Natural product; TMEM16A chloride channel; PKM2; NF-κB

The enzyme PKM2, which determines the last rate-limiting step of glycolysis and is essential for the survival and proliferation of cancer cells, is strongly and selectively inhibited by alkannin. Alkannin's IC50 is 0.3 μM when D-fructose-1,6-bisphosphate (FBP) is not present. Alkannin's IC50 is 0.9 μM when FBP (125 μM) is present. When alkannin is applied to cancer cells that mostly express PKM2, it can effectively limit the cellular glycolytic flow [8]. The rate of cellular lactate generation and glucose consumption is inhibited by alkannin (2.5–20 μM, 1 hour) [8].

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Shikonin, an inhibitor of the TMEM16A chloride channel, with an IC50 of 6.5 μM[1]. Additionally, shikokinin inhibits PKM2 specifically [2]. Additionally, it can stop the nuclear factor-κB (NF-κB) pathway from being activated and inhibit tumor necrosis factor-α (TNF-α). Normal human keratinocytes (NHK) were shown to be considerably less viable (P<0.05) when exposed to shikonin at concentrations greater than 50 μM in comparison to the control. TNF-α-induced NF-κB p65 nuclear translocation was inhibited by shikonin pretreatment for two hours [3]. Cell viability was considerably reduced after 12 hours of treatment with 5 and 7.5 μM shikonin. Both cell lines' inhibitory effects also displayed a time-dependent pattern as compared to the 0 hour group. It was discovered that at the 24- to 48-hour time period, 5 μM shikonin had a stronger inhibitory impact than 2.5 μM shikonin. When U87 and U251 cells were treated with shikonin at 2.5, 5 and 7.5 μM for 24 and 48 hours (p<0.01), their invasiveness was much lower than that of the control group [4].

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In this study, we found that 10 μmol/L Shikonin stimulated the growth of normal human keratinocytes and 1 μmol/L shikonin promoted growth of human dermal fibroblasts. However, shikonin did not directly induce COL1 mRNA expression and PIP production in dermal fibroblasts in vitro. In addition, 1 μmol/L shikonin inhibited translocation of NF-κB p65 from cytoplasm to nucleus induced by tumor necrosis factor-α stimulation in dermal fibroblasts. Furthermore, shikonin inhibited chymotrypsin-like activity of proteasome and was associated with accumulation of phosphorylated inhibitor κB-α in dermal fibroblasts.
Conclusions: These results suggested that Shikonin may promote wound healing via its cell growth promoting activity and suppress skin inflammation via inhibitory activity on proteasome. Thus, shikonin may be a potential therapeutic reagent both in wound healing and inflammatory skin diseases. [3]

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Shikonin is an anthraquinone derivative extracted from the root of lithospermum. Shikonin is traditionally used in the treatment of inflammatory and infectious diseases such as hepatitis. Shikonin also inhibits proliferation and induces apoptosis in various tumors. However, the effect of shikonin on gliomas has not been fully elucidated. In the present study, we aimed to investigate the effects of shikonin on the migration and invasion of human glioblastoma cells as well as the underlying mechanisms. U87 and U251 human glioblastoma cells were treated with shikonin at 2.5, 5, and 7.5 μmol/L and cell viability, migration and invasiveness were assessed with CCK8, scratch wound healing, in vitro Transwell migration, and invasion assays. The expression and activity of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) and the expression of phosphorylated β-catenin (p-β-catenin) and phosphorylated PI3K/Akt were also checked. Results showed that shikonin significantly inhibited the cell proliferation, migration, invasion, and expression of MMP-2 and MMP-9 in U87 and U251 cells. The expression of p-β-catenin showed contrary trends in two cell lines. It was significantly inhibited in U87 cells and promoted in U251 cells. Results in this work indicated that shikonin displayed an inhibitory effect on the migration and invasion of glioma cells by inhibiting the expression and activity of MMP-2 and -9. In addition, shikonin also inhibited the expression of p-PI3K and p-Akt to attenuate cell migration and invasion and MMP-2 and MMP-9 expression in both cell lines, which could be reversed by the PI3K/Akt pathway agonist, insulin-like growth factor-1 (IGF-1).[4]

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Shikonin is a highly lipophilic naphtoquinone found in the roots of Lithospermum erythrorhizon used for its pleiotropic effects in traditional Chinese medicine. Based on its reported antipyretic and anti-inflammatory properties, we investigated whether shikonin suppresses the activation of NLRP3 inflammasome. Inflammasomes are cytosolic protein complexes that serve as scaffolds for recruitment and activation of caspase-1, which, in turn, results in cleavage and secretion of proinflammatory cytokines IL-1β and IL-18. NLRP3 inflammasome activation involves two steps: priming, i.e. the activation of NF-κB pathway, and inflammasome assembly. While shikonin has previously been reported to suppress the priming step, we demonstrated that shikonin also inhibits the second step of inflammasome activation induced by soluble and particulate NLRP3 instigators in primed immortalized murine bone marrow-derived macrophages. Shikonin decreased NLRP3 inflammasome activation in response to nigericin more potently than acetylshikonin. Our results showed that shikonin also inhibits AIM2 inflammasome activation by double stranded DNA. Shikonin inhibited ASC speck formation and caspase-1 activation in murine macrophages and suppressed the activity of isolated caspase-1, demonstrating that it directly targets caspase-1. Complexing shikonin with β-lactoglobulin reduced its toxicity while preserving the inhibitory effect on NLRP3 inflammasome activation, suggesting that shikonin with improved bioavailability might be interesting for therapeutic applications in inflammasome-mediated conditions [7].

When compared to the osteoarthritis group, shikonin significantly prevented the increase in IL-1β and TNF-α expression levels in the osteoarthritis rat model (P<0.01). In the rat model of osteoarthritis, shikonin dramatically reduced the amount of NF-κB protein expression when compared to the osteoarthritis group (P<0.01). In the rat osteoarthritis model treated with shikonin, the induction of iNOS levels was reduced (P<0.01) in comparison to the osteoarthritis group. Shikonin treatment dramatically reduced the increase of COX-2 protein expression in the osteoarthritis rat model when compared to the osteoarthritis group (P<0.01). The increase in caspase-3 activity was much lower in the shikonin-treated osteoarthritis rat model than in the osteoarthritis group (P<0.01). After receiving shikonin treatment, the osteoarthritis group's Akt phosphorylation was dramatically recovered in the rat model of osteoarthritis (P<0.01) [5].

