Natural product; TMEM16A chloride channel (IC50 = 6.5 μM); PKM2; NF-κB

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.

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]
\nICR 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.\n

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\n\nMouse Model of Rotaviral Diarrhea [1]
\nNeonatal 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.\n

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\n\nChemically-induced mouse skin carcinogenesis [2]
\nSixty 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.\n

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\n\nExperimental groups and treatment [5]
\nThe 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).\n

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\n\nELISA analysis [5]
\nFollowing 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.).\n

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\n\nWestern blot analysis [5]
\nFollowing 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.\n

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\n\nCaspase-3 activity analysis [5]
\nFollowing 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
Shikonin and shikonin glycosides are naturally occurring hydroxynaphthoquinone compounds with broad-spectrum wound-healing, antibacterial, anti-inflammatory, and antioxidant activities. In recent years, extensive scientific research has focused on their efficacy against various tumors and their anti-tumor mechanisms. Liposomes have proven to be effective drug carriers, offering significant advantages over traditional formulations, such as controlled release and targeted delivery. Consequently, various liposomal formulations have emerged on the market, some of which are used in anticancer drugs. This study aimed to prepare liposomes loaded with shikonin for the first time, thereby improving the therapeutic index of shikonin. We developed an optimized technique based on thin-film hydration and characterized the liposomes in terms of physicochemical properties, drug encapsulation efficiency, and release profiles. The results showed that shikonin was successfully encapsulated in liposomes using both 1,2-dipalmitoylphosphatidylcholine and lecithin liposomes. The resulting liposomes exhibited good physicochemical properties, high encapsulation efficiency, and satisfactory in vitro release profiles. Furthermore, the in vitro cytotoxicity of liposomes against three human cancer cell lines (breast cancer, glioma, and non-small cell lung cancer) was tested, showing moderate growth-inhibiting activity. Practical Application: Shikonin is a naturally occurring hydroxynaphthoquinone compound. In recent years, extensive scientific research (including in vitro, in vivo, and clinical trials) has been conducted on its efficacy against various tumors and its anti-tumor mechanism. This study aims to prepare and characterize liposomes loaded with shikonin, hoping to use it as a novel shikonin drug delivery system. Compared with traditional dosage forms, liposome formulations have significant advantages, such as controlled release and targeted delivery of anticancer drugs. Therefore, liposomes can reduce the side effects of shikonin, improve its selectivity for cancer cells, and protect shikonin from in vivo biotransformation and instability (oxidation and polymerization). In addition, liposome delivery helps overcome the problem of low water solubility of shikonin, which is a major obstacle to its oral and oral administration, as shikonin cannot dissolve and be further absorbed from receptors. Pharmacokinetic studies have shown that shikonin is rapidly absorbed when administered by gavage and intramuscular injection, and is almost undetectable in plasma after 1 minute. The bioavailability of gavage administration is approximately 34% (Wang et al., 1988). In this study, the doses used for intestinal motility (0.38 mg/kg) and in a rotavirus-infected mouse model (0.69 mg/kg) were both lower than doses considered toxic. [1]

