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Shikonin

Shikonin: A Versatile Natural Compound for Cutting-Edge Research.
Shikonin, a red naphthoquinone pigment derived from the roots of Lithospermum erythrorhizon, has garnered significant attention due to its diverse pharmacological properties.
This small-molecule natural product exhibits a range of biological activities, including anti-inflammatory, antioxidant, and anticancer effects.
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Most cited protocols related to «Shikonin»

Shikonin derivatives were extracted from both the cells and culture media. For obtaining dye fractions, a powdered sample of lyophilized cells was sonicated with n-hexane. The extraction was done for 15 min at 40°C until the red coloration had faced. The media samples were similarly extracted with n-hexane. The extracts were evaporated from the extract solution under reduced pressure. Dry residue was dissolved in methanol and analyzed in a DIONEX HPLC system (Sunnyvale, CA), equipped with an automated sample injector (ASI-100), and UVD 340S detector using the following conditions: gradient elution—acetonitrile (40–0 ml) + 0.04 M orthophosphoric acid (60–100 ml); flow rate, 1.5 ml min−1; column, EC 250/4.6 Nucleosil 120–127 mm C18 (Macherey-Nagel, Düren, Germany), and monitoring eluent at 215, 278, 514, and 320 nm. Shikonin (Wako, Tokyo, Japan) and its two derivatives ACS and isobutyrylshikonin (IBS), isolated previously from natural roots of Lithospermum canescens (Pietrosiuk and Wiedenfeld 2005 (link)), were used as standards and analyzed under the same conditions. Peaks were assigned by spiking the samples with the standards and comparison of the retention times and UV spectra.
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Publication 2012
acetonitrile Cells Culture Media derivatives High-Performance Liquid Chromatographies isobutyrylshikonin Lithospermum Methanol n-hexane phosphoric acid Plant Roots Pressure Retention (Psychology) shikonin
Purified rabbit 20S proteasome (35 ng) was incubated with 40 μmol/L of fluorogenic peptide substrate Suc-LLVY-AMC (for the proteasomal chymotrypsin-like activities) in 100 μl assay buffer (20 mM Tris-HCl, pH 7.5) in the presence of shikonin at different concentrations or the solvent DMSO for 2 h at 37°C, followed by measurement of hydrolysis of the fluorogenic substrates using a Wallac Victor3™ multilabel counter with 355-nm excitation and 460-nm emission wavelengths.
Publication 2009
Biological Assay Buffers Chymotrypsin Fluorogenic Substrate Hydrolysis Multicatalytic Endopeptidase Complex peptide L Rabbits shikonin Solvents Sulfoxide, Dimethyl Tromethamine

