Sustained silencing peanut allergy by xanthopurpurin is associated with suppression of peripheral and bone marrow IgE-producing B cell.

Introduction: Peanut allergy is an immunoglobulin E (IgE) mediated food allergy. Rubia cordifolia L. (R. cordifolia), a Chinese herbal medicine, protects against peanut-induced anaphylaxis by suppressing IgE production in vivo. This study aims to identify IgE-inhibitory compounds from the water extract of R. cordifolia and investigate the underlying mechanisms using in vitro and in vivo models. Methods: Compounds were isolated from R. cordifolia water extract and their bioactivity on IgE production was assessed using a human myeloma U266 cell line. The purified active compound, xanthopurpurin (XPP), was identified by LC-MS and NMR. Peanut-allergic C3H/HeJ mice were orally administered with or without XPP at 200µg or 400µg per mouse per day for 4 weeks. Serum peanut-specific IgE levels, symptom scores, body temperatures, and plasma histamine levels were measured at challenge. Cytokines in splenocyte cultures were determined by ELISA, and IgE + B cells were analyzed by flow cytometry. Acute and sub-chronic toxicity were evaluated. IL-4 promoter DNA methylation, RNA-Seq, and qPCR analysis were performed to determine the regulatory mechanisms of XPP. Results: XPP significantly and dose-dependently suppressed the IgE production in U266 cells. XPP significantly reduced peanut-specific IgE (>80%, p <0.01), and plasma histamine levels and protected the mice against peanut-allergic reactions in both early and late treatment experiments (p < 0.05, n=9). XPP showed a strong protective effect even 5 weeks after discontinuing the treatment. XPP significantly reduced the IL-4 level without affecting IgG or IgA and IFN-γ production. Flow cytometry data showed that XPP reduced peripheral and bone marrow IgE + B cells compared to the untreated group. XPP increased IL-4 promoter methylation. RNA-Seq and RT-PCR experiments revealed that XPP regulated the gene expression of CCND1, DUSP4, SDC1, ETS1, PTPRC, and IL6R, which are related to plasma cell IgE production. All safety testing results were in the normal range. Conclusions: XPP successfully protected peanut-allergic mice against peanut anaphylaxis by suppressing IgE production. XPP suppresses murine IgE-producing B cell numbers and inhibits IgE production and associated genes in human plasma cells. XPP may be a potential therapy for IgE-mediated food allergy.

Chemical constituents from the aerial parts of Rubia cordifolia L. with their NO inhibitory activity.

A new naphthoquinone derivative (1) together with twenty-three known compounds (2-24), were isolated from the aerial parts of Rubia cordifolia L. Their structures were elucidated on the basis of NMR and HR-ESIMS data. Compounds 1-13 were assessed for their inhibitory effects on NO production in LPS-stimulated RAW 264.7 macrophage cells. Compounds 2-6 exhibited significant inhibitory activities with IC50 values of 21.37, 13.81, 24.56, 20.32, and 30.08 µmol·L-1, respectively.

Changes in marker secondary metabolites revealed the medicinal parts, harvest time, and possible synthetic sites of Rubia cordifolia L.

Rubia cordifolia L. is a significant medicinal plant. To investigate the changes of marker metabolites of R. cordifolia, the purpurin, mollugin, carbon, nitrogen contents, and the expression of genes involved in anthraquinones synthesis were examined. The findings indicated that the two secondary metabolites were only detected in stems and roots. Root purpurin content was 5-26 times higher than in stems, and root mollugin content was 92 times higher than in stems in June. These findings suggest that the potential of the roots as a medicinal part. The roots were found to have highest purpurin content in October (2.406 mg g-1), whereas the mollugin content was highest in August (6.193 mg g-1). However, the purpurin content in August was only 0.029 mg g-1 lower than that in October, making August a suitable harvest period for R. cordifolia. The expression 1-deoxy-D-xylulose 5-phosphate synthase (dxs) and 1-deoxy-D-xylulose-5-phosphate reductorisomerase (dxr) genes in roots showed an upward trend. However, the expression level of dxr gene was significantly higher than dxs with the range of 60-518 times higher, indicating the important role of dxr gene. Through correlation and redundancy analyses, it was found that mollugin showed positive correlation with carbon contents and carbon-nitrogen ratio of aerial parts. Additionally, purpurin showed a positive correlation with the expression of both genes. As a result, mollugin is likely to be synthesized in the aerial parts and then stored in the roots, whereas purpurin might be synthesized in the stems and roots. These findings could provide cultivation guidelines for R. cordifolia.