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Secretory diarrhea remains a global health burden and causes major mortality in children. There have been some focuses on antidiarrheal therapies that may reduce fluid losses and intestinal motility in diarrheal diseases. In the present study, we identified Shikonin as an inhibitor of TMEM16A chloride channel activity using cell-based fluorescent-quenching assay. The IC50 value of shikonin was 6.5 μM. Short-circuit current measurements demonstrated that shikonin inhibited Eact-induced Cl(-) current in a dose-dependent manner, with IC50 value of 1.5 μM. Short-circuit current measurement showed that shikonin exhibited inhibitory effect against CCh-induced Cl(-) currents in mouse colonic epithelia but did not affect cytoplasmic Ca(2+) concentration as well as the other major enterocyte chloride channel conductance regulator. Characterization study found that shikonin inhibited basolateral K(+) channel activity without affecting Na(+)/K(+)-ATPase activities. In vivo studies revealed that shikonin significantly delayed intestinal motility in mice and reduced stool water content in a neonatal mice model of rotaviral diarrhea without affecting the viral infection process in vivo. Taken together, the results suggested that shikonin inhibited enterocyte calcium-activated chloride channels, the inhibitory effect was partially through inhbition of basolateral K(+) channel activity, and shikonin could be a lead compound in the treatment of rotaviral secretory diarrhea. [1]

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The M2 isoform of pyruvate kinase M2 (PKM2) has been shown to be up-regulated in human skin cancers. To test whether PKM2 may be a target for chemoprevention, Shikonin, a natural product from the root of Lithospermum erythrorhizon and a specific inhibitor of PKM2, was used in a chemically-induced mouse skin carcinogenesis study. The results revealed that shikonin treatment suppressed skin tumor formation. Morphological examinations and immunohistochemical staining of the skin epidermal tissues suggested that shikonin inhibited cell proliferation without inducing apoptosis. Although shikonin alone suppressed PKM2 activity, it did not suppress tumor promoter-induced PKM2 activation in the skin epidermal tissues at the end of the skin carcinogenesis study. To reveal the potential chemopreventive mechanism of shikonin, an antibody microarray analysis was performed, and the results showed that the transcription factor ATF2 and its downstream target Cdk4 were up-regulated by chemical carcinogen treatment; whereas these up-regulations were suppressed by shikonin. In a promotable skin cell model, the nuclear levels of ATF2 were increased during tumor promotion, whereas this increase was inhibited by shikonin. Furthermore, knockdown of ATF2 decreased the expression levels of Cdk4 and Fra-1 (a key subunit of the activator protein 1. In summary, these results suggest that shikonin, rather than inhibiting PKM2 in vivo, suppresses the ATF2 pathway in skin carcinogenesis. [2]

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Shikonin has previously been shown to have antitumor, anti-inflammatory, antiviral and extensive pharmacological effects. The aim of the present study was to explore whether the protective effect of shikonin is mediated via the inhibition of inflammation and chondrocyte apoptosis, and to elucidate the potential molecular mechanisms in a rat model of osteoarthritis. A model of osteoarthritis was established in healthy male Sprague-Dawley rats and 10 mg/kg/day shikonin was administered intraperitoneally for 4 days. It was found that shikonin treatment significantly inhibited inflammatory reactions in the rats with osteoarthritis. Osteoarthritis was found to significantly increase interleukin (IL)-1β, tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS) levels compared with those in the sham group. However, shikonin treatment significantly inhibited the increases in IL-1β, TNF-α and iNOS levels in the rats with osteoarthritis. Furthermore, caspase-3 activity and cyclooxygenase (COX)-2 protein expression were significantly increased and phosphorylated Akt protein expression was greatly suppressed in rats with osteoarthritis when compared with the sham group. Shikonin administration attenuated the changes in caspase-3 activity and COX-2 expression and Akt phosphorylation in rats with osteoarthritis. These results indicate that shikonin inhibits inflammation and chondrocyte apoptosis by regulating the phosphoinositide 3-kinase/Akt signaling pathway in a rat model of osteoarthritis [5].

Iodide Influx Fluorescent Assay [1]
To measure the inhibition of TMEM16A by Shikonin, FRT cells expressing TMEM16A were seeded in a black walled clear bottom 96 well plate until confluent. The cells were then washed three times with PBS followed by incubation with different concentrations of shikonin for 10 min. Fluorescence data were recorded with a FLUOstar Galaxy microplate reader equipped with HQ 535/30M (535 ± 15 nm) emission and HQ500/20X (500 ± 10 nm) excitation filters, and syringe pumps. Fluorescence was recorded continuously for 14 s, and ATP (200 μM) were pumped into the system along with iodide at 2 s. Iodide influx rates (d[I–]/dt) were computed as described in previous study (Kristidis et al., 1992).

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Cell-based proteasome activity assay [3]
Approximately, 4,000 HDFs/well in a white-walled 96-well plate were treated with 0.1% DMSO, 1 μmol/L Shikonin or 10 μmol/L lactacystin at 37°C for 2 h, and then stimulated with 50 ng/ml TNF-α for 30 min. Cells were then incubated with proteasome-Glo™ cell-based assay reagent for 15 min according to the manufacturer's protocol. Luminescence generated from each reaction was detected by Centro LB 960 microplate luminometer.

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Caspase-1 Activity Assay [7]
Potential inhibitors of caspase-1 were analyzed using Caspase-1 Inhibitor Drug Screening Kit from BioVision. Shikonin and positive inhibition control (Z-VAD-FMK) were prepared in PBS and applied to black 96-well fluorescence plate. Active caspase-1 was added, followed by caspase-1 substrate YVAD-AFC. After 45 min incubation at 37°C, fluorescence of samples was measured using SinergyMx plate reader.

Western Blot Analysis[8]
Cell Types: MCF-7 and A549 express PKM2 but not PKM1 and PKL
Tested Concentrations: 0-20 μM
Incubation Duration: 1 hour
Experimental Results: Inhibition of cellular glycolysis rate in a concentration-dependent manner.