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Publication 2011
anti-IgG Cell Nucleus Cells Goat Hoechst33342 Immunoglobulins Macrophage Microscopy, Confocal Rabbits Rhodamine Serum Albumin, Bovine shikonin Sulfoxide, Dimethyl tetramethylrhodamine isothiocyanate Transcription Factor RelA Triton X-100
For the L. erythrorhizon root periderm and vascular tissues RNA-seq experiment, 3-month-old Siebold & Zucc. plants grown in soil under standard greenhouse conditions were harvested. Roots were collected from nine individual plants and divided into three groups, each containing three unique individuals. The periderm and vascular tissues were isolated by peeling the periderm from the roots (Fig. S5a), and the prepared portions from the three individuals in each group were pooled. Tissues were frozen in liquid nitrogen, ground by mortar and pestle, and 100 mg was used to analyze total shikonin content each sample (Fig. S5b). From the same sets of samples, RNA was extracted as described below, quantified, and DNase-treated (NEB) according to the manufacturer’s instructions. A total of six cDNA libraries from the three biological replicates prepared from each of the L. erythrorhizon periderm and vascular tissue pools, were constructed using a ribominus TruSeq Stranded Total RNA library prep kit (Illumina, San Diego, CA), and 101-bp paired-end reads were generated via Illumina HiSeq 2500 at the Purdue Genomics Center, with at least 67 million reads per library. Sequence quality was assessed by FastQC (v. 0.10.0; http://www.bioinformatics.babraham.ac.uk). The raw data were submitted to the Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra/) and are available at the NCBI Sequence Read Archive (PRJNA596998).
The experimental design for the RNA-seq experiment comparing L. erythrorhizon hairy roots sampled in B5 in the light and M9 in the dark was based on a previous report of observed rapid increases in expression of shikonin precursor pathway genes, and in PGT, within 2 h after switching L. erythrorhizon cell cultures from growth in B5 in the light to growth in M9 in darkness40 (link). In this study, several cultures from three independently generated L. erythrorhizon hairy root lines were started in liquid Gamborg B5 media containing 3% sucrose at 28 °C in the light (~100 µE m−2 s−1). After 2 weeks, hairy roots from three cultures for each of the three lines (n = 3 biological replicates per line) were harvested and pooled to represent the B5 light-treated samples. The remaining hairy root cultures were transferred to M9 media and darkness. After 2 h, hairy roots from three cultures for each of the three lines (n = 3 biological replicates per line) were harvested and pooled to represent the M9 dark-treated samples. Samples were frozen in liquid nitrogen, ground by mortar and pestle, and RNA was extracted as described below. Six cDNA libraries were generated with a TruSeq Stranded mRNA library prep kit (Illumina, San Diego, CA) and were sequenced on an Illumina NovaSeq 6000 at the Purdue Genomics Center. Sequence quality assessment were performed as described above for the periderm and vascular tissues RNA-seq experiment. The raw data were submitted to the Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra/) and are available at the NCBI Sequence Read Archive (PRJNA596998).
Additionally, unstranded RNA-seq data of L. erythrorhizon whole roots and aerial tissue from an unknown accession was downloaded from the NCBI SRA (experiments SRR3957230 and SRR3957231) to include in the gene expression analysis. Gene abundance estimates of PGT and PGT-like genes (Fig. 3b, Table S9) were measured using Kallisto v0.45.069 (link) and normalized for library depth using DESeq270 (link). Differential expression status was determined using the EdgeR v3.24.371 (link) package. For the EdgeR analysis, raw counts were normalized into effective library sizes using the trimmed mean of M-values (TMM) method72 (link), and exact tests were conducted using a trended dispersion value and a double tail reject region. A false discovery rate was calculated using the Benjamini–Hochberg procedure73 . Genes with a log2-fold change in abundance greater than 1 and false discovery rate less than 0.05 were considered as differentially represented.
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Publication 2020
The IC50 values of the
testing compounds against various SARS-CoV-2, EV-A71, and EV-D68 proteases
in the presence or in the absence of 4 mM DTT were measured with a
common protocol as the following: First, 100 μL of protease
(SARS-CoV-2 Mpro at 100 nM; SARS-CoV-2 PLpro at 200 nM; EV-A71 2Apro at 3 μM; EV-A71 3Cpro at 2 μM; EV-D68 2Apro at 1 μM; or
EV-D68 3Cpro at 100 nM) was incubated with various concentrations
of testing inhibitors at 30 °C for 30 min in its reaction buffer
in a 96-well plate, and then the reaction was initiated by adding
FRET substrate (SARS-CoV-2 Mpro and PLpro substrates
at 10 μM; EV-A71 and EV-D68 substrates at 20 μM). The
reaction was monitored for 2 h, and the initial velocity was calculated
using the data from the first 15 min by linear regression. The IC50 was calculated by plotting the initial velocity against
various concentrations of testing inhibitor by using a four parameters
dose–response curve in Prism (v8.0) software. The reaction
buffers used were as follows:

SARS-CoV-2 Mpro reaction buffer: 20 mM HEPES,
pH 6.5, 120 mM NaCl, 0.4 mM EDTA, and 20% glycerol

SARS-CoV-2 PLpro reaction buffer: 50 mM HEPES,
pH7.5, 0.01% triton X-100

EV-A71 2Apro reaction buffer: 50 mM Tris
pH 7.0, 150 mM NaCl, 10% glyceol

EV-A71
3Cpro reaction buffer: 50 mM Tris
pH 7.0, 150 mM NaCl, 1 mM EDTA, 10% glycerol

