a purple pigment on a 3rd century BC oinochoe from Canosa di Puglia present in the British Museum collection. Further investigation was carried out to elucidate both the nature of this pigment and its possible method of manufacture. In a practice typical of the period, Egyptian blue and a pink lake were found to be physically mixed to produce the purple pigment observed on the object. High pressure liquid chromatography coupled with mass spectrometry (HPLC-MS) revealed that the main colourant of the pink lake was derived from madder (Rubia spp.). However, the samples investigated also contained markers for insect-derived colourants from cochineal (Porphyrophora spp., in partial agreement with recent archaeological findings from Thessaloniki) and lac (Kerria Lacca Kerr). Despite allusions to its existence in ancient sources, evidence for the use of lac dye as a colourant (in a lake pigment) from Antiquity has never previously been found. In addition, markers indicative of tannins were also found in the mixture. This was interpreted as an indication of the reuse of a colourant that has been extracted from previously dyed textiles. Although common practice in the manufacture of red lakes from the 14th to 17th centuries, this may be the first evidence for this method to produce lake pigments found on an object from Antiquity. In addition to confirming the power of HPLC-MS in terms of the sensitivity and specificity of information, that in many cases allows species differentiation, these discoveries raise many questions, not least of which is the real extent of use of insect-derived colourants, as part of the polychrome palette of these votive objects and ancient polychromy in general. The implications of this use in the wider context of pigment manufacture in Antiquity, particularly in terms of the trade in raw materials, must also be considered.

Four previously undescribed naphthohydroquinone dimers named rubipodanones A-D, together with 19 known quinones containing three known napthohydroquinone dimers named rubioncolin C, methyl 5-hydroxy-dinaphtho[1,2-2′,3′]furan-7,12-dione-6-carboxylate and rubialatin B, were isolated from the roots and rhizomes of Rubia podantha. Their structures and absolute configurations were determined mainly by NMR, X-ray diffraction, and computational methods. Rubipodanones C and D, the glycosides of rubipodanone A and a pair of C-3 epimer, are the first identified dimeric napthohydroquinone glycosides from the Rubia plants. All naphthohydroquinone dimers were evaluated for their cytotoxicities against ten tumor cell lines and effects on the tumor-associated NF-κB signaling pathway, and rubioncolin C showed the best cytotoxicity with IC50 value of 1.53 μM and NF-κB inhibitory activity with IC50 value of 2.97 μM. These results also demonstrated that the key roles of C-3 configuration and sugar group for biological activities of rubipodanone C.