Biosynthesis of copper oxide nanoparticles using Rubia cordifolia bark extract: characterization, antibacterial, antioxidant, larvicidal and photocatalytic activities.

Rubia cordifolia represents the pivotal plant resource belonging to traditional Chinese medicine and Indian Ayurveda. The present study aims to synthesize biocompatible copper oxide nanoparticles (CuONPs) using R. cordifolia bark extracts, characterize the incumbent chemical transitions, and explore their biomedical and environmental applications. The absorbance peak between 250 and 300 nm clearly demonstrates the formation of CuONPs in the UV-visible spectrum. Fourier transform infrared spectroscopy results showed the presence of functional groups essential for copper ion reduction. Field emission scanning electron microscopy (FE-SEM) and dynamic light scattering analysis revealed that the CuONPs are spherical-shaped with a mean particle size of 50.72 nm. Additionally, the zeta potential demonstrates its robustness at 11.2 mV. X-ray diffraction pattern showed mixed phases (Cu, Cu2O, and CuO) of cubic monoclinic crystalline nature. CuONPs exhibited noticeable antibacterial activity against Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and Bacillus cereus) pathogenic bacteria. Bacterial cell damages were affirmed through FE-SEM imaging when treated with CuONPs. Further, CuONPs demonstrated considerable antioxidant activities by quenching free radicals such as DPPH (60.75%), ABTs (70.88%), nitric oxide (65.48%) and reducing power (71.44%) in a dose-dependent way. CuONPs showed significant larvicidal activity against Aedes aegypti (65 ± 8.66%), Anopheles stephensi (80 ± 13.69%), and Culex quinquefasciatus (72 ± 13.04%) mosquito larvae. The photocatalytic activity of the CuONPs demonstrates the methylene blue (81.84%) and crystal violet (64.0%) dye degradation potentials, indicating the environmental bioremediation efficacy. Hence the present study is the first report in accounting for the versatile applications of the phyto-CuONPs. Moreover, the green synthesis of CuONPS has future applications in designing the drug for life-threatening diseases and various environmental issues.

Research Note: Effect of Rubia cordifolia L. processed Terminalia chebula Retz polysaccharide on the histological structure and apoptosis in the spleen in immunosuppressed Chinese yellow quail.

