Abstract!

The naphthoquinone shikonin is the main activeprinciple of Zicao, a traditional Chinese herbalmedicine made from the dried root of Lithosper-mum erythrorhizon. Studies carried out over thepast 30 years have provided a scientific basis forthe use of Zicao which has been long employedin folk medicine to treat a variety of inflammatoryand infectious diseases. In particular, shikonin hasbeen shown to possess many diverse properties,including antioxidant, anti-inflammatory, antith-rombotic, antimicrobial, and wound healing ef-fects. The fact that shikonin shows so many bene-ficial properties has increased the interest in thismolecule dramatically, especially in the past fewyears. The aim of this review is to provide an up-date of the new data published on shikonin,whose wide spectrum of pharmacological effectsas well as pharmacokinetic properties and toxic-ity make it a highly interesting target molecule.

Abbreviations!

ABTS: 2,2′-azino-bis(3-ethylbenzothiazo-line-6-sulphonic acid)

Adv3: adenovirus type 3Akt: protein kinase BASK1: apoptosis signal-regulating kinase 1cAMP: cyclic adenosine monophosphateCC50: cytotoxic concentration 50C/EBP: CCAAT/enhancer binding proteinCCR: chemokine receptorchi-aDDnSs: chimeric advanced drug delivery

nano systems

c-Maf: c-musculoaponeuroticfibrosarcoma

COX: cyclooxygenaseDPPH: 1,1-diphenyl-2-picryl-hydrazylEC50: effective concentration 50ERK: extracellular signal-regulated

kinaseGLUT4: glucose transporter type 4HCV: hepatitis C virusHIV: human immunodeficiency virusIC50: inhibitory concentration 50IκB: inhibitor of NF-κBIL: interleukiniNOS: inducible nitric oxide synthaseLC50: lethal concentration 50LDL: low-density lipoproteinLPS: lipopolysaccharideLRS: lactated Ringerʼs solutionMIC: minimal inhibitory concentrationMPO: myeloperoxidaseNAT: N-acetyltransferaseNF-κB: nuclear factor κBPPAR: peroxisome proliferator-activated

receptorRIP: receptor interacting proteinROS: reactive oxygen speciesSREBP: sterol regulatory element-binding

proteinSTAT3: signal transducer and activator of

transcription-3TATA box-binding protein:

transcription factor IID proteincomplex

T‑bet: T‑box expressed in T cellsTNF: tumor necrosis factorTPA: 12-O-tetradecanoylphorbol-13-

acetate

Pharmacological Properties of Shikonin –A Review of Literature since 2002

Authors Isabel Andújar, José Luis Ríos, Rosa María Giner, María Carmen Recio

Affiliation Departament de Farmacologia, Facultat de Farmàcia, Universitat de València, Valencia, Spain

Key wordsl" anti‑infectiousl" anti‑inflammatoryl" antioxidantl" glucose metabolisml" wound healingl" shikoninl" Lithospermum erythrorhizonl" Boraginaceae

received May 23, 2013revised July 29, 2013accepted Sept. 16, 2013

BibliographyDOI http://dx.doi.org/10.1055/s-0033-1350934Published online October 23,2013Planta Med 2013; 79:1685–1697 © Georg ThiemeVerlag KG Stuttgart · New York ·ISSN 0032‑0943

CorrespondenceProf. Dr. María Carmen RecioDepartament de FarmacologiaFacultat de FarmàciaUniversitat de ValènciaAv. Vicent Andrés Estellés s/n46100 Burjassot, ValenciaSpainPhone: + 34963543283Fax: + 34963544943maria.c.recio@uv.es

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Fig. 1 Chemical structures of shikonin derivatives.

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Introduction!

Sources of shikonin and pharmacological effects ofLithospermum erythrorhizon extractsShikonin is one of the active principles of Zicao, the root extract ofLithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae), widelyused in traditional Chinese medicine for its anti-inflammatoryproperties [1]. The first derivative of this extract to be describedwas acetylshikonin, which was isolated from the root ofL. erythrorhizon by Kuroda and Majima in 1922. These samechemists later isolated other derivatives, including shikonin, andidentified many of their chemical properties. However, becauseof its physical and chemical similarities to naphthazarin, Kurodaand Majimaʼs original description of shikoninʼs structure was in-accurate. Some years later, in 1936, Brockmann and Liebigs eluci-dated the correct structure of this molecule, whichwas identifiedas 5,8-dihydroxy-2-[(1R)-1-hydroxy-4-methyl-3-pentenyl]-1,4-naphthoquinone (l" Fig. 1). They were also the first to identifyshikoninʼs enantiomer alkannin [2]. Although mostly found inthe root of L. erythrorhizon, shikonin, along with alkannin andother derivatives, can also be found in the roots of other speciesof the Boraginaceae family. These species include those from thegenera Arnebia (such as A. euchroma), Echium (E. lycopsis), Eri-trichium, and Onosma, among others [2].Studies carried out over the past three decades have demonstrat-ed that many effects associated with the use of Zicao in Chinesemedicine have a scientific basis, with shikonin and its derivativesbeing the principal substances responsible for the pharmacologi-cal properties of this crude drug.Until 2002, only 86 articles on shikonin could be found inPubmed. Since then, however, over 200 more articles have beenpublished. This exponential increase in interest in shikonin maybe motivated by the fact that this molecule has been demonstrat-ed to have interesting pharmacological properties, includinganti-inflammatory, anticancer, antimicrobial, and wound healingeffects. This review, therefore, is focused on the current knowl-edge of this naphthoquinone, which, because of its wide spec-trum of effects and numerous molecular targets, is a potentiallyhighly interesting target molecule.

Andújar I et al. Pharmacological Properties of… Planta Med 2013; 79: 1685–1697

The pharmacokinetics and bioavailability of shikoninBased on the long history of Zicao as an effective treatment for avariety of inflammatory and infectious diseases, various studieshave been carried out to identify its primary active principles,with all of them indicating that shikonin and its derivatives arethe main active components [1]. Previous reviews [1,2] had al-ready reported on the known pharmacological properties of shi-konin as well as on its pharmacokinetic behavior and toxicity.Unfortunately, studies on the toxicity of this molecule in differentspecies and with different administration routes are still missingand should be carried out.Due to its high lipophilicity, shikonin is generally used in creamsand ointments, that is, oil-based preparations; indeed, its insolu-bility in water is usually the cause of its low bioavailability [3].Moreover, shikonin and its derivatives are also thermolabile andprone to oxidation and polymerization [3–5]. One study using3H-shikonin in mice showed that shikonin was rapidly absorbedafter oral and intramuscular administration, with a half-life inplasma of 8.79 h and a distribution volume of 8.91 L/kg. After in-travenous injection, most of the excreted radioactivity corre-sponded to the transformed metabolite. In order to improve thestability and water solubility of shikonin, several approacheshave been tried. Thus, in 1996, Chen et al. [6] were able to achievean approximate 200-fold increase in the solubility, photostability,and in vitro permeability of shikonin through the formation of a1:1 inclusion complex with hydroxypropyl-β-cyclodextrin. In2004, Assimopoulou and Papageorgiou [5] confirmed these re-sults in a study in which they explored the inclusion of shikonin,alkannin, and their derivatives in β-cyclodextrin and hydroxy-propyl-β-cyclodextrin. More recently, Albreht et al. [3] were ableto achieve a 181-fold increase in the solubility of shikonin inaqueous media in the presence of β-lactoglobulin at a concentra-tion of 3.1mg/mL. Because they are generally recognized as safe,proteins such as β-lactoglobulin are widely used in the food andpharmaceutical industries. One shikonin molecule binds cova-lently to β-lactoglobulin via Cys121, whereas the remaining pig-ment molecules most probably bind to the protein via non-cova-lent interactions. In order to facilitate cellular intake, Xia et al. [7]prepared shikonin-containing liposomes using shikonin, soybeanphospholipid and cholesterol, and dehydrated alcohol. The lipo-