EV-D68 2Apro reaction buffer: same as EV-A71
2Apro reaction buffer

EV-D68
3Cpro reaction buffer: same as EV-A71
3Cpro reaction buffer

Publication 2020
Buffers Edetic Acid Enterovirus 71, Human HEPES Human Enterovirus 68 inhibitors papain-like protease, SARS coronavirus Peptide Hydrolases prisma SARS-CoV-2 Sodium Chloride

Most recents protocols related to «Shikonin»

Shikonin was provided by Pharmacrea Kobe Co., Ltd. (Hyogo, Japan) and β-1,3-1,6 glucan was provided by Osaka Soda Co., Ltd. (Osaka, Japan). Inclusion dispersion of shikonin in β-1,3-1,6 glucan was performed using a wet-milling method by Fuji Pigment Co., Ltd. (Hyogo, Japan). Briefly, β-1,3-1,6 glucan (3.0 g) and arginine (3.0 g) were mixed with 94 mL of demineralized water and stirred using a homo disper (PRIMIX Corporation, Hyogo, Japan) at 100 g for 3 h at 85 °C to obtain a 3.0% (w/v) of β-1,3-1,6 glucan solution. The glucan solution (0.52 g) was diluted with 9.47 mL deionized water and mixed with 0.015 g shikonin extract (powder). The solution was dispersed using a paint conditioner (Red Devil Inc., Pryor, OK, USA) with zirconia beads as the medium for 2 h to give a shikonin dispersion.
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Publication 2024
To explore the roles of shikonin in cardiac function, cardiac injury and survival of DOX-treated mice, mice were subjected to a low-dose DOX treatment with or without shikonin. DOX administration resulted in deterioration of cardiac function in mice, as verified by the decreased ejection fraction (EF), shortening fraction (FS) and pressure decay (dp/dt) in left ventricles, which were all increased in mice treated with shikonin (Fig. 1B–F). Previous studies indicated that DOX application significantly decreased the body weight in cancer patients29 (link), but intriguingly, we found that shikonin attenuated DOX-induced body weight loss in mice (Fig. 1G), which raises the possibility for its clinical use. Meanwhile, we also found that DOX injection decreased the ratio of heart weight to tibia length (HW/TL), which were significantly alleviated by shikonin administration (Fig. 1H). We measured cTnT, CK-MB, and LDH levels to evaluate cardiac injury and function. These biomarkers are commonly used indicators of myocardial damage and provided valuable information on the protective effects of shikonin against doxorubicin-induced cardiotoxicity. And we observed that shikonin significantly reversed the increased levels cTnT, CK-MB and LDH in DOX-treated mice (Fig. 1I–K). Additionally, we observed that Shikonin treatment led to an increase in the survival rate of the mice and a significant increase in their body weight (Fig. S1A–C). More importantly, we found that administration of shikonin showed no hepatic toxicity in mice, as evaluated by the serum concentrations of liver enzymes (Fig. S1D,E). And shikonin did not affect heart rates (Fig. S1F). Altogether, these findings demonstrate that shikonin protects against DOX-induced myocardial damage and dysfunction.
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Publication 2024
Next, we explored the precise mechanism by which shikonin activated Nrf2. One of the essential elements of the Hippo signaling system, which regulates cell survival and death to control tissue growth, is Mst130 (link). Mst1 is well known for being a pro-apoptotic molecule, and by being suppressed, it reduces the generation of ROS, which lessens cell apoptosis31 (link). Recent research has revealed that Mst1 controls both autophagy and apoptosis in cardiomyocytes32 (link),33 (link). We detected the expression of Mst1 and found that the high expression of Mst1 induced by DOX was significantly decreased after shikonin administration (Fig. 7A,B). Compared with the control groups, mice treated with DOX displayed decreased phosphorylation of AMPK and AKT in the hearts, but shikonin couldn’t increase the phosphorylation of AMPK and AKT (Fig. 7A,C). To Verify the hypothesis that shikonin activated Nrf2 via Mst1, NRCMs were infected with adenovirus to overexpress Mst1 (Fig. S2C). In the cells infected with GFP, shikonin could reverse the low expression of Nrf2 induced by DOX, but in the cells overexpressing Mst1, shikonin had no effect on the low expression of Nrf2 induced by DOX (Fig. 7D,E). Further detection of ROS level showed that shikonin decreased the level of ROS induced by DOX, and overexpression of Mst1 completely offsets the protective effect of shikonin on cardiomyocyte oxidative stress (Fig. 7F). In addition, NRCMs exposed to DOX had decreased cell viability and after shikonin administration the cell viability was increased. However, overexpression of Mst1 abolished the protection of shikonin against DOX-induced cell death (Fig. 7G).