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Immunofluorescence studies [3]
Cells were seeded onto coverslips in six-well plates and allowed to attach overnight in a medium containing 5% FBS. After cells were starved in serum-free medium for 24 h, cells were pretreated with 1 μmol/L Shikonin or 0.1% DMSO for 2 h prior to stimulation with TNF-α (50 ng/ml). Then, after medium was removed, cells were rinsed with phosphate-buffered saline and fixed with methanol for 8 min at 4°C. A blocking step was performed with 1% BSA in PBS for 30 min at room temperature. Cells were immunostained with anti-NF-κB p65 (C-20) antibody (1:100 dilution) in 1% BSA/PBS for 2 h at room temperature, followed by incubation with FITC-conjugated anti-rabbit IgG pAb (1:100 dilution) for 1 h at room temperature. Slides were observed with BX 51TRF fluorescence microscope.

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Immunoblot analysis [3]
Human dermal fibroblasts were pretreated with 1 μmol/L Shikonin or 0.1% DMSO for 2 h, and then stimulated with 50 ng/ml TNF-α for 30 min. Then, cytoplasmic proteins were extracted by nuclear extract kit according to the manufacturer's instructions. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 5–20% gradient gel, and transferred onto nitrocellulose membrane by a semi-dry transfer method using iBlot® system.

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Cell Proliferation Assay [4]
Cell proliferation was measured with CCK8 assay kit according to the literature. Briefly, U87 and U251 cells were seeded into 96-well plates at a density of 1 × 104 cells per well in standard DMEM and incubated for 24 h under standard conditions (37 °C and 5 % CO2). Our previous data showed that the IC50 values of Shikonin at 24 h were 1.84 ± 0.34 μmol/L for U251 cells and 2.02 ± 0.44 μmol/L for U87 cells. Therefore, the concentrations used in this study were 2.5, 5, and 7.5 μmol/L. Then the medium was replaced with either blank, serum-free DMEM or DMEM containing shikonin at concentrations of 2.5, 5, and 7.5 μmol/L. The total volume in each well was 200 μL. Glioma cells were incubated in these solutions for 0, 12, 24, 36, 48, or 72 h followed by treatment with 20 μL of CCK8 in each well for another 1.5 h at 37 °C. Finally, the plates were shaken softly and the optical density was recorded at 570 nm (OD570) using an ELISA plate reader. At least three independent experiments were performed. The inhibition rate was calculated using the following formula: (ControlOD570—Experimental group OD570)/ControlOD570 × 100%.

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In Vitro Migration Assay [4]
The migratory capacity of human glioblastoma cells was evaluated in 24-well plates with Transwell inserts of 8-μm pore size according to the literature. Parental U87 or U251 cells were trypsinized and resuspended in serum-free DMEM at the density of 5 × 105/mL and 200 μL of cell suspension was added into the upper chambers. Five hundred microliters of conditioned medium (DMEM medium supplemented with 10% FBS) were placed in the lower chambers, serving as the revulsant of cell migration. Serum-free DMEM served as a negative control. Shikonin was added in the suspension of parental U87 cells or U251 cells at the concentration of 2.5, 5, or 7.5 μmol/L. PIRES2-p-β-catenin, shRNA-p-β-catenin, LY294002 (20 μmol/L) or shikonin (5 μmol/L) combined with PI3K/Akt agonist insulin-like growth factors-1 (IGF-1) (20 μg/mL, Proteintech) were also added. After incubation for 24 or 48 h, the inserts were taken out and cells remaining on the upper surface of the filters were carefully removed with a cotton wool swab. The cells migrating to the underside surface were gently washed once with PBS and fixed with methanol and glacial acetic acid (mixed at 3:1) for 30 min at room temperature and stained in Giemsa stain for 15 min. The average number of migrating cells was counted in six random high-power fields (×400).

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Scratch Wound Healing Assay [4]
A scratch wound healing assay was performed to evaluate the migration ability of glioblastoma cells, as described previously. Briefly, cells were seeded into six-well plates at a density of 1.0 × 105/well until they reached 80% confluence. The scratching wounds were created in the monolayer of confluent U87 or U251 cells with a pipette tip. The width of the wounds was assessed to be the same at the beginning of the experiments. The wells were rinsed with PBS three times to remove floating cells and debris. To test the effects of Shikonin on the migration of human glioblastoma cells, parental U87 or U251 cells were seeded in serum-free DMEM with or without shikonin (2.5, 5, or 7.5 μmol/L). Then these cells were incubated for 0–48 h. The culture plates were incubated at 37 °C and in 5% CO2. Wound healing was measured and recorded photographically over time using phase-contrast microscopy at 0, 24, and 48 h.

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In Vitro Invasion Assay [4]
The effects of Shikonin on the invasion of human glioblastoma cells were checked using Transwell invasion assay with inserts of 8-μm pore size, as described previously. The membranes of Transwell filter inserts were coated with Matrigel diluted with medium at the ratio of 1:7. Parental U87 or U251 cells were prepared as described above. Five hundred microliters of DMEM supplemented with 10% FBS were placed in the lower chambers. Serum-free DMEM served as a negative control. Shikonin (2.5, 5, or 7.5 μmol/L), pIRES2-p-β-catenin, shRNA-p-β-catenin, LY294002 (20 μmol/L) or shikonin (5 μmol/L) combined with IGF-1 (20 μg/mL, Proteintech: Chicago, IL, USA) was added in the suspension of cells in the upper chamber. After incubation for 0–48 h, the inserts were taken out and prepared for observation under a microscope as described above. The average number of invasive cells was counted in six random high-power fields (×400).

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Western Blot Assay [4]
In order to determine the expression of p-β-catenin, Western blot assay was performed. U87 or U251 cells were treated with Shikonin at the concentrations of 2.5, 5, and 7.5 μmol/L for 48 h. The cells were washed three times with ice-cold PBS to stop the stimulation. Then, the cells were collected and lysed in ice-cold radio immunoprecipitation assay lysis buffer containing 50 mmol/L Tris (PH 7.4), 150 mmol/L NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mmol/L sodium orthovanadate, 50 mmol/L sodium fluoride, and 1 mmol/L EDTA for 30 min. Then the pellet was disrupted with an ultrasonic crusher and samples were centrifuged at 17,000 rpm for 60 min at 4 °C. The supernatant was collected as the soluble fraction and transferred to a new tube. The sample tubes were heated in a boiling water bath immediately for 5 min to denature the proteins. The protein concentration of the soluble material was determined with BCA protein assay kit.