This study observed that shikonin reduced fecal water content in rotavirus-infected neonatal mice, possibly through its antisecretory effect, which involves inhibiting the activity of the CaCCGI chloride channel. Although TMEM16A is present in intestinal cells, some researchers have proposed that diarrhea induced by the rotavirus non-structural protein NSP4 is primarily mediated by the activation of TMEM16A in intestinal epithelial cells. A study using the small-molecule TMEM16A inhibitor T16Ainh-A01 showed that TMEM16A is only a minor component of intestinal epithelial CaCC (Namkung et al., 2011). Our previous research indicated that TMEM16A and CaCCGI have different properties because the lignans cobuxin and eucalyptol have different effects on TMEM16A and CaCCGI; the former inhibits TMEM16A, while the latter activates CaCCGI (Jiang et al., 2015). This study demonstrates that shikonin inhibits CaCCGI-mediated short-circuit currents in both cell culture models and isolated mouse colons. Furthermore, in vivo studies have shown that shikonin can reduce water content in a neonatal mouse diarrhea model without affecting the rotavirus infection process (Figure 6). These findings support the view that the main pathway for watery diarrhea caused by rotavirus infection is through NSP4 activation of CaCCGI, rather than TMEM16A, thereby exacerbating fluid accumulation. In addition, the inhibitory effect of shikonin on TMEM16A can slow gastrointestinal motility, prolonging fluid absorption time and further reducing net fluid secretion. Despite its many benefits, shikonin is not without toxicity. Intraperitoneal injection of shikonin has been shown to produce some toxicity, with an LD50 of 20 mg/kg (Sankawa et al., 1977). Pharmacokinetic studies have shown that shikonin is rapidly absorbed after gavage and intramuscular injection, with almost undetectable shikonin in plasma after 1 minute, and the bioavailability of gavage administration is approximately 34% (Wang et al., 1988). In this study, the doses used for intestinal motility (0.38 mg/kg) and the mouse rotavirus infection model (0.69 mg/kg) were both lower than doses considered toxic. [1]

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479503tmousetLD50toralt>1 gm/kgttNippon Yakurigaku Zasshi. Japanese Journal of Pharmacology., 73(193), 1977 [PMID:560339]
479503trabbittLD50tintravenoust16 mg/kgttPakistan Journal of Pharmacology., 3(1-2)(43), 1986
479503tmousetLD50tintraperitonealt20 mg/kgtBEHAVIORAL: CHANGES IN MOTOR ACTIVITY (SPECIFIC ASSA); Behavioral Science: Ataxia, Japanese Journal of Pharmacology, 73(193), 1977 [PMID:560339]

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Antidotes and First Aid Treatment
/SRP:/ Immediate First Aid: Ensure adequate decontamination has been performed. If the patient stops breathing, begin artificial respiration immediately, preferably using a demand ventilator, bag-valve-mask, or simple breathing mask, following training instructions. Perform cardiopulmonary resuscitation (CPR) if necessary. Immediately flush contaminated eyes with running water. Do not induce vomiting. If vomiting occurs, tilt the patient forward or place them in the left lateral decubitus position (head down if possible) to maintain an open airway and prevent aspiration. Keep the patient calm and maintain normal body temperature. Seek medical attention. /Class A and B Poisons/
/SRP:/ Basic Treatment: Establish a patent airway (using an oropharyngeal or nasopharyngeal airway if necessary). Suction if necessary. Observe for signs of respiratory failure and provide assisted ventilation if necessary. Administer oxygen using a non-invasive breathing mask at a flow rate of 10 to 15 liters per minute. Monitor for pulmonary edema and treat as needed… Monitor for shock and treat as needed… Anticipate seizures and treat as needed… If eyes are contaminated, flush them immediately with water. During transport, continuously flush each eye with 0.9% normal saline (NS)... Do not use emetics. If swallowed, rinse mouth and dilute with 5 ml/kg body weight to 200 ml of water, provided the patient is able to swallow, has a strong gag reflex, and is not drooling... After cleansing, cover skin burns with a dry, sterile dressing... /Class A and B Poisons/
/SRP:/ Advanced Treatment: For patients with altered mental status, severe pulmonary edema, or severe respiratory distress, consider oropharyngeal or nasopharyngeal endotracheal intubation to control the airway. Positive pressure ventilation using a bag-valve-mask may be effective. Consider medical treatment for pulmonary edema... Consider the use of a β-receptor agonist (such as salbutamol) for severe bronchospasm... Monitor heart rhythm and treat arrhythmias as needed... Initiate intravenous infusion of 5% glucose solution (D5W) /SRP: "Keep patent", minimum flow rate/. If signs of hypovolemia appear, administer 0.9% normal saline (NS) or lactated Ringer's solution. For hypotension accompanied by signs of hypovolemia, administer fluids with caution. Be alert for signs of fluid overload… Use diazepam or lorazepam to treat seizures… Use promecaine hydrochloride to assist eye irrigation… /Toxins A and B/ Currance, PL Clements, B., Bronstein, AC (eds.).; Emergency Care for Hazardous Substance Exposure. 3rd ed., Elsevier Mosby Publishers, St. Louis, Missouri, 2005, pp. 160-161