Mst1 downregulation was responsible for shikonin-mediated activation on Nrf2. (A–C) Western blot and quantitative analysis showing the protein levels of p-AMPK, t-AMPK, p-AKT, t-AKT and Mst1 in vivo (n = 6). Original blots/gels are presented in Supplementary Fig. S8. (D,E) Western blot and quantitative analysis showing the Nrf2 expression after Ad-Mst1administration (n = 6). Original blots/gels are presented in Supplementary Fig. S9. (F) ROS level (n = 6). (G) CCK-8 assay for cell viability (n = 6). **p < 0.01, ****p < 0.0001, significantly different as indicated. ns not significant.

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Publication 2024
Next, we detected proteins that are representative of inflammation and apoptosis. In our research, we examined the levels of p65 and TNF-α to understand the inflammatory response in the context of doxorubicin-induced cardiotoxicity. These indicators served as crucial markers in elucidating the inflammatory pathways involved in our experimental model. Western bolt analysis indicated that DOX promoted the phosphorylation and nuclear accumulation of p65, and shikonin administration mostly reversed this alteration (Fig. 3A,B). Cardiac TNF-α levels detected by ELISA showed that shikonin decreased the elevlated cardiac TNF-α induced by DOX (Fig. 3D). The mRNA expression of cardiac inflammation biomarkers were also examined, and the result showed that DOX administration promoted the mRNA expression of pro-inflammatory genes (il-6, il-1β, mcp-1 and tnf-α), which were prevented by shikonin treatment (Fig. 3E). In our study, we investigated the expression of key apoptotic regulators, including Bax and Bcl-2, along with assessing caspase-3 activity, to elucidate the underlying mechanisms of cell apoptosis in response to doxorubicin-induced cardiotoxicity. Shikonin attenuated DOX-induced upregulation of Bax and the down-regulation of Bcl-2 (Fig. 3A,C). Besides, shikonin significantly reduced the level of apoptotic cardiomyocytes in DOX-treated mice, as evidenced by Tunel staining and caspase3 activity (Fig. 3F–H).

Shikonin attenuated DOX-induced cardiomyocyte inflammation and apoptosis. (A–C) Western blot and quantitative analysis showing the protein levels of p-P65, t-P65, Nuc-P65, Bax, Bcl-2 in four groups (n = 6). Original blots/gels are presented in Supplementary Fig. S4. (D) Cardiac TNF-α levels as detected by ELISA (n = 8). (E) The relative mRNA levels of il-6, il-1β, tnf-α, and mcp-1 normalized to gapdh in mice (n = 8). (F) Activity of caspase-3 of mice in four groups (n = 8). (G,H) Myocardial apoptosis measured by TUNEL staining in heart sections (n = 8, bar = 50 μm). **p < 0.01, ****p < 0.0001, significantly different as indicated.

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Publication 2024
Oxidative stress was involved in the development of DOX-induced cardiotoxicity. Western blots showed that shikonin significantly reversed the up-regulation of NADPH oxidase subunit p67phox and down-regulation of SOD2 expression induced by dox treatment (Fig. 2A,B). In our study, we assessed several key oxidative stress indicators, including MDA level, 4-HNE level, GSH level, and total SOD activity, to comprehensively evaluate the oxidative status. Additionally, we measured NADPH oxidase activity as a crucial indicator of oxidative stress pathway activation. Consistent with the molecular changes, we observed that shikonin decreased the excessive MDA level, 4-HNE and NADPH oxidase activity caused by DOX, and increased the low GSH level and total SOD activity caused by DOX (Fig. 2C–G). In addition, the mRNA level of NADPH oxidase subunit (p67phox, p22phox and gp91phox) decreased significantly after shikonin administration (Fig. 2H).