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somes were used to study the angiogenic suppression and cellu-lar uptake by the MTT assay, Transwell tests, as well as chick cho-rioallantoic membrane and Matrigel plug assays, which demon-strated that shikonin liposomes decreased toxicity, rate of inhibi-tion of migration, and angiogenic suppression compared to freeshikonin.A different approach involves the encapsulation of shikonin. As-simopoulou et al. [8] found that the combination of ethylcellulose(viscosity = 10 cp) as a matrix, mastic gum as a core additive, lowdichloromethane volume, and low sodium dodecyl sulfate con-centration results in microcapsules with the best characteristicsin terms of efficiency, loading, release, and particle size distribu-tion. Huang et al. [9] studied different alternatives for microen-capsulation and established the best proportions for stabilityand coacervation processes as a surfactant/oil ratio of 1/10 and agelatin/oil ratio of 1/5 with a pH condition of 4–6. They also ob-served that one core microcapsule prepared with 6% glycerolcould be a good potentially nontoxic crosslinking material forthe applications of encapsulated extracts containing shikonin,because it has the same crosslinking effects as those used to ob-tain gelatin/acacia microcapsules.Other biodegradable and/or biocompatible polymeric materialsare the electrospun poly(ε-caprolactone)/poly(trimethylene car-bonate) ultrafine composite fiber mats, prepared by Han et al.[10] who demonstrated their potential as effective carriers forshikonin delivery. After demonstrating the drug stability, highdrug-loading efficacy, and the stability of the shikonin-loadedcomposite fibers, they studied the in vitro release and observedthat the initial rapid release of shikonin is followed by a plateauafter 11 h. This release behavior could be used as a drug deliverysystem but also in the treatment of wound healing or dermal bac-terial infections due to the free radical scavenging properties andthe antibacterial activity (which will be later discussed in this re-view) of the shikonin-loaded fiber mats.This interest in topical or transdermal biomaterial containing shi-konin was later ratified by Kontogiannopoulos et al. [11]. Theyprepared different biocompatible polymers to produce electro-spun fiber mats containing shikonin in various amounts. Shiko-ninwas incorporated and did not affect fibers morphology. In ad-dition, they conclude that the prepared shikonin-loaded e-spunsubmicron-sized fiber mats are advantageous compared to con-ventional wound healing devices. One year later, the same au-thors [12] used chi-aDDnSs, which are mixed nanosystems com-bining different biomaterials that can offer advantages as drugcarriers. They applied this system for the characterization of theshikonin-loaded chi-aDDnSs, which was performed by measur-ing their particle size distribution, ζ-potential, drug encapsula-tion efficiency, stability, and the in vitro release profile. Theseproperties gave promising results, which confirmed the interestof this kind of formulations, and the authors hypothesize that itcould be used as a road map for designing in vivo experiments.

Shikonin in Inflammation!

The anti-inflammatory effect of shikonin has been studied foryears in animal models of inflammation, and its potential mech-anisms have been investigated in vitro. The reviews published byChen et al. [1] and Papageorgiou et al. [2] list the most relevantstudies conducted up to 2002, concluding that the anti-inflam-matory effects of shikonin and some of its derivatives can be at-tributed to several distinct mechanisms of action, for example,

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inhibition of the biosynthesis of leukotriene B4, suppression ofmast cell degranulation, inhibition of the respiratory burst inneutrophils, alteration of phosphatidylinositol-mediated signal-ing, or blockade of chemokine binding to the CCR-1. Accordingto Kourounakis et al. [13], the naphthazarin structure of shikoninand its derivatives makes them free radical scavengers. This maybe one of the mechanisms of action that gives shikonin its anti-inflammatory properties. It was also demonstrated that shikoninhas a better potency as a COX inhibitor than alkannin, but alsohigher cytotoxicity and pro-oxidant activity [14]. However, re-cent papers have demonstrated the implication of other media-tors as well as other signaling pathways, thus broadening shiko-ninʼs possible mechanisms of action and therapeutic potential.

Shikonin in classic anti-inflammatory mouse modelsThe dichloromethane extract of L. erythrorhizon was studied byZeng [15] in two classic models of anti-inflammatory activity:mouse ear edema induced by application of TPA and mouse pawedema induced by subcutaneous injection of carrageenan intothe plantar foot. In both cases, the extract was observed to inhibitthe edema. The researchers identified the prenylnaphtoquinonespresent in this extract as the probable active agents. The activityof other shikonin derivatives as anti-inflammatories has been al-so established by other authors, such as Kundakovic et al. [16],who described the effects of β,β-dimethylacrylshikonin, isovaler-ylshikonin, and acetylshikonin on a carrageenan-induced rat pawedema test, the last one being the best anti-inflammatory agent,with an ED50 of 6.91mg/kg (p.o.).In addition, the n-hexane extract obtained from the roots ofL. erythrorhizon, which contains shikonin, acetyl shikonin, isobu-tyryl shikonin, β,β-dimethylacryl shikonin, and isovaleryl shiko-nin as the most relevant compounds, was tested on the transcrip-tional activation of the TNF-α promoter in mouse abdominal skintransfected with plasmid pTNFP‑Luc via gene gun delivery as amodel of inflammation [17]. This hexane extract (100 µg of eachtest agent/site/mouse) significantly inhibited (49%) the tran-scriptional activity of the TNF-α promoter in comparison withthe untreated controls in the in vivo system. All the tested com-pounds inhibited TNF-α promoter activity in a dose-dependentmanner, but shikonin (77–96% inhibition at 50–200 µg/site/mouse) and isobutyryl shikonin (61–89%) exhibited the highestinhibition of the transcriptional activation of the human TNF-αpromoter through interference with the basal transcription ma-chinery, inhibiting the binding of transcription factor IID proteincomplex (TATA box-binding protein) to the TATA box. In contrast,shikonin did not inhibit the phosphorylation of ERK1/2 or NF-κBp65 induced by particle-mediated injury/stress in mouse skin[17].Other studies have been carried out using the active principleshikonin. Andújar et al. [18] studied the effect of shikonin onmouse ear edema induced by TPA and found that it exerted anti-inflammatory activity, as demonstrated by the reduction of theedema, the inhibition of COX-2 by 70%, and the complete inhibi-tion of iNOS. Topical treatment with shikonin significantly de-creased TPA-induced translocation of protein kinase Cα alongwith the phosphorylation and activation of ERK1/2, the nucleartranslocation of NF-κB, and the TPA-induced NF-κB‑DNA-bindingactivity in mouse skin. These results were confirmed in vitro inRAW 264.7 cells, where shikonin (1 µM) significantly inhibitedthe binding of NF-κB to DNA in a dose-dependent manner as wellas the nuclear translocation of p65 through a mechanism involv-ing the downregulation of the inhibitor of NF-κB (IκB)α phospho-

dújar I et al. Pharmacological Properties of… Planta Med 2013; 79: 1685–1697

Fig. 2 Suggested targets for shikonin in inflam-mation. CREB (cAMP response element-bindingprotein); DAG (diacylglicerol); IKK (IκB kinase); IP3(inositol trisphosphate); IP3R (inositol trisphosphatereceptor); NEMO (NF-κB essential modulator); PIP2(phosphatidylinositol 4,5-bisphosphate); PKC (pro-tein kinase C); PLC (phospholipase C); RACK (recep-tor for activated C-kinase).

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rylation. In a complementary study, Cheng et al. [19] investigatedthe effect of a mixture of shikonin/alkannin and their derivativesin RAW 264.7 macrophages stimulated with LPS. They concludedthat this particular mixture inhibited LPS-induced nitric oxideand prostaglandin E2 production, iNOS protein expression, andERK, IκBα, and NF-κB activation. In all cases, the effect of the shi-konin/alkannin mixture was higher than that of the other deriva-tives.In contrast to Staniforth et al. [17], who were unable to demon-strate that shikonin inhibited both ERK and NF-κB activation invivo, these two studies, carried out by two independent groups[18,19], have demonstrated this inhibition both in vitro and invivo. Since the administration of shikonin in vivo was topical inall of the studies, the discrepancies in the results are probablydue to the different doses tested: Staniforth et al. [17] used shiko-nin at a dose of 100 µg/site/mouse, while Andújar et al. [18] cal-culated an ID50 = 1.0mg per ear.Lu et al. [20] studied the effect of shikonin in two other classicmouse models widely used in research on drugs with anti-in-flammatory activity: xylene-induced auricle swelling and aceticacid-induced capillary permeability increase. The authors con-cluded that shikonin efficiently inhibited auricle swelling in adose-dependent manner, with 4mg/kg of shikonin yielding thesame effect as dexamethasone at 2.5mg/kg. In vitro, they demon-strated that shikonin inhibited TNF-α production in LPS-stimu-lated rat primary macrophages as well as NF-κB translocationfrom the cytoplasm to the nucleus. Moreover, at 1 µM shikoninblocked NF-κB nuclear translocation via inhibition of protea-some-mediated IκBα degradation and induced cell death by in-hibiting the proteasome in macrophage cells. The principal tar-gets studied for shikonin are summarized in l" Fig. 2.