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Human Toxicity Excerpt
/Alternatives and In Vitro Tests/ Shikonin has the potential to prevent or treat amine-induced bladder transitional cell carcinoma. /Researchers/ assessed its efficacy by measuring the levels of acetylated 2-aminofluorene (AF), the levels of AF-DNA adducts, /N-acetyltransferase (NAT)/mRNA changes, and the levels of NAT enzymes. T24 human bladder cancer cells were incubated with 30 μM AF and different concentrations of shikonin for varying durations. After treatment with shikonin (16 μM), T24 cells were collected and used in two experiments: 1) T24 cells were incubated with 22.5 μM AF and shikonin (0, 16 μM) (co-treatment) for 6, 12, 18, 24, and 48 hours; 2) T24 cells were incubated with different concentrations of AF and shikonin (0, 16 μM) for 24 hours, and the contents of AF and AAF were determined by high-performance liquid chromatography (HPLC). Then, different concentrations of AF and shikonin were added to the prepared human T24 cell cytosol to determine the kinetic constant of NAT. Next, the content of AF-DNA adducts in shikonin-treated and untreated human T24 cells was detected and determined. The last two steps included determining the content of NAT antigen-antibody complexes after shikonin treatment and untreatment, and assessing the effect of shikonin on the NAT gene. Higher concentrations of shikonin can reduce AF acetylation levels. Studies have found that at the same shikonin concentration, the longer the culture time, the greater the difference in AF acetylation levels. Furthermore, the increase in AAF is directly proportional to the incubation time. In the presence of 16 μM shikonin, the N-acetylation level of AF decreased by 72-84%. At all tested AF concentrations, shikonin reduced the production of AAF in human T24 cells. The Km and Vmax values of cytoplasmic NAT decreased after the addition of shikonin. Finally, at all tested AF concentrations, shikonin reduced the production of AAF and the formation of AF-DNA adducts in human 724 cells. The percentage of antibody-stained cells changed significantly after shikonin treatment, especially at higher shikonin concentrations. With increasing shikonin concentration (16-24 μM), NAT1 mRNA levels and the NAT1/β-actin ratio decreased significantly. Shikonin Affects NAT Activity, Gene Expression (NAT1 mRNA), AF-DNA Adduct Formation, and NAT Antigen-Antibody Formation in Human Bladder Tumor T24 Cells…PMID: 15011747
/Alternative and In Vitro Assays/ Shikonin, isolated from the root of the traditional Chinese medicine Lithospermum erythrorhizon, possesses anti-inflammatory properties. /Researchers/ evaluated the chemotherapeutic potential of shikonin and investigated its possible mechanism of action against human skin tumors in tissue culture. Compared with SV-40 transfected keratinocytes, shikonin preferentially inhibited the growth of human epidermal-like cancer cells in a concentration- and time-dependent manner, indicating its anti-proliferative effect on this cancer cell line. Furthermore, shikonin reduced the phosphorylation levels of EGFR, ERK1/2, and protein tyrosine kinases, while increasing the phosphorylation level of JNK1/2. Overall, shikonin treatment was associated with increased intracellular phosphorylation levels of apoptosis-related proteins and decreased levels of proliferation-related proteins in human epidermal-like cancer cells. PMID:14568164
/Alternatives and In Vitro Assays/ This study explored the potential of shikonin as an anti-hepatocellular carcinoma drug and established an in vitro human hepatocellular carcinoma model. The HepG2 cell line was used as the hepatocellular carcinoma model. The inhibitory effect of shikonin on HepG2 cell growth was detected by the MTT assay. To investigate the potential mechanism of shikonin's inhibition of cell growth, this study examined the cell cycle distribution, DNA fragmentation, mitochondrial membrane potential disruption, and the expression of Bax and Bcl-2 in HepG2 cells. The apoptosis-inducing activity of shikonin was investigated by flow cytometry and electron microscopy, respectively, to detect Annexin V signaling and CD95 expression. The results showed that shikonin inhibited the growth of HepG2 cells in a dose-dependent manner, with an IC50 value (50% inhibition concentration) of 4.30 mg/mL. Shikonin inhibited cell growth in a dose-dependent manner and arrested the HepG2 cell cycle in the S phase. Changes in mitochondrial morphology and a dose-dependent decrease in mitochondrial membrane potential were observed in different concentrations of shikonin treatment groups. Western blot analysis showed that shikonin inhibited Bcl-2 expression and induced Bax expression. Shikonin enhanced Annexin V signaling and CD95 (Fas/APO) expression, leading to apoptosis in HepG2 cells. Furthermore, electron microscopy revealed that after 48 hours of shikonin treatment, HepG2 cells exhibited pathological changes associated with apoptosis, including chromatin clumping, nuclear pyknosis, microvilli disappearance, mitochondrial vacuolar degeneration, reduction of rough endoplasmic reticulum, and lysis of free ribosomes. Shikonin may inhibit HepG2 cells by inducing early apoptosis and can restore apoptosis sensitivity through CD95, therefore it should be considered a candidate drug for the prevention or treatment of human liver cancer. PMID:21164560
/Alternatives and In Vitro Assays/ Shikonin (SK) is a key bioactive component isolated and identified from the herb Lithospermum erythrorhizon (comfrey). This study investigated the anti-estrogenic activity of SK in breast cancer cells (MCF-7, T47D, and MDA-MB-231 cells). In human breast cancer cells, we observed that SK treatment inhibited the growth of estrogen receptor α (ERα)-positive breast cancer cells, but had no inhibitory effect on ERα-negative breast cancer cells. Combined treatment with SK and estrogen inhibited the growth of estrogen-dependent cells. This study explored the potential molecular mechanisms by which SK inhibits estrogen effects…SK had no effect on ERα mRNA expression, but reduced its protein level. This effect is related to increased ERα ubiquitination leading to its degradation. The results indicate that SK downregulates ERα protein through a proteasome-mediated pathway. …SK treatment inhibited estrogen-induced estrogen response element reporter gene activity. Furthermore, SK inhibited ERα recruitment at estrogen-dependent gene promoters, thereby suppressing gene expression. Finally, combination therapy with SK enhanced the sensitivity of breast cancer cells to endocrine therapy…PMID: 19760501