Intestinal Motility Measurement [1]
ICR mice were starved for 24 h and then orally administered Shikonin (5.8 μg). After 30 min, 20 mg of 10% activated charcoal diluted in 5% gum arabic was administered orally. After another 30 min, the animals were sacrificed and the small intestines were removed. Peristaltic index was calculated as the ratio of the length that activated charcoal had traveled to the total length of the small intestine.

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Mouse Model of Rotaviral Diarrhea [1]
Neonatal ICR mice (age 4–7 days, weight 2–3 g) were inoculated with 30 μL rotavirus (virus titer 1.2 × 107 pfu/mL) by oral gavage using a polyethylene tube (0.6 mm outer diameter, 0.3 mm inner diameter) and an insulin syringe. The mice were then returned to their mothers and allowed to suckle. Stool samples were collected daily by gentle palpation of the abdomen. In one set of the experiments, the Shikonin-treated group received Shikonin orally (0.4 and 1.7 μg in 30 μL PBS) the day before virus inoculation, and three times a day until day 3. Control mice received 30 μL PBS alone. The positive control (CaCCinh-A01) mice received 34 μg (in 20 μL PBS) CaCCinh-A01 by intraperitoneal injection on the day before virus inoculation and twice a day thereafter until day 3. In another set of experiments, shikonin-treated group received shikonin in PBS on the next day of virus inoculation, and three times a day until day 3. Negative control mice received 30 μL PBS alone. The positive control mice received CaCCinh-A01 by intraperitoneal injection the day before virus inoculation and twice a day thereafter until day 3.

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Chemically-induced mouse skin carcinogenesis [2]
Sixty 6–8-week old female DBA/2 mice (which are relatively sensitive to skin carcinogenesis) were divided into 4 groups: DMSO, DMBA/TPA, SKN, SKN+DMBA/TPA. The DMSO group (5 mice) received DMSO treatment as the vehicle control; the DMBA/TPA group received a single topical application of 200 nmol DMBA for 2 weeks, following by a single topical application of 5 μg TPA (12-O-tetradecanoylphorbol-13-acetate), once per day, three times per week for 14 weeks. The SKN group received topical application of Shikonin at 10 μg following the same schedule for DMBA/TPA treatments. The SKN+DMBA/TPA groups received shikonin (SKN) treatment first followed by TPA treatment 30 min later. At the end of the skin carcinogenesis study, mice were euthanized by pentobarbital (150 mg/kg, i.p.). The skin samples from experimental sites were collected and submitted for biochemical and morphologic analysis as described in the following.

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Experimental groups and treatment [5]
The rats were randomly assigned to three groups: Sham-operated group (n=10), osteoarthritis model group (n=10) and sShikonin-treated group (n=10). In the sham-operated group, the right knee joint of the anesthetized rat was only exposed under sterile conditions, and the rats were treated with 0.1 ml/100 g physiological saline (i.p.). In the osteoarthritis model group, osteoarthritis model rats were treated with 0.1 ml/100 g physiological saline (i.p.). In the shikonin-treated group, osteoarthritis model rats were treated with 10 mg/kg shikonin (i.p.) once daily for 4 days after osteoarthritis modeling (14,15).

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ELISA analysis [5]
Following treatment with 10 mg/kg Shikonin, peripheral blood was collected from the abdominal aorta of rats in each group (n=10). The blood was centrifuged at 12,000 × g for 10 min at 4°C and the supernatant was analyzed for IL-1β, TNF-α and iNOS using ELISA assay kits according to the manufacturer's protocol (Beijing 4A Biotech Co., Ltd.).

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Western blot analysis [5]
Following the treatment with 10 mg/kg Shikonin, rats were anesthetized with 50 mg/kg pentobarbital intraperitoneally (i.p.), sacrificed by decapitation, and samples of arthrotic tissue were collected (n=10 per group). The samples were homogenized with radioimmunoprecipitation assay (RIPA) lysis buffer. The homogenate was centrifuged at 12,000 × g for 10 min at 4°C and analyzed using a bicinchoninic acid (BCA) assay kit. Approximately 50 µg protein was separated by electrophoresis on a 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel and then transferred onto a nitrocellulose filter membrane. Proteins were detected using mouse anti-nuclear factor (NF)-κB p65 (sc-29311; 1:500), anti-cyclooxygenase (COX)-2 (sc-23984; 1:300), anti-Akt (sc-8312; 1:500) and anti-phosphorylated-Akt (anti-p-Akt; sc-135650; 1:1,000) and anti-β-actin (BB-2101-1; 1:5,000) followed by horseradish peroxidase-conjugated goat antimouse antibody (sc-2777; 1:5,000). The relative quantities of protein expression were measured using AlphaEase FC software.

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Caspase-3 activity analysis [5]
Following the 4-day treatment with 10 mg/kg Shikonin, rats were sacrificed and osteoarthritis samples were collected. The samples were homogenized with RIPA lysis buffer. The homogenate was centrifuged at 12,000 × g for 10 min at 4°C and analyzed using a BCA assay kit. Protein (20 µg) was mixed with the substrate Ac-DEVD-pNA in reaction buffer, and incubated at 37°C for 2 h in the dark. The absorption was then detected at a wavelength of 405 nm.