Shikonin attenuated DOX-induced oxidative stress in the hearts. (A,B) Western blot and quantitative analysis showing the protein levels of p67 phox and SOD2 in vehicle and shikonin treated mice (n = 8). Original blots/gels are presented in Supplementary Fig. S3. (C–E) Levels of 4-hydroxynonenal (4-HNE), endogenous antioxidants (GSH) content and malondialdehyde (MDA) in mice myocardium (n = 8). (F) NADPH oxidase activity (n = 8). (G) Total SOD activity in the myocardium (n = 8). H, NADPH oxidase subunits mRNA expression by real time RT-PCR (n = 8). ***p < 0.001, ****p < 0.0001, significantly different as indicated.

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Publication 2024

Top products related to «Shikonin»

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Shikonin is a natural pigment derived from the roots of the Lithospermum erythrorhizon plant. It is a reddish-purple compound with a distinct color and chemical structure. Shikonin serves as a versatile laboratory reagent and is commonly used in various research applications.
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DMSO is a versatile organic solvent commonly used in laboratory settings. It has a high boiling point, low viscosity, and the ability to dissolve a wide range of polar and non-polar compounds. DMSO's core function is as a solvent, allowing for the effective dissolution and handling of various chemical substances during research and experimentation.
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DMEM (Dulbecco's Modified Eagle's Medium) is a cell culture medium formulated to support the growth and maintenance of a variety of cell types, including mammalian cells. It provides essential nutrients, amino acids, vitamins, and other components necessary for cell proliferation and survival in an in vitro environment.
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The FACSCalibur flow cytometer is a compact and versatile instrument designed for multiparameter analysis of cells and particles. It employs laser-based technology to rapidly measure and analyze the physical and fluorescent characteristics of cells or other particles as they flow in a fluid stream. The FACSCalibur can detect and quantify a wide range of cellular properties, making it a valuable tool for various applications in biology, immunology, and clinical research.
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The FACSCalibur is a flow cytometry system designed for multi-parameter analysis of cells and other particles. It features a blue (488 nm) and a red (635 nm) laser for excitation of fluorescent dyes. The instrument is capable of detecting forward scatter, side scatter, and up to four fluorescent parameters simultaneously.
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Penicillin/streptomycin is a commonly used antibiotic solution for cell culture applications. It contains a combination of penicillin and streptomycin, which are broad-spectrum antibiotics that inhibit the growth of both Gram-positive and Gram-negative bacteria.

More about "Shikonin"

Shikonin, a natural naphthoquinone pigment found in the roots of the Lithospermum erythrorhizon plant, has garnered significant attention for its diverse range of pharmacological properties.
This small-molecule natural product exhibits potent anti-inflammatory, antioxidant, and anticancer effects, making it a subject of intense research interest.
Explore the latest advancements in Shikonin research with the help of PubCompare.ai, a powerful AI-driven platform that optimizes your workflow and enhances reproducibility.
Easily access protocols from literature, preprints, and patents, and leverage AI-driven comparisons to identify the most effective approaches for your Shikonin studies.
Streamline your research process and uncover meaningful insights that drive scientific progress.
Discover how PubCompare.ai can help you locate protocols from various sources, including research articles, preprints, and patents, and compare them using AI-driven analysis to identify the most suitable protocols and products for your Shikonin experiments.
Enhance your research efficiency by leveraging tools like Fetal Bovine Serum (FBS), Dimethyl Sulfoxide (DMSO), TRIzol reagent, Dulbecco's Modified Eagle Medium (DMEM), RPMI 1640 medium, GraphPad Prism 5, and FACSCalibur flow cytometer.
These resources can be seamlessly integrated into your Shikonin research workflow, helping you to achieve greater accuracy, reproducibility, and meaningful insights.
Explore the diverse applications of Shikonin, from its anti-inflammatory and antioxidant properties to its potential anticancer effects.
Collaborate with PubCompare.ai to streamline your research process, access a wealth of protocols, and drive scientific advancement in the field of Shikonin and related natural compounds.