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Excerpt of Non-Human Toxicity
/Experimental Animals: Subchronic or Chronic Pre-Exposure/ /The purpose of this study was/to investigate the anti-inflammatory or immunomodulatory effects of shikonin on early and established collagen-induced arthritis (CIA) in mice. /Mice/ were intraperitoneally injected with shikonin (5 mg/kg) for 10 days during, before, or after the onset of CIA. Arthritis response was visually monitored by macroscopic scoring. The mRNA and protein expression of cytokines in the patella and adjacent synovium of CIA mice were detected by reverse transcription-polymerase chain reaction and Western blotting. Knee joint histology was used to assess the occurrence of cartilage destruction and bone erosion. Treatment with shikonin alone (5 mg/kg) had no effect on macroscopic scores or the incidence of arthritis in early collagen-induced arthritis (CIA). However, in mice with established CIA models, macroscopic scores and cartilage destruction were significantly improved after 10 days of shikonin treatment. Furthermore, compared with the control group, the treatment group showed significantly reduced mRNA levels of Th1 cytokines (tumor necrosis factor-α and interleukin (IL)-12) in the synovial tissue and articular cartilage, while the mRNA and protein levels of Th2 cytokines (IL-10 and IL-4) remained elevated throughout the treatment period. Additionally, in mice with established CIA models, the mRNA and protein levels of the inflammatory cytokine IL-6 were downregulated after shikonin treatment. Compared with the control group, the shikonin group showed reduced mRNA levels of T-box protein (T-bet) expressed by T cells, while GATA-3 mRNA levels were increased. Shikonin treatment in mice with established collagen-induced arthritis (CIA) inhibited the expression of Th1 cytokines and induced the expression of Th2 cytokines. The inhibitory effect of shikonin on Th1 cytokine expression may not only inhibit the Th1 response through the T-bet mechanism but may also induce the production of 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.