It is generally accepted herbal polysaccharide and is a bioactive compound of herbal medicines with immunomodulatory activities. It has a wide range of pharmacological effects. It can be used as a green substitute for antibiotics or as a feed additive in quail breeding. Therefore, the herbal polysaccharide has a broader and safer application prospect. The immunosuppressive disease of quail is one of the most important infectious diseases. It seriously affects the growth, development, and production performance of quail, causing huge economic losses to quail industry. However, there is no report on the effective alleviation of spleen injury in immunosuppressed animals by herbal polysaccharide. Therefore, we established a pathological model of immunosuppressive Chinese yellow quail for the first time, with the Terminalia chebula Retz polysaccharide (TCP) as the control, and histological observation, TUNEL staining were used to study the effects of Rubia cordifolia L. processed Terminalia chebula Retz polysaccharide (RTCP) on splenic tissue structure and apoptosis of immunosuppressed Chinese yellow quail. The experimental results showed that spleen organ index of the cyclophosphamide (CTX) group was significantly lower than these of blank control group, the TCP group and the RTCP group (P < 0.05). And the number of splenic nodules in the CTX group was significantly lower than that in the blank control group (P < 0.01). Compared with the CTX group, the spleen volume of the TCP group and the RTCP group increased, and the number and area of spleen nodules increased. Among them, the spleen nodules in the RTCP group were significantly more higher than that in the CTX group (P < 0.01). Meanwhile, TUNEL staining showed that the TUNEL positive cells in the CTX group were the most significantly higher than those in the blank control group (P < 0.01). TCP group and RTCP group were significantly higher than the blank control group (P < 0.01), but significantly lower than CTX group (P < 0.05). All these results suggested that RTCP could effectively improve CTX-induced spleen damage in immunosuppressed Chinese yellow quails by promoting the recovery of spleen organ index, repairing the spleen tissue structure, and diminishing the apoptosis. Moreover, RTCP is more effective than TCP. The results prove that the efficacy of RTCP in protecting spleen from CTX induced injury was enhanced after processing with Rubia cordifolia L. Therefore, our findings will provide more possibilities to promote the clinical application and development of processed traditional Chinese medicine in the further.

Biodegradable Sodium Alginate/Carrageenan/Cellulose Composite Hydrogel Wound Dressings Containing Herbal Extracts for Promoting Blood Coagulation and Wound Healing.

Accelerating the coagulation process and preventing wound infection are major challenges in the wound care process. Therefore, new multifunctional wound dressings with procoagulant, antibacterial, and antioxidant properties have enormous potential for clinical application. In this work, biodegradable hydrogels containing herbal extracts are prepared for wound dressings. First, the active ingredients are extracted from Amaranthus spinosus (A. spinosus) and Rubia cordifolia (R. cordifolia) and added to the hydrogels prepared from microcrystalline cellulose (MCC), carrageenan, and sodium alginate. Then the composite hydrogels are air-dried to obtain the wound dressings. The wound dressings prepared in this work have good biocompatibility and moisture retention. The mechanical properties of the wound dressings are further improved with the addition of MCC. Besides, the wound dressings have excellent procoagulant, antibacterial, and antioxidant properties due to the presence of R. cordifolia extract. Overall, the most effective group of wound dressings with different ingredient formulations reduces clotting time by 75% and largely inhibits bacterial growth. The wound dressings perform well in the animal wound models to promote wound healing. These results indicate that the hydrogel wound dressings prepared in this work have great potential for medical applications.

A comprehensive review of Rubia cordifolia L.: Traditional uses, phytochemistry, pharmacological activities, and clinical applications.

Rubia cordifolia (family: Rubiaceae) L (R. cordifolia) is a perennial botanical drug climbing vine. As the main part of the traditional Chinese medicine, the rhizome has a long history. A great number of literary studies have reported that it can be used for the improvement of blood circulation, hemostasis, activation of collaterals, etc. When it comes to the wide application of R. cordifolia in traditional medicine, we systematically review its traditional uses, phytochemistry and pharmacological effects. Literatures were systematically searched using several scientific databases, including China National Knowledge Infrastructure (CNKI), Baidu Scholar, PubMed, Web of Science, and other professional websites. Kew Botanical Garden and the iPlant were used for obtaining the scientific names and plant images of R. cordifolia. In addition, other information was also gathered from books including traditional Chinese herbal medicine, the Chinese Pharmacopoeia, and Chinese Materia Medica. So far, many prescriptions containing R. cordifolia have been widely used in the clinical treatment of abnormal uterine bleeding, primary dysmenorrhea and other gynecological diseases, allergic purpura, renal hemorrhage and other diseases. The phytochemistry studies have reported that more than 100 compounds are found in R. cordifolia, such as bicyclic peptides, terpenes, polysaccharides, trace elements, flavonoids, and quinones. Among them, quinones and peptides are the types of components with the highest contents in R. cordifolia. The modern pharmacological studies have revealed that R. cordifolia and its derived components have anti-tumor, anti-oxidative, anti-platelet aggregation, and anti-inflammatory effects. However, most studies are preclinical. The pharmacological mechanism of R. cordifolia has not been thoroughly studied. In addition, there are few pharmacokinetic and toxicity studies of R. cordifolia, therefore the clinical safety data for R. cordifolia is lacking. To sum up, this review for the first time summarizes a systemic and integrated traditional uses, chemical compositions, pharmacological actions and clinical applications of R. cordifolia, which provides the novel and full-scale insight for the drug development, medicinal value, and application of R. cordifolia in the future.