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Shikonin in autoimmune-mediatedinflammatory diseasesShikonin in arthritis: In vivo, shikonin was found to be active in amousemodel of established collagen-induced arthritis, but not inthe early arthritis [21]. Indeed, they observed that shikonin hadno effect on the influx of inflammatory cells and tissue damageduring the early arthritis. However, the intraperitoneal injectionof shikonin (5mg/kg) for 10 days after arthritis had been estab-lished significantly improved the macroscopic appearance anddecreased cartilage destruction, reducing the expression of theTh1 cytokines TNF-α and IL-12 in both the synovial tissue andthe articular cartilage through a mechanism involving T‑bet. Incontrast, the Th2 cytokines IL-10 and IL-4 remained elevatedthroughout treatment due to an increase in GATA-binding pro-tein-3 expression. The expression levels of IL-6 were also inhib-ited.More recently, Kim et al. [22] published a study on the efficacy ofshikonin in protecting the cartilage in a murine model of rheu-matoid arthritis, also induced by collagen. In this case, the au-thors used a dose of 2mg/kg of shikonin administered orally dai-ly for 35 days. As in the previous study, the incidence, bone min-eral density, bone mineral content, and joint histopathology allimproved significantly, with a decrease in inflammation, jointdestruction, and matrix metalloproteinase-1 production and aconcomitant increase in the tissue inhibitor of metalloprotein-ase-1.These two studies suggest that shikonin may be a good candidatefor the development of a cartilage protective medicine. The tran-scription factors GATA-3 and T‑bet regulate the differentiation ofTh cells into either Th1 cells, which are responsible for cell-medi-ated immune defense against intracellular pathogens, or Th2cells, which defend the host against extracellular parasites [23].This polarization of Th cells is important because Th1 cells are

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considered to play a central role in autoimmune diseases. Shiko-ninʼs ability to switch the established polarization by inhibitingT‑bet and increasing GATA-3 expression could be crucial in thetreatment of such diseases for which an effective, definitive ther-apy has yet to be developed.Shikonin in inflammatory bowel disease: Inflammatory boweldisease encompasses several pathologies that share various clin-ical and pathophysiological characteristics with rheumatoid ar-thritis. As immune-mediated inflammatory diseases, they are alltreated with similar disease-modifying drugs, such as metho-trexate, sulfasalazine, or more recently anti-TNF agents [24]. In arecent study, Andújar et al. [25] determined the effect of shikonin(25mg/kg, p.o. twice during the experiment) on a mouse modelof acute ulcerative colitis induced in Balb/C mice by oral adminis-tration of 5% dextran sodium sulfate. The authors found thattreatment with shikonin prevented the shortening of the colo-rectum and decreased weight loss by 5% while improving the ap-pearance of feces and preventing bloody stools. Moreover, MPOactivity was reduced as well as the expression of COX-2, the acti-vation of NF-κB and that of STAT3. Cytokine production (TNF-α,IL-1β, and IL-6) was also inhibited. Similar results were obtainedin primary macrophage cultures. Although these authors did notevaluate the effect of shikonin on T‑bet and GATA-3 expression,the inhibition of Th1 cytokines in this model indicates that a sim-ilar mechanism of action for shikonin is at work in both arthritisand colitis.Shikonin in asthma: The usefulness of shikonin as an antiallergicagent has also been described. Tanako-Ohmuro et al. [26] re-ported that shikonin inhibits in a dose-dependent manner(IC50 = 2.6 µM) histamine release mediated by anti-immunoglob-ulin E antibodies in basophils isolated from the blood of healthyvolunteers. As a target in the signal cascade of IgE-mediated acti-vation, the authors proposed spleen tyrosine kinase. More re-cently, the effects of shikonin on a murine model of asthma andthe maturation of dendritic cells derived from bone marrow havealso been studied [27]. The results demonstrated not only thatshikonin at 0.3 µM is able to inhibit the in vitro maturation ofdendritic cells but also that in vivo (4.5 µg, once daily for 5 days)it inhibits the allergic reaction and airway hyperresponsivenessin asthmatic mice. To gain insight into the mechanism of actionof shikonin as an antiallergic drug, Lee et al. [28] studied the ef-fects of shikonin on TPA plus cAMP-induced Th2 cytokine prod-uction in activated EL-4 murine T-lymphoma cells. These cellsproduce IL-4 and IL-5 but not interferon-γ which is produced byTh2 cell-like cells and is responsible for allergic reactions. Shiko-nin (0.3 µM) directly inhibited the Th2 responses in T cells by re-ducing the expression of IL-4, IL-5, and the transcription factors(GATA)-3 and c-Maf, but not T‑bet. Moreover, shikonin sup-pressed the phosphorylation of p38, IκB kinase β, and IκBα whileinhibiting the subsequent IκBα degradation induced by TPA +cAMP. However, it seemed to have no effect on the TPA + cAMP-induced activation of ERK and c-Jun N-terminal kinase. These re-sults appear to be contradictory with those obtained by Dai et al.[21], Kim et al. [22], and Andújar et al. [25]. However, while theseauthors published their results based on in vivo studies, Lee et al.[28] were working with a Th2-like cell line. Although the impor-tance of in vitro studies in elucidating mechanisms of action isundeniable, it should be remembered that the complex immuneregulatory networks that act throughout the whole organismmight completely modify the expected outcome based on resultsobtained in vitro. Another possible explanation for the aforemen-tioned discrepancies may be the different doses tested. In any

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case, more studies should be carried out to shed light on how dif-ferent doses of shikonin may polarize the immune system to-wards a Th1 or a Th2 response.Shikonin in lupus nephritis: Wang et al. [29] orally administeredshikonin (24 and 40mg/kg/day) to NZB/W F1 mice for 14 weeksand reported that proteinuria was dose-dependently reduced aswell as circulating adhesion molecules. Moreover, they observeda downregulation in intercellular adhesion molecule-1 and vas-cular cell adhesion molecule-1 mRNA expression in the kidneysof the treated mice. The histological examination of the kidneyalso confirmed that renal glomerular lesions were alleviated aftershikonin treatment. The results from this study reveal a possibletherapeutic effect of shikonin on lupus nephritis through amech-anism which the authors hypothesize to be an inhibition of thecytokine-induced cellular adhesion molecules. This cytokine in-hibitionwould be a commonmechanism displayed in other auto-immune models in which shikonin has been studied, althoughWang et al. [29] did not carry out any experiment to confirm this.Effect of shikonin on other animal models: Shikonin has recentlyalso been studied in models of acute pancreatitis [30] and acutelung injury [31]. In the case of the murine model of cerulein-in-duced acute pancreatitis [30], shikonin (50mg/kg) significantlyreduced the pancreatic histological scores and the serum amylaseand lipase activities. Moreover, the production of the cytokinesTNF-α, IL-1β, and IL-6 was reduced, as well as MPO activity. Thiscytokine and MPO inhibition was also accompanied by a reducedNF-κB activity. Shikonin also decreased the productions of theproinflammatory cytokines TNF-α and IL-1β, and the concentra-tions of MPO and NO in acute lung injury induced by LPS in micein a model of acute lung injury [31]. The pretreatment of micewith shikonin significantly suppressed LPS-induced activation ofCOX-2 and iNOS, as well as the NF-κB DNA-binding activity inlung tissues.These molecular mechanisms described all coincide with thoseobserved in the other mouse models reviewed. It seems reason-able, then, to conclude that the most feasible mechanism in-volved in shikoninʼs anti-inflammatory activity is the inhibitionof the NF-κB pathway, as demonstrated by the different in vivoand in vitro studies cited. The downregulation of this pathwayexplains perfectly the other effects observed, such as the inhibi-tion of the production of cytokines, adhesion molecules, and oth-er inflammatory mediators. In fact, using LPS-stimulated THP-1monocytes, Chiu et al. [32] established that shikonin significantlyinhibited the early expression of approximately 50 genes, some ofthem corresponding to relevant cytokines implicated in inflam-mation (TNF-α, IL-1β, IL-4), chemokines and inflammatory mod-ulators. Their results led them to the conclusion that shikoninstrongly inhibits macrophage activation (results which have beenconfirmed by other authors [18–20,25]), chemotaxis, and T-lym-phocyte recruitment. In this same line, Li et al. [33] have recentlyconfirmed that shikonin is able to suppress humanT-cell prolifer-ation, IL-2 and IFN-γ secretion, CD69 and CD25 expression, aswell as cell cycle arrest activated by costimulation of TPA/iono-mycin or OKT-3/CD28 monoclonal antibodies. These effects aremediated by shikonin through the suppression of the NF-κB sig-naling pathway via inhibition of the IKKα/β phosphorylation, Iκ-Bα phosphorylation and degradation, and NF-κB nuclear trans-location by directly decreasing IKKβ activity. Shikonin also sup-pressed JNK phosphorylation in the MAPKs pathway of T cells.The main animal models in which shikonin has been tested aswell as the reported molecular targets for shikonin have beensummarized in two tables (l" Tables 1 and 2).