Shikonin is a hydroxy-1,4-naphthoquinone. It has been reported to exist in plants of the genus Arnebia (such as Arnebia decumbens and Arnebia euchroma) and other organisms with relevant data. See also: Arnebia guttata root (partial); Arnebia euchroma root (partial); Lithospermum erythrorhizon root (partial). Mechanism of Action: Researchers previously developed a gene gun-based in vivo screening system and identified shikonin as a potent inhibitor of tumor necrosis factor-α (TNF-α) gene expression. Here…shikonin selectively inhibits TNF-α expression 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-α, but accumulation of unspliced precursor mRNA, leading to a reduction in functional mRNA. This effect occurs at non-cytotoxic doses of shikonin and is highly specific, as the mRNA production of housekeeping genes and another inflammatory cytokine gene, interleukin-8 (IL-8), remains unaffected. Furthermore, co-treatment with LPS and shikonin increases the final protein production of IL-8 while inhibiting the double-stranded RNA-activated protein kinase (PKR) pathway. Since PKR inactivation has been shown to downregulate TNF-α RNA splicing and interfere with translation, our results suggest that shikonin may differentially regulate cytokine protein expression through PKR pathway inactivation, revealing that the regulation of TNF-α precursor mRNA splicing may constitute a promising target for future anti-inflammatory applications. Shikonin is isolated from the root of the traditional Chinese medicine Lithospermum erythrorhizon and is believed to possess anti-inflammatory properties. Researchers evaluated the chemotherapeutic potential of shikonin and investigated its possible mechanism of action in human skin tumors in tissue culture. Compared with SV-40 transfected keratinocytes, shikonin preferentially inhibited the growth of human epidermal-like cancer cells in a concentration- and time-dependent manner, indicating its anti-proliferative effect on this cancer cell line. Furthermore, shikonin reduced the phosphorylation levels of EGFR, ERK1/2, and protein tyrosine kinases, while increasing the phosphorylation level of JNK1/2. Overall, shikonin treatment was associated with increased intracellular phosphorylation levels of apoptosis-related proteins and decreased levels of proliferation-related proteins in human epidermal-like cancer cells. Previous studies have shown that shikonin, a natural compound isolated from Lithospermum erythrorhizon Sieb. et Zucc., can inhibit adipogenesis and fat accumulation. This study aimed to investigate the molecular mechanism of shikonin's anti-adipogenic effect. We performed gene knockdown experiments using small interfering RNA (siRNA) transfection technology to elucidate the key role of β-catenin in shikonin's anti-adipogenic effect. During adipogenesis in 3T3-L1 preadipocytes derived from mouse embryos, shikonin can prevent the downregulation of β-catenin and increase the level of its transcription product cyclin D1. siRNA-mediated gene knockdown experiments showed that β-catenin is a key mediator of shikonin's anti-adipogenic effect. The decreased expression of shikonin-induced adipogenic major transcription factors (including peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α) and lipid metabolism enzymes (including fatty acid-binding protein 4 and lipoprotein lipase), as well as intracellular fat accumulation, were significantly restored by siRNA-mediated β-catenin gene knockdown. Among genes in the WNT/β-catenin signaling pathway, the expression levels of WNT10B and DVL2 were significantly upregulated, while the expression level of AXIN was downregulated by shikonin. This study demonstrates that shikonin can inhibit lipogenesis by regulating the WNT/β-catenin signaling pathway in vitro, suggesting that the WNT/β-catenin signaling pathway may serve as a potential target for treating obesity and related diseases, for example, through the use of natural compounds such as shikonin. Shikonin is the main component of Lithospermum erythrorhizon (the dried root of the plant Lithospermum), and is widely used due to its anti-inflammatory activity (Chen et al., 2002). Shikonin has been reported to possess antioxidant, antibacterial, antiparasitic, antiviral, and wound-healing activities (Andujar et al., 2013). Shikonin may be used to treat asthma. Takano-Ohmuro et al. (2008) focused on the anti-inflammatory activity of shikonin and explored its application in the treatment of asthma (Takano-Ohmuro et al., 2008). Other researchers have used mouse asthma models to confirm that shikonin can inhibit the maturation of bone marrow-derived dendritic cells in vitro and inhibit allergic reactions and tracheal hyperresponsiveness in vivo (Lee et al., 2010). Since TMEM16A is expressed in airway smooth muscle cells and participates in smooth muscle contraction (Huang et al., 2012), we speculate that TMEM16A may be involved in the shikonin-mediated asthma suppression. In this study, we found that shikonin can inhibit the activity of TMEM16A chloride ion channels, indicating that shikonin may alleviate asthma by inhibiting tracheal smooth muscle contraction. [1] In summary, the results of this study indicate that shikonin can effectively inhibit chemically induced skin carcinogenesis, and its main mechanism is through inhibiting cell proliferation during skin carcinogenesis. The potential target ATF2 discovered in this study will be verified in future experiments. [2] In conclusion, our results indicate that shikonin promotes skin cell proliferation through a currently unknown mechanism, but shikonin does not induce the expression of COL1 in HDFs. Furthermore, shikonin inhibits the NF-κB signaling pathway and proteasome activity in HDFs, suggesting that shikonin has anti-inflammatory effects. Therefore, shikonin may be a potential therapeutic agent for wound healing and the treatment of inflammatory skin diseases. Therefore, shikonin may be best suited for the treatment of refractory inflammatory skin ulcers. [3]