Rubia cordifolia L. ameliorates DSS-induced ulcerative colitis in mice through dual inhibition of NLRP3 inflammasome and IL-6/JAK2/STAT3 pathways.

The aerial part of â€‹Rubia cordifolia â€‹L. has been used as an herbal medicine for a long time with various pharmacological activities, including anti-inflammatory, anticancer, and antibacterial activities. The most notable usage of these was that this herbal medicine had good therapeutic effects on diarrhea caused by various factors. However, the mechanism for the ethanolic extract of â€‹R. cordifolia â€‹L. (RCEE) to treat Ulcerative colitis (UC) effectively is still unclear. In this study, DSS successfully induced UC mice and then intervene using different polar parts of RCEE. The results indicated that RCEE-treatment inhibited colonic combination NLRP3 inflammasome formation and IL-6/JAK2/STAT3 activation in vivo, significantly ameliorating the clinical symptoms, including alleviating colonic mucosal damage and infiltration of macrophages, suppressing the release of inflammatory cytokines, and reducing mortality. Taken together, this study suggests that dual inhibition of NLRP3 inflammasome and IL-6/JAK2/STAT3 pathways activation using RCEE may be a promising therapeutic strategy for preventing the progression of UC.

Chemistry, Biosynthesis, Physicochemical and Biological Properties of Rubiadin: A Promising Natural Anthraquinone for New Drug Discovery and Development.

Anthraquinones (AQs) are found in a variety of consumer products, including foods, nutritional supplements, drugs, and traditional medicines, and have a wide range of pharmacological actions. Rubiadin, a 1,3-dihydroxy-2-methyl anthraquinone, primarily originates from Rubia cordifolia Linn (Rubiaceae). It was first discovered in 1981 and has been reported for many biological activities. However, no review has been reported so far to create awareness about this molecule and its role in future drug discovery. Therefore, the present review aimed to provide comprehensive evidence of Rubiadin's phytochemistry, biosynthesis, physicochemical properties, biological properties and therapeutic potential. Relevant literature was gathered from numerous scientific databases including PubMed, ScienceDirect, Scopus and Google Scholar between 1981 and up-to-date. The distribution of Rubiadin in numerous medicinal plants, as well as its method of isolation, synthesis, characterisation, physiochemical properties and possible biosynthesis pathways, was extensively covered in this review. Following a rigorous screening and tabulating, a thorough description of Rubiadin's biological properties was gathered, which were based on scientific evidences. Rubiadin fits all five of Lipinski's rule for drug-likeness properties. Then, the in depth physiochemical characteristics of Rubiadin were investigated. The simple technique for Rubiadin's isolation from R. cordifolia and the procedure of synthesis was described. Rubiadin is also biosynthesized via the polyketide and chorismate/o-succinylbenzoic acid pathways. Rubiadin is a powerful molecule with anticancer, antiosteoporotic, hepatoprotective, neuroprotective, anti-inflammatory, antidiabetic, antioxidant, antibacterial, antimalarial, antifungal, and antiviral properties. The mechanism of action for the majority of the pharmacological actions reported, however, is unknown. In addition to this review, an in silico molecular docking study was performed against proteins with PDB IDs: 3AOX, 6OLX, 6OSP, and 6SDC to support the anticancer properties of Rubiadin. The toxicity profile, pharmacokinetics and possible structural modifications were also described. Rubiadin was also proven to have the highest binding affinity to the targeted proteins in an in silico study; thus, we believe it may be a potential anticancer molecule. In order to present Rubiadin as a novel candidate for future therapeutic development, advanced studies on preclinical, clinical trials, bioavailability, permeability and administration of safe doses are necessary.