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Table 1 Principal molecular targets implicated in inflammatory diseases thatare inhibited by shikonin.

Molecular targets of inflammation inhibited by shikonin

Cytokines

TNFα [17,20,21,25,30–32]

IL-12 [21]

IL-6 [21,25,30,31]

IL-1β [25,30–32]

IL-2 [33]

IFNγ [33]

MAPK

ERK1/2 [18,19]

JNK [33]

Pro-inflammatory enzymes

COX-2 [18,25,31]

IκBα [18–20,33]

iNOS [18,19,31]

MMP-1 [22]

MPO [25,30,31]

Proteasome [20]

Serum amylase activity [30]

Serum lipase activity [30]

Spleen tyrosine kinase [26]

Transcription factors

NF-κB [18–20,25,30,31,33]

T‑bet [21,22]

STAT3 [25]

Othermediators

NO [30]

Intracellular adhesionmolecule [29]

PGE2 [19]

Vascular cell adhesionmolecule [29]

Table 2 In vivomodels used for the study of the anti-inflammatory activity ofshikonin.

Anti-inflammatory effect of shikonin in vivo

In vivomodel Reference

TPA-inducedmouse ear edema [18]

Xylene-induced auricle swelling [20]

Acetic acid-induced capillary permeability increase [20]

Arthritis [21,22]

Inflammatory bowel disease [25]

Asthma [27]

Lupus nephritis [29]

Acute pancreatitis [30]

Lung injury [31]

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Antioxidant Effects of Shikonin!

The antioxidant properties of shikonin and its derivatives werestudied, and their structure-activity relationship was approxi-mately established by Assimopoulou et al. [34]. All the hydroxy-naphthoquinones studied by these authors showed high radicalscavenging activity against the DPPH radical; therefore, they es-tablished that the presence of the naphthoquinone moiety is es-sential for the activity, while the side chain possibly plays aminorrole. Moreover, in a recently published paper, Jin and Bai [35]studied the radical scavenging activity of shikonin and its deriva-tives by using density functional theory and demonstrated thatthe introduction of acyl groups decreases the ionization potentialvalues compared with those of shikonin. However, Zeng [15] ob-tained a poor activity for shikonin as a DPPH radical scavenger,with an IC50 value of 56.3 µg/mL, whereas the IC50 values againstABTS and Fe2+/ascorbate/rat brain were 1.93 µg/mL and 6.3 µg/mL, respectively. Moreover, shikonin had no activity in the super-oxide and xanthine oxidase tests.An application for shikoninʼs antioxidant properties has beensuggested by Nishizawa et al. [36]: a possible use of shikonin forcigarette filters. These authors quantified the peroxyradicals re-covered from cigarette smoke and compared the scavenging ac-tivity of shikonin vs. other antioxidants, such as ascorbic acid, re-duced glutathione, and epigallocatechin gallate, concluding that,among all the antioxidants studied, shikonin alone exerted thebest scavenging activity. Its effectiveness against peroxyradicalstogether with its heat resistance, non-volatility, and high affinity

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to the filter are all characteristics that made the authors suggestthe possible use of shikonin for cigarette filters.

Neuroprotective Properties of Shikonin!

Shikonin has also been shown to exhibit a neuroprotective effectagainst the damage caused by ischemia/reperfusion in adult maleKunming mice [37]. This finding implies that treatment with thismoleculemay exert a significant protective effect against cerebralbrain injury. Shikonin administered at a dose of 50mg/kg (i.g.)once a day for three days before middle cerebral artery occlusionand once again 2 h after the onset of ischemia resulted in a signif-icant decrease in neurological deficit scores, infarct size, and lev-els of malondialdehyde, carbonyl, and reactive oxygen species. Italso attenuated neuronal damage and the upregulation of super-oxide dismutase, catalase, and glutathione peroxidase activitieswhile reducing the glutathione/glutathione disulfide ratio. Theseresults indicate that the neuroprotective effects of shikoninagainst cerebral ischemia/reperfusion injury may be due to itsantioxidant effects.This neuroprotective activity of shikonin has also been describedby Nam et al. [38]. These authors examined the effect of shikoninand five of its derivatives (acetylshikonin, isobutyrylshikonin,isovalerylshikonin, β,β-dimethylacrylshikonin, and β-hydroxy-isovalerylshikonin) on microglial cells which are the prime effec-tors in immune and inflammatory responses of the central ner-vous system. Their results indicate that two of shikoninʼs deriva-tives (isobutyryl- and isovalerylshikonin) were even more effec-tive than shikonin in repressing microglial LPS-induced activa-tion.Finally, in a recently published paper, Esmaeilzadeh et al. [39]demonstrated that shikonin protected dopaminergic neuronsagainst 6-hydroxydopamine-induced neurotoxicity. In fact, thetreatment of PC12 cells (rat pheochromocytoma) with shikonin(10 µM) protected nearly 70% of the cells against toxicity, via bothglutathione-dependent and direct anti-apoptotic pathways, be-cause shikonin avoided the depletion of intracellular glutathioneand upregulated glutathione-independent superoxide dismutasemRNA. These results are in concordancewith those ofWang et al.[37]. Moreover, shikonin upregulated Bcl-2, downregulated Baxand prevented cell nuclei from undergoing morphologicalchanges typical of apoptosis.

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Cardioprotective Effects of Shikonin!

Two different studies have suggested a possible cardioprotectiveeffect of shikonin that would be related to its anti-inflammatoryand antioxidant effects. To this respect, Andrikopoulos et al. [40]reported that shikonin at 10 µM gave a percentage of protectionagainst the in vitro LDL oxidation of 61%; however, it did notshow a clear dose-dependency. Later, Yoshida et al. [41] did de-scribe a dose-dependent inhibition of the rat thoracic aorta re-laxation in response to acetylcholine (pD'2 value 6.29) and theLPS-induced NO production (82% at 1 µM) in RAW264.7 cells. Be-cause shikonin inhibits all isoforms of NOS (IC50 4–7 µM), it washypothesized that inhibition of NOS is implied in both events. Theanti-inflammatory studies carried out with shikonin all coincidein its ability to inhibit inducible NOS; therefore, it is reasonable tosuspect, as Yoshida et al. [41] did, that shikonin could also havean effect on the endothelial NOS. However, in view of the appar-ent discrepancy of these two studies on the cardiovascular effectof shikonin, more studies should be carried out in this field in or-der to confirm this effect.

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Anticancer Activity of Shikonin!