Absorption, Distribution and Excretion
Alkannin and shikonin are naturally occurring hydroxynaphthoquinones with a well-established spectrum of wound healing, antimicrobial, anti-inflammatory, and antioxidant activities. Recently, extensive scientific effort has been focused on their effectiveness on several tumors and mechanism(s) of antitumor activity. Liposomes have been proved as adequate drug carriers offering significant advantages over conventional formulations, such as controlled release and targeted drug delivery, leading to the appearance of several liposomal formulations in the market, some of them concerning anticancer drugs. The aim of the present study was to prepare shikonin-loaded liposomes for the first time in order to enhance shikonin therapeutic index. An optimized technique based on the thin film hydration method was developed and liposomes characterization was performed in terms of their physicochemical characteristics, drug entrapment efficiency, and release profile. Results indicated the successful incorporation of shikonin into liposomes, using both 1,2-dipalmitoylphosphatidylcholine and egg phosphatidylcholine lipids. Liposomes presented good physicochemical characteristics, high entrapment efficiency and satisfactory in vitro release profile. In vitro cytotoxicity of liposomes was additionally tested against three human cancer cell lines (breast, glioma, and non-small cell lung cancer) showing a moderate growth inhibitory activity. Practical applications: Shikonin is a naturally occurring hydroxynaphthoquinone and extensive scientific research (in vitro, in vivo, and clinical trials) has been conducted during the last years, focusing on its effectiveness on several tumors and mechanism(s) of antitumor action. The purpose of this work was to prepare and characterize shikonin-loaded liposomes as a new drug delivery system for shikonin. Liposomal formulations provide significant advantages over conventional dosage forms, such as controlled release and targeted drug delivery for anticancer agents. Thus, liposomes could reduce shikonin's side effects, enhance selectivity to cancer cells and protect shikonin from internal biotransformations and instability matters (oxidization and polymerization). Furthermore, liposomal delivery helps overcome the low aqueous solubility of shikonin, which is the major barrier to its oral and internal administration, since it cannot be dissolved and further absorbed from the receptor.

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Pharmacokinetics study has shown that shikonin absorption is fast if given by oral gavage and muscle injection, as it is barely detected in the plasma after 1 min, with a oral gavage yielding a bioavailability of about 34% (Wang et al., 1988). In this study, the doses used for intestinal motility (0.38 mg/kg) and rotaviral infection in the mice model (0.69 mg/kg) were below the doses that are considered as toxic.[1]

Reduction of stool water content in rotaviral neonatal mice by shikonin observed in this study probably occurred via an anti-secretory action of shikonin, which involved inhibition of CaCCGI chloride channel activity. Although TMEM16A exists in enterocytes, some investigators have proposed that secretory diarrhea caused by the rotaviral non-structural protein NSP4 is predominantly through the activation of epithelial TMEM16A in the intestine. One study that used the small molecule TMEM16A inhibitor T16Ainh-A01 has demonstrated that TMEM16A constitutes only a minor component of the intestinal epithelial CaCC (Namkung et al., 2011). Our previous work has shown that TMEM16A and CaCCGI have different characteristics, since the lignan compounds kobusin and eudesmin can affect TMEM16A and CaCCGI differently, inhibiting TMEM16A, while activating CaCCGI, (Jiang et al., 2015). We have shown in this study that shikonin was inhibitory toward CaCCGI-mediated short-circuit currents in both cell culture model and isolated mouse colon. In addition, in vivo studies showed that shikonin reduced water content in a neonatal mice diarrhea model without affecting the rotavirus infection process (Figure 6). These findings supported the view that the primary pathway of watery diarrhea associated with rotaviral infection is through activation of CaCCGI rather than TMEM16A by NSP4, thereby enhancing the accumulation of fluid. Furthermore, inhibition of TMEM16A by shikonin delayed gastrointestinal motility, helping to prolong the fluid absorption time to further decrease net fluid secretion.
Despite the many positive benefits of shikonin, it is not without toxicity. Intraperitonal injection of shikonin has been demonstrated to result in some toxicity, with an LD50 of 20 mg/kg (Sankawa et al., 1977). Pharmacokinetics study has shown that shikonin absorption is fast if given by oral gavage and muscle injection, as it is barely detected in the plasma after 1 min, with a oral gavage yielding a bioavailability of about 34% (Wang et al., 1988). In this study, the doses used for intestinal motility (0.38 mg/kg) and rotaviral infection in the mice model (0.69 mg/kg) were below the doses that are considered as toxic.[1]

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479503 mouse LD50 oral >1 gm/kg Nippon Yakurigaku Zasshi. Japanese Journal of Pharmacology., 73(193), 1977 [PMID:560339]
479503 rabbit LD50 intravenous 16 mg/kg Pakistan Journal of Pharmacology., 3(1-2)(43), 1986
479503 mouse LD50 intraperitoneal 20 mg/kg BEHAVIORAL: CHANGES IN MOTOR ACTIVITY (SPECIFIC ASSAY); BEHAVIORAL: ATAXIA Nippon Yakurigaku Zasshi. Japanese Journal of Pharmacology., 73(193), 1977 [PMID:560339]

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Antidote and Emergency Treatment
/SRP:/ Immediate first aid: Ensure that adequate decontamination has been carried out. If patient is not breathing, start artificial respiration, preferably with a demand valve resuscitator, bag-valve-mask device, or pocket mask, as trained. Perform CPR if necessary. Immediately flush contaminated eyes with gently flowing water. Do not induce vomiting. If vomiting occurs, lean patient forward or place on the left side (head-down position, if possible) to maintain an open airway and prevent aspiration. Keep patient quiet and maintain normal body temperature. Obtain medical attention. /Poisons A and B/

/SRP:/ Basic treatment: Establish a patent airway (oropharyngeal or nasopharyngeal airway, if needed). Suction if necessary. Watch for signs of respiratory insufficiency and assist ventilations if needed. Administer oxygen by nonrebreather mask at 10 to 15 L/min. Monitor for pulmonary edema and treat if necessary ... . Monitor for shock and treat if necessary ... . Anticipate seizures and treat if necessary ... . For eye contamination, flush eyes immediately with water. Irrigate each eye continuously with 0.9% saline (NS) during transport ... . Do not use emetics. For ingestion, rinse mouth and administer 5 mL/kg up to 200 mL of water for dilution if the patient can swallow, has a strong gag reflex, and does not drool ... . Cover skin burns with dry sterile dressings after decontamination ... . /Poisons A and B/