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Shikonin weakens the proliferation, migration and invasion of human glioblastoma cells by inhibiting MMP-2 and MMP-9. In p53 wild-type glioma cells, this mechanism is associated with the downregulation of phosphorylated β-catenin Y333 and p-PI3K/p-Akt expression. In p53 mutant glioma cells, it is associated with the inhibition of the PI3K/Akt pathway. [4]

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In summary, the results of this study confirm that shikonin inhibits inflammation and chondrocyte apoptosis in a rat model of osteoarthritis by regulating the PI3K/Akt signaling pathway. These findings suggest that shikonin has the potential to treat osteoarthritis. [5]

C16H16O5

288.3

288.099

C, 66.66; H, 5.59; O, 27.75

517-89-5

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

479503

Brown to reddish brown solid powder

1.4±0.1 g/cm3

567.4±50.0 °C at 760 mmHg

147ºC

311.0±26.6 °C

0.0±1.6 mmHg at 25°C

1.642

4.35

3

5

3

21

501

1

CC(=CC[C@H](C1=CC(=O)C2=C(C=CC(=C2C1=O)O)O)O)C

NEZONWMXZKDMKF-SNVBAGLBSA-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/t10-/m1/s1

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

Tokyo Violet; 517-89-5; Isoarnebin 4; Tokyo Violet; (R)-(+)-Shikonin; Shikonin S; (+)-Shikonin; UNII-3IK6592UBW; Shikonin

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)

DMSO : ~125 mg/mL (~433.58 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.21 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.21 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (7.21 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30 mg/mL (104.06 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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