The anticancer effects of L. erythrorhizon have been demonstrat-ed in vitro in B16F10 murine melanoma cells [42]. The n-hexaneextract was found to inhibit the growth of melanoma cells withan IC50 of 2.73 µg/mL. Moreover, L. erythrorhizon treatment at adose of 10mg/kg for 21 days in experimental mice implantedwith tumors resulted in significant reductions in tumor growth(43%) and weight (36%). Five naphthoquinone compounds (shi-konin, deoxyshikonin, β-hydroxyisovalerylshikonin, acetylshiko-nin, and isobutyrylshikonin) purified from the extract were de-scribed as being responsible for its anticancer activity. In a similarstudy, Kretschmer et al. [43] described the ability of a petroleumether-soluble extract of the roots of Onosma paniculata (contain-ing several shikonin derivatives, namely, β-hydroxyisovalerylshi-konin, acetylshikonin, dimethylacrylshikonin, and amixture of α-methylbutyrylshikonin and isovalerylshikonin) to exert strongcytotoxicity against eight cancer cell lines and MRC-5 lung fibro-blasts. However, the isolated shikonin molecule has been moreextensively studied than the root extract as an anticancer agent.A wide spectrum of anticancer mechanisms of action have beendescribed for shikonin: suppression of NF-κB-regulated geneproducts [44], inhibition of proteasome activity [45], ROS gener-ation [46], inhibition of tumor-specific pyruvate kinase-M2 [47,48], cell cycle arrest [49], or induction of necroptosis [50], amongothers. Since a review on the anticancer activity of shikonin hasrecently been published [51], the present review will only focuson the very recently published papers, therefore being an updateof the previously published paper. In this sense, Huang et al. [52]have only recently published their results on shikoninʼs ability (ata dose of 3 µM) to induce cell death in C6 and U87 glioma cellsthrough a mechanism that involved the induction of necroptosis,with the participation of the RIP-1 pathway and an increase inoxidative stress. In this same line, Piao et al. [53] tried to examinein more depth the molecular mechanisms involved in cell deathinitiation by shikonin, both in caspase-dependent and caspase-independent cell death. In their study they demonstrate that shi-konin at 1 µM induced caspase-dependent apoptosis in U937cells after 6 h with an increase in DNA fragmentation, intracellu-lar ROS, lowmitochondrial membrane potential, andwith the ex-

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pression of Noxa and tBid proapoptotic proteins. Necroptosis wasalso detected but with a higher dose of shikonin (10 µM). At10 µM, shikonin induced a greater release of cytochrome c fromthe mitochondria and of lactate dehydrogenase, but Noxa andtBidwere not affected. Interestingly, exogenous glutathione com-pletely inhibited 1 µM apoptosis and converted 10 µM necropto-sis to apoptosis. In other words, shikonin depletes glutathionedose-dependently, and therefore the oxidative stress induced byshikonin plays an important role in cell death initiation. More-over, these authors analyzed the gene expression during theseprocesses and found that 353 genes were significantly regulatedby 1 µM shikonin (that is, during apoptosis) and 85 genes by10 µM shikonin treatment (that is, in necroptosis induction).Among these genes, the transcription factor 3 and DNA-dam-age-inducible transcript 3 were highly expressed at shikonin-in-duced apoptosis (1 µM), while TNF expression mainly increasedat 10 µM treatment.In addition, Ahn et al. [54] also confirmed the ROS-mediated in-duction of apoptosis in several cancer cell lines (cervical cancercell line HeLa, colon cancer cell line Hct116, hepatocellular carci-noma cell line Hep3B, and a lung cancer cell line A549). Their re-sults indicate that shikonin exerts a cytotoxic effect in these celllines, having a 50% growth inhibition range of 0.5–3.0 µM. More-over, they demonstrated that elevated ROS levels due to pro-longed exposure to shikonin activated ASK1 and p38 MAPK anddownregulated p21Cip1, which results in G1 arrest, all theseevents leading to the promotion of apoptosis. These two studies[53,54] give a deeper insight into the possible mechanism of ac-tion by which shikonin induces cell death through a pro-oxidantmechanism. Zhang et al. [55] also justify some properties of shi-konin on the basis of its pro-oxidative effects. In this sense, work-ing with HL-60 cells, they demonstrated that treatment with shi-konin reduced the glutathione/glutathione disulfide ratio, andthe nonenzymatic antioxidant, 4,5-dihydroxybenzene-1,3-disul-fonate, showed a remarkable recovery effect on the shikonin-in-duced decrease in glutathione. Also, when the enzymatic antiox-idant and ROS scavengers, superoxide dismutase and catalasewere added to cell cultures prior to shikonin, a similar trend wasobserved in the suppression of ROS production but also in therestoration of glutathione. Based on these results, the authorsconclude that shikonin has a specific role of redox homeostasisin regulating HL-60 cell differentiation.A very recently published paper describes the antimetastasisproperties of shikonin [56]. The authors treated non-small celllung cancer A549 cells with shikonin 8 µM for 24 h and demon-strated that shikonin significantly inhibited cell proliferation.When cells were treated with a dose inferior to 2.0 µM for 24 h,cell adhesion to the extracellular matrix was significantly sup-pressed, as well as invasion and migration.Moreover, they showed that the mechanisms by which shikoninwas able to exert this antimetastasis effect involved a reductionin the expression of integrin β1 at the mRNA and protein levelsand a repression of the phosphorylation of ERK1/2.These results, together with others previously reviewed [51],demonstrate that shikonin is able to inhibit or modulate a widespectrum of cellular targets associated with cancer. Since multi-ple mechanisms are involved in the development of cancer, amolecule such as shikonin seems to be a promising anticanceragent.

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Antimicrobial Activity!

Antibacterial activityIn general, shikonin and its derivatives have been shown to be ac-tive against gram-positive bacteria such as Staphylococcus aureus,Enterococcus faecium, and Bacillus subtilis at MICs ranging from0.30 to 6.25mg/mL, as well as against various species of lactic ac-id bacteria. In contrast, they are inactive against gram-negativebacteria such as Escherichia coli, Pseudomonas aeruginosa, andMicrococcus luteus [2,57], although a study published in 2011 byDing et al. [58] demonstrated that at a dose of 200 µM, shikonininhibits biofilm formation by P. aeruginosa and Stenotrophomo-nas maltophilia. Initially it was thought that shikoninʼs antibacte-rial activity derived from a bacteriostatic effect, but a kineticstudy revealed that the compound possesses bactericidal proper-ties [2]. In the same study, it was suggested that shikonin could bea useful therapeutic agent against methicillin-resistant S. aureus.In 2002, Shen et al. [59] analyzed the activity of shikonin andsome derivatives against methicillin-resistant S. aureus and van-comycin-resistant E. faecium and E. faecalis. In this test, shikoninshowed an MIC of 6.25 µg/mL against S. aureus (both methicillin-resistant and not resistant), and 50 and 25 µg/mL against E. faeci-cum and E. faecalis, respectively. This study corroborates shiko-ninʼs inhibitory activity against S. aureus, however, the ester de-rivatives showed greater potency, especially its methylbutyrylderivative which had MIC values of 1.56 µg/mL against the threeresistant microorganisms tested. Recently, Li et al. [60] publisheda paper in which they recorded an MIC value of 125 µg/mLagainst S. aureus, but these authors did not study the effect of shi-konin on the methicillin-resistant strain. The MIC difference be-tween both studies is remarkable: Li et al.ʼs [60] results are 20times higher than Shen et al.ʼs [59], therefore more studies toconfirm which is the correct MIC should be carried out. In anycase, it is worth mentioning that the two authors used differentapproaches to determine the MIC.In 2004, Kuo et al. [61] demonstrated that shikonin inhibits thegrowth of Helicobacter pylori in a dose-dependent manner(IC50 = 18 µM). Some enzymes of enteric bacteria, including Kleb-siella pneumoniae, Salmonella group B, S. aureus, E. coli, and H. py-lori, are known to exhibit NAT activity, which contributes to themetabolic activation of chemical carcinogens. In the aforemen-tioned study, the authors were also able to demonstrate the abil-ity of shikonin (4 µM) to inhibit NAT-mediated N-acetylation.