/SRP:/ Advanced treatment: Consider orotracheal or nasotracheal intubation for airway control in the patient who is unconscious, has severe pulmonary edema, or is in severe respiratory distress. Positive-pressure ventilation techniques with a bag valve mask device may be beneficial. Consider drug therapy for pulmonary edema ... . Consider administering a beta agonist such as albuterol for severe bronchospasm ... . Monitor cardiac rhythm and treat arrhythmias as necessary ... . Start IV administration of D5W /SRP: "To keep open", minimal flow rate/. Use 0.9% saline (NS) or lactated Ringer's if signs of hypovolemia are present. For hypotension with signs of hypovolemia, administer fluid cautiously. Watch for signs of fluid overload ... . Treat seizures with diazepam or lorazepam ... . Use proparacaine hydrochloride to assist eye irrigation ... . /Poisons A and B/ Currance, P.L. Clements, B., Bronstein, A.C. (Eds).; Emergency Care For Hazardous Materials Exposure. 3Rd edition, Elsevier Mosby, St. Louis, MO 2005, p. 160-1

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Human Toxicity Excerpts
/ALTERNATIVE and IN VITRO TESTS/ Shikonin has the potential to prevent, or be used in the treatment of bladder transitional cell carcinoma induced by arylamines. /Investigators/ evaluated its effectiveness by measuring the amount of acetylated 2-aminofluorene (AF), AF-DNA adducts, changes of / N-acetyltransferase (NAT)/ mRNA and the amount of NAT enzyme. T24 human bladder cancer cells were incubated with 30 uM AF with different concentrations of shikonin for various times. T24 cells treated with shikonin (16 uM) were then harvested and used in 2 experiments: 1). T24 cells were incubated with 22.5 uM AF and shikonin (0, 16 uM) (co-treatment) for 6, 12, 18, 24 and 48 hr). T24 cells were incubated with various concentrations of AF and shikonin (0, 16 uM) for 24 hr AF and AAF were measured by HPLC. Then in the prepared human T24 cell cytosols different concentrations of AF and shikonin were added to measure the kinetic constants of NAT. Next, AF-DNA adducts in human T24 cells with or without treatment with shikonin were detected and measured. The final two steps included measuring the NAT Ag-Ab complex after treatment with and without shikonin and evaluating the effect of shikonin on the NAT genes. Higher concentrations of shikonin induced decreasing AF acetylation. /It was/ found that the longer the culture period, the greater the difference in AF acetylation in the same shikonin concentrations. It was also noted that increase in AAF was proportional to incubation time. In the presence of 16 uM of shikonin, N-acetylation of AF decreased by up to 72-84%. Shikonin decreased the amount of AAF production in human T24 cells in all examined AF doses. Both Km and Vmax values in the cytosolic NAT decreased after the addition of shikonin to the cytosol. Finally, shikonin decreased the amount of AAF production and AF-DNA adducts formation in human 724 cells in all examined AF doses. The percentage of cells stained by antibody was significantly different after treatment with shikonin, especially with the higher shikonin concentrations. The NAT1 mRNA level and the NAT1/beta-actin ratio decreased significantly with higher concentrations (16-24 uM) of shikonin. Shikonin affected NAT activity, gene expression (NAT1 mRNA), AF-DNA adducts formation and formation of NAT Ag-Ab in human bladder tumor T24 cells... PMID:15011747

/ALTERNATIVE and IN VITRO TESTS/ Shikonin isolated from the roots of the Chinese herb Lithospermum erythrorhizon has been associated with anti-inflammatory properties. /Investigators/ evaluated shikonin's chemotherapeutic potential and investigated its possible mechanism of action in a human cutaneous neoplasm in tissue culture. Shikonin preferentially inhibits the growth of human epidermoid carcinoma cells concentration- and time-dependently compared to SV-40 transfected keratinocytes, demonstrating its anti-proliferative effects against this cancer cell line. Additionally, shikonin decreased phosphorylated levels of EGFR, ERK1/2 and protein tyrosine kinases, while increasing phosphorylated JNK1/2 levels. Overall, shikonin treatment was associated with increased intracellular levels of phosphorylated apoptosis-related proteins, and decreased levels of proteins associated with proliferation in human epidermoid carcinoma cells. PMID:14568164

/ALTERNATIVE and IN VITRO TESTS/ This study investigated the potential of shikonin as an anticancer agent against liver cancer and an in vitro human hepatoma cancer model system. The HepG2 cell line was the hepatoma cancer model in the present study. The inhibitory effect of shikonin on the growth of HepG2 cells was measured by MTT assay. To explore the underlying mechanism of cell growth inhibition of shikonin, the cell cycle distribution, DNA fragmentation, mitochondrial membrane potential disruption, and expression of Bax and Bcl-2 were measured in HepG2 cells. The activity of shikonin in inducing apoptosis was investigated through the detection of Annexin V signal and CD95 expression by flow cytometry and electron microscopy, respectively. Shikonin inhibited the growth of HepG2 cells in a dose-dependent manner. The IC50 value (inhibiting cell growth by 50%) was 4.30 mg/mL. Shikonin inhibited cell growth in a dose-dependent manner and blocked HepG2 cell cycle progression at the S phase. The changes in mitochondrial morphology, dose-dependently decreased in mitochondrial membrane potential, were observed in different concentrations of the drug treatment group. Western blot analysis showed that cajanol inhibited Bcl-2 expression and induced Bax expression. ...shikonin increases Annexin V signal and CD95 (Fas/APO) expression, resulting in apoptotic cell death of HepG2 cells. In addition, lump formation of intranuclear chromatin, pyknosis of cell nucleus, deletion of microvillus, vacuolar degeneration of mitochondria, reduction of rough endoplasmic reticulum, and resolution of free ribosome, etc., associated with apoptosis were discovered by electron microscopy in HepG2 cells after 48 hr treatment. Shikonin inhibited HepG2 cells, possibly through the pathway of inducing early apoptosis, and was beneficial for restoring the apoptotic sensitivity of HepG2 cells by CD95, and should therefore be considered as a candidate agent for the prevention or treatment of human hepatoma. PMID:21164560