H2O-Induced Hydrophobic Interactions in MS-Guided Counter-Current Chromatography Separation of Anti-Cancer Mollugin from Rubia cordifolia.

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.

TMARg, a Novel Anthraquinone Isolated from Rubia cordifolia Nakai, Increases Osteogenesis and Mineralization through BMP2 and ß-Catenin Signaling.

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 ß-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 ß-catenin signaling pathways, and suggest that TMARg might be a potential phytomedicine for the treatment of bone diseases.

Revealing the mechanisms and the material basis of Rubia cordifolia L. on abnormal uterine bleeding with uniting simultaneous determination of four components and systematic pharmacology approach-experimental validation.

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.

Anti-adipogenic and anti-obesity activities of purpurin in 3T3-L1 preadipocyte cells and in mice fed a high-fat diet.

BACKGROUND: The body responds to overnutrition by converting stem cells to adipocytes. In vitro and in vivo studies have shown polyphenols and other natural compounds to be anti-adipogenic, presumably due in part to their antioxidant properties. Purpurin is a highly antioxidative anthraquinone and previous studies on anthraquinones have reported numerous biological activities in cells and animals. Anthraquinones have also been used to stimulate osteoblast differentiation, an inversely-related process to that of adipocyte differentiation. We propose that due to its high antioxidative properties, purpurin administration might attenuate adipogenesis in cells and in mice. METHODS: Our study will test the effect purpurin has on adipogenesis using both in vitro and in vivo models. The in vitro model consists of tracking with various biomarkers, the differentiation of pre-adipocyte to adipocytes in cell culture. The compound will then be tested in mice fed a high-fat diet. Murine 3T3-L1 preadipocyte cells were stimulated to differentiate in the presence or absence of purpurin. The following cellular parameters were measured: intracellular reactive oxygen species (ROS), membrane potential of the mitochondria, ATP production, activation of AMPK (adenosine 5'-monophosphate-activated protein kinase), insulin-induced lipid accumulation, triglyceride accumulation, and expression of PPARγ (peroxisome proliferator activated receptor-γ) and C/EBPα (CCAAT enhancer binding protein α). In vivo, mice were fed high fat diets supplemented with various levels of purpurin. Data collected from the animals included anthropometric data, glucose tolerance test results, and postmortem plasma glucose, lipid levels, and organ examinations. RESULTS: The administration of purpurin at 50 and 100 µM in 3T3-L1 cells, and at 40 and 80 mg/kg in mice proved to be a sensitive range: the lower concentrations affected several measured parameters, whereas at the higher doses purpurin consistently mitigated biomarkers associated with adipogenesis, and weight gain in mice. Purpurin appears to be an effective antiadipogenic compound. CONCLUSION: The anthraquinone purpurin has potent in vitro anti-adipogenic effects in cells and in vivo anti-obesity effects in mice consuming a high-fat diet. Differentiation of 3T3-L1 cells was dose-dependently inhibited by purpurin, apparently by AMPK activation. Mice on a high-fat diet experienced a dose-dependent reduction in induced weight gain of up to 55%.

Network pharmacology-based research on the active component and mechanism of the antihepatoma effect of Rubia cordifolia L.