Antifungal activityThere is less consensus in the literature with regard to the anti-fungal properties of shikonin, alkannin, and their derivatives. Asrecorded by Papageorgiou et al. [2], the antidermatophytic prop-erties of shikonin and deoxyshikonin were tested against fivefungi: Saccharomyces sake, Trichophyton rubrum, T. mentagro-phytes, T. tonsulans var. sulfureum, Microsporum gypseum, andEpidermophyton fluccosum (MIC < 25mg/mL). Both were foundto exert a fungistatic rather than fungicidal action, with deoxy-shikonin generally being the more active compound. An oint-ment containing deoxyshikonin as the main component to con-trol athleteʼs foot was patented, along with other antimicrobialpreparations containing mainly shikonin. Papageorgiou et al. [2]reviewed conflicting studies regarding the activity of shikoninand deoxyshikonin against Candida albicans; while several re-searchers found shikonin and its derivatives to be inactive againstC. albicans, others claimed it was active. In 2002, a paper pub-lished by Sasaki et al. [62] comparing the effects of shikonin and

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the standard antifungal fluconazole showed that the fungicidalactivity of shikonin (MIC = 4mg/mL) was 4 times higher againstCandida krusei, 2 times higher against Saccharomyces cerevisiae,and the same as that of fluconazole against C. glabrata. Deoxyshi-konin also exhibited a more potent activity against C. krusei andS. cerevisiae than fluconazole. While other derivatives testedproved to be less active than the standard, all showed some activ-ity. More recently, Miao et al. [63] demonstrated that shikonin isactive against C. albicans, including several fluconazole-resistantstrains, against which shikonin (MIC80 = 4 µg/mL) was shown tobe > 16 times more potent than the standard (MIC80 > 64 µg/mL).Moreover, these same authors found that treatment with shiko-nin increased the generation of endogenous reactive oxygen spe-cies while decreasing mitochondrial membrane potential. Theyalso demonstrated that antioxidants such as N-acetylcysteineand glutathione reduced the antifungal activity of shikoninagainst C. albicans. Their analyses identified 9 differentially ex-pressed genes related to glycolysis-related genes (CDC19 andHXK2), fermentation-related genes (ALD5 and ADH1), antioxidantdefense-related genes (SOD2 and SOD5), a thioredoxin reductase-related gene (TRR1), a mitochondrial respiratory electron trans-port chain-related gene (MRF1), and a reduced NADPH oxidore-ductase-related gene (EBP1). These results suggest that mito-chondrial aerobic respiration shifts and increases in endogenousreactive oxygen species may contribute to the action of shikoninagainst C. albicans.This antifungal activity has many potential clinical applications,especially when combined with shikoninʼs anti-inflammatoryand antibacterial activity, whichmay be useful to treat mouth ul-cers and oral candidiasis resulting from the use of cytotoxictherapies and immunosuppressants, human immunodeficiencyvirus (HIV) infection, or opportunistic fungal infections in indi-viduals in whom the use of corticosteroids is contraindicated.

Antiparasitic activityMichaelakis et al. [64] evaluated the concentration-dependent ef-fects of three pure synthetic natural products (alkannin, shiko-nin, and shikalkin) and three acetylated derivatives of shikoninagainst Culex pipiens (Culicidae: Diptera). As the authors note,previous studies had found that both shikonin and shikonin an-gelate exhibited a considerable larvicidal effect against Aedes ae-gypti larvae, with shikonin giving an LC50 value of 7 µg/µL whilethat of shikonin angelate was about 14 µg/µL. Larvicidal bioassaysrevealed that 100% mortality was achieved with 10 µg/µL of shi-konin, whereas higher doses were needed for other naphtoqui-nones: 30 µg/µL for alkannin, 20 µg/µL for shikalkin. The assaysshowed that a relatively high mortality rate (approximately17%) could be reached with shikonin concentrations as low as2 µg/µL. Naphthoquinones showed a higher larvicidal effect thanother naturally-occurring biocides such as thymol and carvacrol,which have LD50 values of 36 µg/µL and 37.6 µg/µL, respectively.In this same study, the authors synthesized three acetylated de-rivatives of shikonin in order to elucidate the structure-activityrelationships of shikonin-type compounds with larvicidal activ-ity against C. pipiens. Their results showed that although hydrox-ylic groups seem to play a secondary role in the efficacy of thesecompounds, the quinonemoiety is essential. In this context, Ali etal. [65] demonstrated that shikonin exhibits the highest toxicityfor intracellular persisting Leishmania major GFP parasites, withan IC50 value of 1.9 µM. Moreover, when comparing shikoninwith its parent analogue, naphtazarin, they concluded that thepresence of the 1′-hydroxy-3′-isohexenyl substituent at C-3 en-

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hanced the activity and that esterification of the side chain hy-droxyl reduced the antileishmanial activity. The presence of thefree hydroxyl group is apparently a major contributing factor inshikoninʼs antileishmanial activities.

Antiviral activityA recent upsurge of studies on the antiviral activity of shikoninhave also produced promising results, with shikonin reportedlyexhibiting in vitro activity against HIV type 1 [66,67]. In particu-lar, Chen et al. [66] examined the effects of shikonin against theHIV-1 receptor antagonist activity of multiple chemokines. AtnM concentrations, shikonin inhibited monocyte chemotaxisand calcium flux in response to a variety of chemokines. It down-regulated surface expression and mRNA expression of CCR5, aprimary HIV-1 co-receptor, in macrophages to a greater degreethan the other receptors. Additionally, shikonin inhibited thereplication of both amultidrug-resistant strain and pediatric clin-ical isolates of HIV in human peripheral blood mononuclear cells,with IC50 values ranging from 96 to 366 nM. Shikonin also effec-tively inhibited the replication of the HIV Ba-L isolate in mono-cytes/macrophages (IC50 = 470 nM). These results suggest thatboth the anti-HIV and the anti-inflammatory activities of shiko-nin may be related to its interference with chemokine receptorexpression and function. The authors therefore consider shiko-nin, a naturally occurring, low-molecular-weight pan-chemokinereceptor inhibitor, to be a potential lead molecule for the designand development of therapeutic agents against HIV.In 2011, Gao et al. [68] reported on the antiviral effects of shiko-nin against AdV3. These authors found that shikonin inhibitedgrowth of AdV3 in a concentration-dependent manner (0.0156–1 µM), with a virus inhibition rate of 24–69%. Specifically, expres-sion of the hexon protein in AdV3 was higher in the virus controlgroup than in the shikonin-treated groups, while the rate of shi-konin-treated HeLa cell apoptosis decreased significantly withincreasing concentrations of the drug. These results indicate thatshikonin possesses anti-AdV3 capabilities and that the potentialantiviral mechanism most likely involves the inhibition of bothapoptosis and hexon protein expression of AdV.More recently, several phytochemicals from Arnebia euchroma,including shikonin, have been assessed for their activity againstthe HCV [60]. Among the compounds tested, shikonin exhibitedthe most potent anti-HCV activity, with an effective concentra-tion (EC)50 value of 25 ng/mL, far lower than that of the positivecontrol ribavirin (EC50 = 2.6 µg/mL), and a CC50 of 1.1 µg/mL. Shi-konin thus seems to be an HCV replication inhibitor and maytherefore be a good candidate for further optimization and drugdevelopment.

Shikonin in Wound Healing!

Medicinal use of the root of Lithospermum erythrorhizon datesback to the second century, but the first written reference to itstherapeutic qualities can be found in a traditional Chinese medi-cal text from 1596, Pen Tsʼao Kang Mu [4]. This medical compen-dium recommends using the root for the treatment of burns, analulcers, hemorrhoids, and skin wounds. It was not until 1976 thatthis property of shikoninwas confirmed experimentally by Papa-georgiou et al. [4]. Subsequent experimental animal models andclinical studies have demonstrated the efficacy of shikonin andalkannin esters in wound healing, leading to the appearance of anumber of pharmaceutical formulations: Histoplastin Red®

