Shikonin, a major ingredient in the traditional Chinese herb Lithospermumerythrorhizon, exhibits multiple biological functions including antimicrobial, anti-inflammatory, and antitumor effects. It has recently been reported that shikonin displays antitumor properties in many cancers. This study was aimed to investigate whether shikonin could inhibit oral squamous carcinoma cell (OSCC) growth via mechanisms of apoptosis and cell cycle arrest. The effects of shikonin on the viability and growth of OSCC cell line, SCC25 cells were assessed by MTT assay and clonogenic assays, respectively. Hoechst staining and DNA electrophoresis indicated that the shikonin-treated SCC25 cells were undergoing apoptosis. Western blotting, immunocytochemistry, confocal microscopy, flow cytometry, MMP activity, and proteasome activity also supported the finding that shikonin induces apoptosis. Shikonin treatment of SCC25 cells resulted in a time- and dose-dependent decrease in cell viability, inhibition of cell growth, and increase in apoptotic cell death. The treated SCC25 cells showed several lines of apoptotic manifestation as follows: nuclear condensation; DNA fragmentation; reduced MMP and proteasome activity; decrease in DNA contents; release of cytochrome c into cytosol; translocation of AIF and DFF40 (CAD) onto the nuclei; a significant shift in Bax/Bcl-2 ratio; and activation of caspase-9, -7, -6, and -3, as well as PARP, lamin A/C, and DFF45 (ICAD). Shikonin treatment also resulted in down-regulation of the G1 cell cycle-related proteins and up-regulation of p27KIP1. Taken together, our present findings demonstrate that shikonin strongly inhibits cell proliferation by modulating the expression of the G1 cell cycle-related proteins, and that it induces apoptosis via the proteasome, mitochondria, and caspase cascades in SCC25 cells.

Lithospermum erythrorhyzon has been used as a red fooddye and traditional Chinese medicine to treat wounds, skin diseases and burns. Platelet activation plays an important role in thrombosis and haemostasis. Here, we studied the inhibition of platelet activation and its active compound from the root of Lithospermum erythrorhyzon. Its ethyl acetate extract inhibited the aggregation of washed rabbit platelets induced by collagen or thrombin. Five naphthoquinone pigments, shikonin, acetylshikonin, isobutylshikonin, α-methyl-n-butylshikonin and (β,β-dimethylacrylshikonin were isolated by means of high pressure liquid chromatography. The structures were determined by comparison of their proton nuclear magnetic resonance spectra. The potency of their inhibition was in the following order : β,β-dimethylacrylshikonin≥α-methyl-n-butylshikonin$gt;isobutylshikonin$gt;acetylshikonin$gt;shikonin. It is suggested that the size of the aliphatic hydroxy group of shikonin is important for the enhancement of potency.

This study investigates the effect of supercritical fluid extract (CMPB803-C) of Lithospermum erythrorhizon,shikonin and acetylshikonin isolated from Lithospermum erythrorhizon on IL-1β-induced chondrocytes and monosodiumiodoacetate (MIA)-induced osteoarthritis in rat. Shikonin (50μM) and acetylshikonin (3μM) treatment reduced signifi-cantly the mRNA expression and enzyme activity of matrix metalloproteinase (MMP)-1, −3 and −13 in IL-1β-inducedSW1353 chondrosarcoma cells. The chondro-protective effects of CMPB803-C and acetylshikonin were than analyzed in arat OA model using a single intra-articular injection of MIA (1㎎) in the right knee joint. CMPB803-C (200㎎/㎏) or ace-tylshikonin (5㎎/㎏) was orally administered daily for two weeks starting after 1 week of MIA injection. In the histologicalobservation, CMPB803-C and acetylshikonin clearly improved OA lesions being comparable to or better that control group.Our results demonstrated that CMPB803-C and acetylshikonin as active compound of Lithospermum erythrorhizon have astrong chondro-protective effect in OA rats, which likely attributes to its anti-inflammatory activity and inhibition ofMMPs production.

This study was conducted to evaluate the quality variation of Lithospermum radix on the pigment contents and antioxidant activities according to different growth stages and areas of cultivation. Acetylshikonin contents showed the tendency to decrease gradually from the middle of July (0.28%) to the end of August (0.05%) and then the content was increased again to the end of October (0.25%). Shikonin content was detected as small amount of about 0.009% during the period. The weight of plants was increased from the end of September to the end of October and showed the highest value as 19.8 g on October 25. ROS scavenging activity was the highest in the early of October as IC50 value of 0.11 μg/mL. Lithospermum radix of September showed lower ROS scavenging activities than those of other growth stages as IC50 value of 1.02 and 0.49 μg/mL on September 9 and September 27, respectively. Among 17 areas cultivated Lithospermum radix, 10 areas (59%) showed 0.05-0.10% of acetylshikonin contents and 3 areas (18%) were measured to contain 0.16-0.26% of acetylshikonin.

The effects of basal media, carbon, nitrogen, phosphate and some major macro elements on growth and shikonin production in Lithospermum erythrorhizon hairy root culture were studied. Among examined media, growth of hairy root cultured in B5 liquid medium was rapid, whereas shikonin production was high in MS liquid medium. Under B5 basal medium, sucrose concentration for optimal growth and shikonin production was 9% and 4% respectively. The growth and shikonin production on pH changes in B5 medium resulted little effect in pH 5.8 to pH 8.8 ranges, whereas growth was decreased dramatically in both above 8.8 and under 5.8. Nitrogen source and concentration effected on the growth and shikonin production. The highest growth rate was in B5 medium (50 mM KNO3 and 1 mM NaH2PO4), whereas the highest shikonin production was in the condition supplemented with 5 mM KNO3 and 10 mM NaH2PO4.