/ALTERNATIVE and IN VITRO TESTS/ Shikonin (SK) has been isolated and identified as a key bioactive component in an herbal plant, Shikon (gromwell). /This study/ investigated antiestrogen activity of SK in breast cancer cells /MCF-7, T47D and MDA-MB-231 cells/. In human breast cancer cells, we observed that treatment with SK inhibits tumor cell growth in estrogen receptor alpha (ERalpha)-positive, but not ERalpha-negative breast cancer cells. Estrogen-dependent cell growth was inhibited by co-treatment with SK. A potential molecular mechanism by which SK inhibits estrogen action was explored... SK has no effect on ERalpha mRNA expression, but decreases its protein level. This effect is associated with an increase in ubiquitinated ERalpha for degradation. /The/ results suggest that SK downregulates ERalpha protein through a proteasome-mediated pathway. ...treatment with SK inhibits estrogen-induced estrogen response elements reporter gene activity. Furthermore, SK inhibits recruitment of ERalpha at the estrogen-dependent gene promoters, and subsequently suppresses gene expression. Finally, co-treatment with SK enhanced sensitivity of breast cancer cells to endocrine therapy... PMID:19760501

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Non-Human Toxicity Excerpts
/LABORATORY ANIMALS: Subchronic or Prechronic Exposure/ /The objective of this study was / to investigate the anti-inflammatory or immunomodulatory effect of shikonin on early stage and established murine collagen-induced arthritis (CIA). /Mice/ were injected intraperitoneally with shikonin (5 mg/kg) for 10 days along, before, or after the onset of CIA. The arthritis response was monitored visually by macroscopic scoring. Reverse transcription-polymerase chain reaction and western blotting were employed to determine the mRNA and protein expression of cytokine in patella with adjacent synovium in CIA /mice/. Histology of knee was used to assess the occurrence of cartilage destruction and bone erosion. Shikonin (5 mg/kg) treatment along had no effect on macroscopic score and incidence of arthritis on early stage of CIA. However, a pronounced amelioration of macroscopic score and cartilage destruction was found in mouse treated with shikonin on established CIA for 10 days. Moreover, The mRNA levels of Th1 cytokines [tumor necrosis factor-alpha and interleukin (IL)-12] was significantly inhibited both in the synovial tissue and in the articular cartilage in treated groups compared with those in control groups, whereas the mRNA and protein levels of Th2 cytokines (IL-10 and IL-4) remained elevated throughout the treatment period. Moreover, the inflammatory cytokine, the mRNA and protein levels of IL-6 were down-regulated in mice with established CIA after treatment with shikonin. T-box expressed in T cells (T-bet) mRNA levels were decreased in shikonin compared with control group, and GATA-3 mRNA levels were higher than that in control group. Shikonin treatment on established CIA can inhibit Th1 cytokines expression and induce Th2 cytokines expression in mice with established CIA. The inhibited effect of shikonin on Th1 cytokines expression may be mediated not only by inhibiting Th1 responses through T-bet mechanism, but also by inducing anti-inflammatory mediators such as IL-10 and IL-4 through a GATA-3 dependent mechanism. PMID:18781399

[1]. Shikonin Inhibits Intestinal Calcium-Activated Chloride Channels and Prevents Rotaviral Diarrhea. Front Pharmacol. 2016 Aug 23;7:270.

[2]. Shikonin Suppresses Skin Carcinogenesis via Inhibiting Cell Proliferation. PLoS One. 2015 May 11;10(5):e0126459.

[3]. Shikonin Promotes Skin Cell Proliferation and Inhibits Nuclear Factor-κB Translocation via Proteasome Inhibition In Vitro. Chin Med J (Engl). 2015 Aug 20;128(16):2228-33.

[4]. Shikonin Inhibits the Migration and Invasion of Human Glioblastoma Cells by Targeting Phosphorylated β-Catenin and Phosphorylated PI3K/Akt: A Potential Mechanism for the Anti-Glioma Efficacy of a Traditional Chinese Herbal Medicine. Int J Mol Sci. 2015 Oct 9;16(10):23823-48.

[5]. Shikonin inhibits inflammation and chondrocyte apoptosis by regulation of the PI3K/Akt signaling pathway in a rat model of osteoarthritis. Exp Ther Med. 2016 Oct;12(4):2735-2740.

[6]. Mechanisms associated with biogenesis of exosomes in cancer. Mol Cancer. 2019 Mar 30;18(1):52.

[7]. Shikonin Suppresses NLRP3 and AIM2 Inflammasomes by Direct Inhibition of Caspase-1. PLoS One. 2016 Jul 28;11(7):e0159826.

[8]. Shikonin and its analogs inhibit cancer cell glycolysis by targeting tumor pyruvate kinase-M2. Oncogene. 2011 Oct 20;30(42):4297-306.

C.I. Natural Red 20 is a naphthoquinone.
C.I. Natural Red 20 has been reported in Arnebia guttata, Arnebia decumbens, and other organisms with data available.
See also: Lithospermum officinale whole (annotation moved to); Shikonin (annotation moved to).

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Shikonin is a hydroxy-1,4-naphthoquinone.
Shikonin has been reported in Arnebia decumbens, Arnebia euchroma, and other organisms with data available.
See also: Arnebia guttata root (part of); Arnebia euchroma root (part of); Lithospermum erythrorhizon root (part of).