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(Chropi), Epouloderm® (Farmalex), and Helixderm® (PNG Geroly-matos). Histoplastin Red® was tested in patients with chronic legulcers that had resisted previous attempts at treatment. After 5 to6 weeks of application, complete healing, or at least a considera-ble reduction of the ulcer, was observed. The success rate was80% with no inflammatory processes recorded during treatment[4]. Later studies confirmed the efficacy of this ointment for thetreatment of decubitus ulcers, leprosy, anal fissures, and burns,against which, according to the authors, it was more effectivethan Betadine® (povidone-iodine) and Fucidine® (fusidic acid)[4].In a recent study conducted by Karayannopoulou et al. [69], theeffect of an alkannin/shikonin-based ointment on second inten-tion wound healing of surgically created, acute, non-contami-nated wounds in dog skin was compared with that of LRS. Thehistological findings point to the ability of the ointment to pro-mote angiogenesis, collagen production, and epithelialization ofthe lesions, although the time to complete wound closure wasnot significantly higher than in the control group (which was giv-en only LRS). These results contradict those of previous humanclinical trials, which had demonstrated the compoundʼs efficacyin clinical cases of chronic, severe, or contaminated wounds. Theauthors explained this difference by arguing that LRS is a lavagesolution capable of achieving closure of an open wound once itis clean; however, in contaminatedwounds, the use of antisepticsor antibiotics in the lavage solution may damage tissues. There-fore, in the case of contaminated wounds, the use of an alkan-nin/shikonin ointment may be more beneficial.Hsiao et al. [70] recently generated a biochemical and proteomicplatform to establish the molecular basis of the wound healingprocess induced by L. erythrorhizon and shikonin. In their studythey found that both the ethanol extracts of L. erythrorhizon andshikonin (100 nM) were able to promote cell proliferation in fi-broblasts by up to 25% and decrease cell migration. The proteo-mic analysis identified twenty-two differentially expressed pro-teins when fibroblast cells were treated with the extract or withshikonin; these proteins were categorized into six hierarchicalclusters entailing antioxidant activity, apoptosis, cell mobility,proliferation, metabolism of abnormal proteins, and collagen se-cretion. This indicates that the efficacy of L. erythrorhizon inwound healing may derive from a synergistic effect on a numberof factors. Assays by these same authors using an animal modelconfirmed that the ethanol extract of L. erythrorhizon was ableto accelerate wound healing on the back flank of SD rats.More recently, Andújar et al. [71] demonstrated that shikonin(1 µM) promoted wound healing in intestinal epithelial cells(IEC-18) through a mechanism that involved the increase of cellmigration and TGFβ1 induction, without increasing cell prolifera-tion. However, the apparently contradictory results to those pub-lished by Hsiao et al. [70] could be explained by the fact that thecell lines studied are different, and, although both fibroblasts andIEC are implicated in wound healing processes, they have differ-ent roles. In any case, the wound healing effects of the root ofL. erythrorhizon have been known for centuries in traditional Chi-nese medicine; therefore, although the data published today mayseem contradictory, a deeper study of this property is worthdoing in order to elucidate the mechanism of action involved inthis demonstrated activity of shikonin.

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Fig. 3 Suggested targets for shikonin in glucosemetabolism. C/EBPα (CCAAT/enhancer bindingprotein α); GLUT4 (glucose transporter type 4); IRS(insulin receptor substrate protein); PI3K (phospha-tidylinositol 3-kinase); PPARγ (peroxisome prolifera-tor-activated receptor γ); PTEN (phosphatase andtensin homolog); SREBP1C (sterol regulatory ele-ment-binding protein 1C). Black continuous linemeans activated by shikonin; dotted line means in-hibited by shikonin.

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Shikonin and Glucose Metabolism!

Recently, Gwon et al. [72] demonstrated the antiobesity effects ofL. erythrorhizon on C57BL/6J mice fedwith a high-fat diet and jus-tified these effects by a mechanism mediated by the suppressionof lipogenic genes in the liver and adipogenic transcription fac-tors in white adipose tissue. They also established that acetylshi-konin is, at least in part, responsible for these effects, but they didnot study other derivatives present in the active extract, such asshikonin.Other authors who have studied the antiobesity effect of shikoninhave tried to elucidate its mechanism of actionwith regard to theadipogenesis of 3 T3-L1 cells [73]. Shikonin (IC50 = 1.1 µM) wasfound to inhibit fat droplet formation and triglyceride accumula-tion in 3 T3-L1 adipocytes through amechanism involving the in-hibition of genes implicated in lipid metabolism, such as FABP4and LPL. Shikonin also inhibited the ability of PPARγ and C/EBPα,the major transcription factors of adipogenesis, to bind to theirtarget DNA sequences. Among the upstream regulators of adipo-genesis, only SREBP1C was found to be downregulated by shiko-nin, which suggests that shikonin first inhibits the expression ofSREBP1C and subsequently the expression of PPARγ and C/EBPα,resulting in the suppression of lipid metabolizing enzymes andthe reduction of fat accumulation. In a more recent paper [74],these same authors demonstrated that shikonin inhibits adipo-genesis in vitro through the activation of the WNT/β-cateninpathway tomaintain the nuclear levels of β-catenin, thereby sup-pressing the expression of PPARγ, C/EBPα, and intracellular fat ac-cumulation. The suggested targets for shikonin in glucose metab-olism are compiled in l" Fig. 3.Kamei et al. [75] reported that treatment with 10–50 µM of shi-konin stimulated glucose uptake in 3 T3-L1 cells, not through theinsulin receptor, insulin receptor substrate proteins, or phospha-tidylinositol 3-kinase, but through an unidentified tyrosine ki-nase-mediated mechanism, resulting in the activation of Akt ki-

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nase and the stimulation of glucose uptake by GLUT4 transloca-tion. Moreover, in Chinese hamster ovary cells transfected withthe insulin receptor, shikonin (30 µM) inhibited phosphataseand the tensin homolog, which may explain some of its insulin-like actions, especially increased glucose uptake [76]. Comparingthe results obtained by Kamei et al. [75] and Nigorikawa et al.[76] to those published by Lee et al. [73,74], at least two possibletargets for shikonin seem likely: (i) a target for the activation ofglucose uptake and (ii) a target for the inhibition of fat accumula-tion, with the latter being efficiently inhibited at much lowerconcentrations.In contrast to these findings, Öberg et al. [77] recently demon-strated that shikonin (1 µM) increased glucose uptake in L6 myo-tubes to the same extent as insulin without affecting Akt-phos-phorylation. These results seem to indicate that shikonin in-creases glucose uptake and, as a consequence, the intracellularlevels of calcium in myotubes through an insulin-independentpathway. These authors concluded that shikonin enhancesGLUT4-translocation from intracellular vesicles to the cell surfacein L6 myoblasts, which is the same end mechanism as that usedby insulin, but with a different intracellular signal (Akt-phos-phorylation). For this reason, the authors hypothesize that shiko-nin may also affect glucose uptake in insulin-insensitive muscles.In addition, Öberg et al. [77] also demonstrated the beneficial ef-fect of shikonin in vivo on spontaneously diabetic Goto-Kakizakirats. When the rats were treated with 10mg/kg of shikonin (i.p.,once daily for 4 days), their levels of glucose in plasma decreasedsignificantly. Moreover, an insulin sensitivity test (0.5 U/kg insu-lin, s. c.) found significantly lower glucose levels in the shikonin-treated group. The authors concluded that shikonin has a poten-tially beneficial effect on glucose metabolism, which makes it ofconsiderable interest for developing novel treatments againsttype 2 diabetes.

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Conclusion!

Modern science has gone a long way in demonstrating that themedicinal properties attributed by ancient folk traditions to theroots of L. erythrorhizon and other species of the Boraginaceaefamily have a scientific basis, lending credence to the use of thesemedicinal plants as a valuable source of modern drugs. In thissense, shikonin is being highlighted as a promising moleculedue to all the beneficial effects that have been associated with itsuse. In fact, a molecule that is able to act simultaneously on vari-ous targets represents a great advantage in the treatment of dif-ferent diseases. For example, the antimicrobial properties of shi-konin are an added benefit to its wound healing properties sincea drug that can both close a wound while preventing infectionand swelling is highly beneficial. This example can be extendedto the case of an inflammatory pathology such as ulcerative coli-tis, in which the anti-inflammatory activity of shikonin canclearly improve the evolution of the disease. Moreover, this par-ticular type of disorder produces defects in the intestinal epithe-lial barrier, which in turn lead to an increase in the concentrationof anaerobic bacteriawithN-acetyltransferase activity, which canactivate certain carcinogens. While shikonin exerts no antimicro-bial activity against these bacteria, it is able to inhibit the enzymeand therefore play a protective role. Moreover, the wound-heal-ing effects of this molecule also contribute to the treatment, sincethe sooner the intestinal ulcers heal, the sooner the patient re-covers. There are even economic benefits when one drug has thesame effect as three. Still, uncertainties remain, since shikoninshows different effects depending on the dose tested. Futurestudies should thus be developed to clarify and define not onlythe molecular targets for shikonin and some of its derivatives,but also the clinical potential of these molecules.