Rubia cordifolia L. is widely used in Asia and its antihepatoma effect has been proved by in vitro and in vivo experiments. However, there are few studies on its specific mechanism. In the present study, the network pharmacology method was used to construct the component/target/pathway molecular regulatory network for the antihepatoma effect of Rubia cordifolia L. to explore the effective components of Rubia cordifolia L. and its potential mechanism. The chemical components of Rubia cordifolia L. were identified through literature and databases, and the components were evaluated and screened by drug likeness and pharmacokinetic characteristics (ADMET). The targets of active components were predicted according to the reverse pharmacophore matching model. The hepatic carcinoma-related genes were found in databases, and antihepatoma-related gene targets were selected through comparison. The functions of target genes and related pathways were analyzed and screened using the Database for Annotation, Visualization and Integrated Discovery, and the component/target/pathways network of antihepatoma effect of Rubia cordifolia L. was constructed using Cytoscape software. Finally, 16 active compounds were screened from Rubia cordifolia L., and 39 gene targets, including AKT1, mitogen-activated protein kinase 1, and epidermal growth factor receptor, were involved. Rubia cordifolia L. also affected the hepatitis B, phosphoinositide-3-kinase-protein kinase B, and mitogen-activated protein kinase signaling pathways. Many direct-acting tumor-related signaling pathways and indirect-acting hepatitis pathways inhibit the generation of liver cancer. The present study provided a scientific basis for further elucidating the mechanism of Rubia cordifolia L. against liver cancer.

Characterization of Rubia cordifolia L. root extract and its evaluation of cardioprotective effect in Wistar rat model.

OBJECTIVES: Rubia cordifolia L. (RC) is a well-known and highly valuable medicinal plant in the Ayurvedic system. The present study involves evaluating antioxidant and cardioprotective property of RC root extract. MATERIALS AND METHODS: The characterization of RC root extract was carried out using standard phytochemical and biochemical analysis. The functional groups were analyzed by Fourier transform infrared (FTIR) spectroscopy and phytotherapeutic compounds were identified using high-resolution mass spectrometry (HR-MS). Cardioprotective activity of RC root extract was investigated against cyclophosphamide (CP; 100 mg/kg, i.p)-induced cardiotoxicity in male albino Wistar rats. RC (100, 200, and 400 mg/kg, p.o) or silymarin (100 mg/kg, p.o) was administered immediately after CP on the 1st day and the next consecutive 10 days. Biochemical and histopathological analysis was performed to observe the cardioprotective effects of RC root extract. RESULTS: Phytochemical analysis revealed the presence of secondary metabolites that include alkaloids, flavonoids, saponins, and anthraquinones in RC root extract. FTIR analysis revealed the presence of several functional groups. Based on HR-MS analysis, eight major phytotherapeutic compounds were identified in methanol root extract of RC. Biochemical analysis in CP-induced rat model administered with RC extract revealed significantly enhanced levels of antioxidant markers such as superoxide dismutase, catalase, and glutathione S-transferase. Histopathological study showed that the rat model treated with the root extract had reduced the cardiac injury. CONCLUSION: Our results have shown that the RC extract contains various antioxidant compounds with cardioprotective effect. Treatment with RC root extract could significantly protect CP-induced rats from cardiac tissue injury by restoring the antioxidant markers.

Evaluation of analgesic and anti-inflammatory activities of Rubia cordifolia L. by spectrum-effect relationships.

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.

ROS-mediated cytotoxic activity of ZnO and CeO2 nanoparticles synthesized using the Rubia cordifolia L. leaf extract on MG-63 human osteosarcoma cell lines.

In the present scenario, the synthesis and characterization of zinc oxide (ZnO) and cerium oxide (CeO2) nanoparticles (NPs) through biological routes using green reducing agents are quite interesting to explore various biomedical and pharmaceutical applications, particularly for the treatment of cancer. This study was focused on the phytosynthesis of ZnO and CeO2 NPs using the leaf extract of Rubia cordifolia L. The active principles present in the plant extract were liable for rapid reduction of Zn and Ce ions to metallic nanocrystals. ZnO and CeO2 NPs were characterized by UV-visible spectroscopy, X-ray diffraction analysis (XRD), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), energy dispersive X-ray spectroscopy (EDAX), and photoluminescence (PL) techniques. ZnO and CeO2 NPs were partially agglomerated with a net-like structure. Biomedical activities of ZnO and CeO2 NPs were tested against MG-63 human osteosarcoma cells using MTT and reactive oxygen species (ROS) quantification assays. In treated cells, loss of cell membrane integrity, oxidative stress, and apoptosis was observed and it is well correlated with cellular damage immediately after induction. Overall, this study shed light on the anti-cancer potential of ZnO and CeO2 NPs on MG-63 human osteosarcoma cells through differential ROS production pathways, describing the potential role of greener synthesis.