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Mechanism of Action
/Investigators/ previously developed a gene-gun-based in vivo screening system and identified shikonin as a potent suppressor of tumor necrosis factor-alpha (TNF-alpha) gene expression. Here... shikonin selectively inhibits the expression of TNF-alpha at the RNA splicing level. Treatment of lipopolysaccharide-stimulated human primary monocytes and THP-1 cells with shikonin resulted in normal transcriptional induction of TNF-alpha, but unspliced pre-mRNA accumulated at the expense of functional mRNA. This effect occurred with noncytotoxic doses of shikonin and was highly specific, because mRNA production of neither a housekeeping gene nor another inflammatory cytokine gene, interleukin-8 (IL-8), was affected. Moreover, cotreatment with lipopolysaccharide (LPS) and shikonin increased the endpoint protein production of IL-8, accompanied by suppressed activation of the double-stranded RNA-activated protein kinase (PKR) pathway. Because PKR inactivation has been shown to down-regulate the splicing process of TNF-alpha RNA and interfere with translation, our findings suggest that shikonin may achieve differential modulation of cytokine protein expression through inactivation of the PKR pathway and reveal that regulation of TNF-alpha pre-mRNA splicing may constitute a promising target for future anti-inflammatory application.
Shikonin isolated from the roots of the Chinese herb Lithospermum erythrorhizon has been associated with anti-inflammatory properties. /Investigators/ evaluated shikonin's chemotherapeutic potential and investigated its possible mechanism of action in a human cutaneous neoplasm in tissue culture. Shikonin preferentially inhibits the growth of human epidermoid carcinoma cells concentration- and time-dependently compared to SV-40 transfected keratinocytes, demonstrating its anti-proliferative effects against this cancer cell line. Additionally, shikonin decreased phosphorylated levels of EGFR, ERK1/2 and protein tyrosine kinases, while increasing phosphorylated JNK1/2 levels. Overall, shikonin treatment was associated with increased intracellular levels of phosphorylated apoptosis-related proteins, and decreased levels of proteins associated with proliferation in human epidermoid carcinoma cells.
... /A previous study showed/ that shikonin, a natural compound isolated from Lithospermun erythrorhizon Sieb. Et Zucc, inhibits adipogenesis and fat accumulation. This study was conducted to investigate the molecular mechanism of the anti-adipogenic effects of shikonin. Gene knockdown experiments using small interfering RNA (siRNA) transfection were conducted to elucidate the crucial role of beta-catenin in the anti-adipogenic effects of shikonin. Shikonin prevented the down-regulation of beta-catenin and increased the level of its transcriptional product, cyclin D1, during adipogenesis of 3T3-L1 cells, preadipocytes originally derived from mouse embryo. beta-catenin was a crucial mediator of the anti-adipogenic effects of shikonin, as determined by siRNA-mediated knockdown. Shikonin-induced reductions of the major transcription factors of adipogenesis including peroxisome proliferator-activated receptor gamma and CCAAT/enhancer binding protein alpha, and lipid metabolizing enzymes including fatty acid binding protein 4 and lipoprotein lipase, as well as intracellular fat accumulation, were all significantly recovered by siRNA-mediated knockdown of beta-catenin. Among the genes located in the WNT/beta-catenin pathway, the levels of WNT10B and DVL2 were significantly up-regulated, whereas the level of AXIN was down-regulated by shikonin treatment. This study ...shows that shikonin inhibits adipogenesis by the modulation of WNT/beta-catenin pathway in vitro, and also suggests that WNT/beta-catenin pathway can be used as a therapeutic target for obesity and related diseases using a natural compound like shikonin...

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As the major component of Zicao, the dried root of Lithospermum erythrorhizon, shikonin has been broadly used due to its anti-inflammatory activity (Chen et al., 2002). Shikonin has been reported to have antioxidant, antibacterial, antiparasitic, antiviral and wound-healing activities (Andujar et al., 2013). Shikonin might be used in the treatment of asthma. Takano-Ohmuro et al. (2008) has explored the use of shikonin in asthma by focusing on its anti-inflammatory activity (Takano-Ohmuro et al., 2008). Other investigators have used a mouse asthma model to demonstrate that shikonin inhibits bone marrow-derived dendritic cell maturation in vitro, and allergic action as well as tracheal hyperresponsiveness in vivo (Lee et al., 2010). Since TMEM16A is expressed in airway smooth muscle cells and it participates in smooth muscle contraction (Huang et al., 2012), we assumed that TMEM16A may be involved in shikonin-mediated inhibition of asthma. In our study, shikonin was found to inhibit TMEM16A chloride channel activity, indicating that shikonin might alleviate asthma by inhibiting smooth muscle contraction in the trachea.[1]

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In summary, the results from this study suggest that shikonin is effective in inhibiting chemically-induced skin carcinogenesis which is mediated largely by inhibiting cell proliferation during skin carcinogenesis. The potential target identified in this study, ATF2, will be examined in future experiments.[2]

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In conclusion, our findings indicated that shikonin promoted proliferation of skin cells by a currently unknown mechanism, while shikonin did not induce COL1 expression in HDFs. In addition, shikonin inhibited NF-κB signaling pathway and proteasome activity in HDFs, which suggested an anti-inflammatory effect of shikonin. Therefore, shikonin may be a potential therapeutic agent both in wound healing and in the treatment of inflammatory skin diseases. Thus, shikonin may be most suitable in the treatment of refractory inflammatory skin ulcers.[3]

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Shikonin attenuates the proliferation, migration, and invasion capability of human glioblastoma cells by inhibiting MMP-2, MMP-9. In p53 wild-type glioma cells, the mechanism is associated with downregulated phosphorylated β-catenin Y333 and p-PI3K/p-Akt expression. In p53 mutant glioma cells, it correlates to an inhibited PI3K/Akt pathway.[4]

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In conclusion, the results of the present study confirmed that shikonin inhibits inflammation and chondrocyte apoptosis by regulating the PI3K/Akt signaling pathway in a rat model of osteoarthritis. These findings suggest that shikonin has therapeutic potential for osteoarthritis.[5]

C16H16O5

288.29524

288.099

C, 66.66; H, 5.59; O, 27.75

23444-65-7

Shikonin;517-89-5;(-)-Alkannin;517-88-4; (-)-Alkannin;517-88-4;Alkannin;23444-65-7;(Rac)-Shikonin;54952-43-1

5208

Typically exists as solid at room temperature

1.4±0.1 g/cm3

567.4±50.0 °C at 760 mmHg

149°C

311.0±26.6 °C

0.0±1.6 mmHg at 25°C

1.642

4.35

3

5

3

21

501

0

C/C(=C/CC(C1=CC(=O)C2=C(C=CC(O)=C2C1=O)O)O)/C

NEZONWMXZKDMKF-UHFFFAOYSA-N

InChI=1S/C16H16O5/c1-8(2)3-4-10(17)9-7-13(20)14-11(18)5-6-12(19)15(14)16(9)21/h3,5-7,10,17-19H,4H2,1-2H3

5,8-dihydroxy-2-(1-hydroxy-4-methylpent-3-enyl)naphthalene-1,4-dione

Alkannin; 517-88-4; Anchusin; Alkanna Red; Anchusa acid; Alkannin (VAN); Anchusin (VAN); ...; 23444-65-7;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)