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Acknowledgements!

The authors thank the Spanish government (MICIIN and FEDER,grant SAF2009-10059-C03-01) for financial support. I.A. thanksthe Generalitat Valenciana for her fellowship (grant BFPI/2008/040).

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Conflict of Interest!

The authors state no conflict of interest.

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10 Han J, Chen TX, Branford-White CJ, Zhu LM. Electrospun shikonin-loaded PCL/PTMC composite fiber mats with potential biomedical ap-plications. Int J Pharm 2009; 382: 215–221

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19 Cheng YW, Chang CY, Lin KL, Hu CM, Lin CH, Kang JJ. Shikonin deriva-tives inhibited LPS-induced NOS in RAW 264.7 cells via downregula-tion of MAPK/NF-κB signaling. J Ethnopharmacol 2008; 120: 264–271

20 Lu L, Qin A, Huang H, Zhou P, Zhang C, Liu N, Li S, Wen G, Zhang C, DongW, Wang X, Dou QP, Liu J. Shikonin extracted from medicinal Chineseherbs exerts anti-inflammatory effect via proteasome inhibition. EurJ Pharmacol 2011; 658: 242–247

21 Dai Q, Fang J, Zhang FS. Dual role of shikonin in early and late stages ofcollagen type II arthritis. Mol Biol Rep 2009; 36: 1597–1604

22 Kim YO, Hong SJ, Yim SV. The efficacy of shikonin on cartilage protectionin a mouse model of rheumatoid arthritis. Korean J Physiol Pharmacol2010; 14: 199–204

23 Chakir H, Wang H, Lefebvre DE, Webb J, Scott FW. T‑bet/GATA‑3 ratio asa measure of the Th1/Th2 cytokine profile in mixed cell populations:predominant role of GATA‑3. J Immunol Methods 2003; 278: 157–169

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26 Takano-Ohmuro H, Yoshida LS, Yuda Y, Morioka K, Kitani S. Shikonin in-hibits IgE-mediated histamine release by human basophils and Sky ki-nase activity. Inflamm Res 2008; 57: 484–488

27 Lee CC, Wang CN, Lai YT, Kang JJ, Liao JW, Chiang BL, Chen HC, Cheng YW.Shikonin inhibits maturation of bone marrow-derived dendritic cellsand suppresses allergic airway inflammation in a murine model ofasthma. Br J Pharmacol 2010; 161: 1496–1511

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29 Wang XC, Feng J, Huang F, Fan YS, Wang YY, Cao LY, Wen CP. Effects ofshikonin isolated from zicao on lupus nephritis in NZB/W F1 mice. BiolPharm Bull 2009; 32: 1565–1570

30 Xiong J, Ni J, Hu G, Shen J, Zhao Y, Yang L, Shen J, Yin G, Chen C, Yu G, Hu Y,Xing M, Wan R, Wang X. Shikonin ameliorates cerulein-induced acutepancreatitis in mice. J Ethnopharmacol 2013; 145: 573–580

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34 Assimopoulou AN, Papageorgiou VP. Radical scavenging activity of Alka-nna tinctoria root extracts and their main constituents, hydroxynaph-thoquinones. Phytother Res 2005; 19: 141–147

35 Jin R, Bai Y. Theoretical investigation of the radical scavenging activityof shikonin and acylshikonin derivatives. J Mol Model 2012; 18: 1401–1408

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37 Wang Z, Liu T, Gan L,Wang T, Yuan X, Zhang B, Chen H, Zheng Q. Shikoninprotects mouse brain against cerebral ischemia/reperfusion injurythrough its antioxidant activity. Eur J Pharmacol 2010; 643: 211–217

38 Nam KN, Son MS, Park JH, Lee EH. Shikonins attenuate microglial in-flammatory responses by inhibition of ERK, Akt, and NF-κB: neuropro-tective implications. Neuropharmacology 2008; 55: 819–825

39 Esmaeilzadeh E, Gardaneh M, Gharib E, Sabouni F. Shikonin protects do-paminergic cell line PC12 against 6-hydroxydopamine-mediated neu-rotoxicity via both glutathione-dependent and independent pathwaysand by inhibiting apoptosis. Neurochem Res 2013; 38: 1590–1604

40 Andrikopoulos NK, Kaliora AC, Assimopoulou AN, Papapeorgiou VP. Bio-logical activity of some naturally occurring resins, gums and pigmentsagainst in vitro LDL oxidation. Phytother Res 2003; 17: 501–507

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47 Chen J, Xie J, Jiang Z, Wang B, Wang Y, Hu X. Shikonin and its analogs in-hibit cancer cell glycolysis by targeting tumor pyruvate kinase-M2. On-cogene 2011; 30: 4297–4306

48 Li W, Liu J, Zhao Y. PKM2 inhibitor Shikonin suppresses TPA-inducedmitochondrial malfunction and proliferation of skin epidermal JB6cells. Mol Carcinogen 2012; DOI: 10.1002/mc.21988

49 Yingkun N, Lvsong Z, Huimin Y. Shikonin inhibits the proliferation andinduces the apoptosis of human HepG2 cells. Can J Physiol Pharmacol2010; 88: 1138–1146

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52 Huang C, Luo Y, Zhao J, Yang F, Zhao H, FanW, Ge P. Shikonin kills gliomacells through necroptosis mediated by RIP‑1. PLoS ONE 2013; 8:e66326

53 Piao JL, Cui ZG, Furusawa Y, Ahmed K, Rehman MU, Tambuchi Y, Kado-waki M, Kondo T. The molecular mechanisms and gene expressionprofiling for shikonin-induced apoptotic and necroptotic cell death inU937 cells. Chembiol Interact 2013; DOI: 10.1016/j.cbi.2013.06.011

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67 Min BS, Miyashiro H, Hattori M. Inhibitory effects of quinones on RNaseH activity associated with HIV‑1 reverse transcriptase. Phytother Res2002; 16: S57–S62

68 Gao H, Liu L, Qu ZY, Wei FX, Wang SQ, Chen G, Qin L, Jiang FY, Wang YC,Shang L, Gao CY. Anti-adenovirus activities of shikonin, a component ofChinese herbal medicine in vitro. Biol Pharm Bull 2011; 34: 197–202

69 Karayannopoulou M, Tsioli V, Loukopoulos P, Anagnostou TL, GiannakasN, Savvas I, Papazoglou LG, Kaldrymidou E. Evaluation of the effective-ness of an ointment based on Alkannins/Shikonins on second intentionwound healing in the dog. Can J Vet Res 2011; 75: 42–48

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73 Lee H, Kang R, Yoon Y. Shikonin inhibits fat accumulation in 3 T3-L1 adi-pocytes. Phytother Res 2010; 24: 344–351

74 Lee H, Bae S, Kim K, Kim W, Chung SI, Yang Y, Yoon Y. Shikonin inhibitsadipogenesis by modulation of the WNT/β-catenin pathway. Life Sci2011; 88: 294–301

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75 Kamei R, Kitagawa Y, Kadokura M, Hattori F, Hazeki O, Ebina Y, Nishi-hara T, Oikawa S. Shikonin stimulates glucose uptake in 3 T3-L1 adipo-cytes via an insulin-independent tyrosine kinase pathway. BiochemBiophys Res Commun 2002; 292: 642–651

76 Nigorikawa K, Yoshikawa K, Sasaki T, Iida E, Tsukamoto M, Murakami H,Maehama T, Hazeki K, Hazeki O. A naphthoquinone derivative, shiko-nin, has insulin-like actions by inhibiting both phosphatase and tensinhomolog deleted on chromosome 10 and tyrosine phosphatases. MolPharmacol 2006; 70: 1143–1149

77 Öberg AI, Yassin K, Csikasz RI, Dehvari N, Shabalina IG, Hutchinson DS,Wilcke M, Östenson CG, Bengtsson T. Shikonin increases glucose uptakein skeletal muscle cells and improves plasma glucose levels in diabeticGoto-Kakizaki rats. PLoS One 2011; 6: e22510

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