Anti-thrombotic and pro-angiogenic effects of Rubia cordifolia extract in zebrafish.

ETHNOPHARMACOLOGICAL RELEVANCE: 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. AIM OF STUDY: 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. MATERIAL AND METHODS: 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. RESULTS: 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 (P < 0.01, P < 0.001) 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 (P < 0.01, P < 0.001) 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 (P < 0.05, P < 0.01, P < 0.001). CONCLUSIONS: 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.

[Preparation and in vitro release evaluation of slow releasing intrauterine silicone rubber bar made of Panax notoginseng and Rubia cordifolia].

To prepare the intrauterine slow release silicone rubber bar made of Panax notoginseng and Rubia cordifolia, and finish its preliminary evaluation of in vitro releasing properties. The open mill method was used for plasticating of silicone rubber. The process parameters of the silicone rubber and drugs mixing were optimized by orthogonal test. The parameters of silicone rubber vulcanization was optimized by single factor test. The preliminary evaluation of in vitro release performance of the silicone rubber bar was conducted with ginsenoside Rg1, ginsenoside Rb1, notoginsenoside R1, purpurin and rubimaillin as the indexes. The results showed that optimum technologic parameters for silicone rubber and drugs mixing：the roller spacing 2 mm; speed ratio 1∶1.2; front roller temperature 55-60 ℃; rear roll temperature 50-55 ℃; and mixing time 20 min. The optimum parameters for silicone rubber vulcanization：temperature 90 ℃, and time 60 min. The studies on release process in vitro revealed that the release process of silicone rubber bar was in line with the Higuchi equations. After 90 days, the cumulative release of ginsenoside Rg1, ginsenoside Rb1 and notoginsenoside R1 was 46.7%, and the cumulative release of purpurin and rubimaillin was 51.9%. The preparation method can be applied to the preparation of silicone rubber bar, with slow release characteristics.

UPLC-QTOF-MS Identification of the Chemical Constituents in Rat Plasma and Urine after Oral Administration of Rubia cordifolia L. Extract.

An effective ultra-performance liquid chromatography coupled with the quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF/MS) method was developed for analysing the chemical constituents in rat plasma and urine after the oral administration of Rubia cordifolia L. extract. Under the optimized conditions, nine of 11 prototypes in rat plasma and four prototypes in urine were identified or characterized by comparing the retention time, accurate mass, fragmentation patterns, reference compounds, and literature data. In total, six metabolites, including alizarin-1-O-ß-glucuronide, alizarin-2-O-ß-glucuronide, alizarin-1-O-sulfation, alizarin-2-O-sulfation, purpurin-1-O-ß-glucuronide, and purpurin-3-O-ß-glucuronide, were identified in rat plasma, which were confirmed by lavaging standard solutions. Purpurin was found to be able to be transformed into alizarin based on the results in which alizarin was detected in rat plasma after the oral administration of a purpurin solution. In total, four metabolites were found in rat urine, but their chemical structures were not confirmed. The results indicate that the metabolic pathway of alizarin involves glucuronidation and sulfation, with the purpurins having undergone glucuronidation. The components absorbed into the blood, and the metabolites have the opportunity to become bioactive constituents. The experimental results would supply a helpful chemical basis for further research on the mechanism of actions of Rubia cordifolia L.