Digitaria ciliaris is widely reported to be a problematic weed in agricultural areas and is mainly used as an indicator plant for the development of herbicides. However, its bioactivities on skin regeneration and wound healing have not been investigated. In the present study, we investigated the effects of D. ciliaris flower absolute on skin wound healing and skin regeneration-related events, that is, proliferation, migration, and collagen biosynthesis, in human fibroblasts and keratinocytes. For this study, we extracted absolute from the D. ciliaris flower by solvent extraction and identified the composition of D. ciliaris flower absolute using GC/MS analysis. We also tested the effect of D. ciliaris flower absolute in CCD986sk fibroblasts and/or HaCaT keratinocytes using the WST assay and 5-bromo-2′-deoxyuridine incorporation assay, Boyden chamber assay, ELISA, sprouting assay, and immunoblotting. GC/MS analysis of D. ciliaris flower absolute revealed that it contained 15 compounds. The absolute increased the proliferations of keratinocytes and fibroblasts and the migration of fibroblasts but did not affect cell viabilities. In addition, it enhanced the syntheses of type I and IV collagen in fibroblasts, but not in keratinocytes. The sprouting assay showed increased sprout outgrowth of fibroblasts. In addition, D. ciliaris flower absolute induced the phosphorylation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase in fibroblasts. These results indicate that D. ciliaris flower absolute may promote skin wound healing/regeneration by inducing the proliferation, migration, and collagen synthesis of fibroblasts, as well as the proliferation of keratinocytes. Therefore, D. ciliaris flower absolute may be a potential natural source for cosmetic or pharmaceutical agents that promote skin wound healing/regeneration.

In the present study, the chemical compositions and skin whitening-related antioxidant and anti-melanogenic effect of essential oils (EOs) extracted from Chrysanthemum borealeMakino (CBM) (CBMEOs) at vegetative, pre-flowering and full-flowering are investigated and contrasted among the three stages. The yields and components of the CBMEOs were different at each stage. The CBMEOs increased DPPH and ABTs scavenging activities and attenuated the α-melanocyte stimulating hormone (α-MSH)-induced tyrosinase activity and melanin synthesis in B16BL6 cells. Among CBMEO components, eugenol had the highest DPPH and ABTs scavenging activities and cuminaldehyde was the strongest inhibitor of α-MSH-induced tyrosinase activity and melanin synthesis. The CBMEOs in each stage showed the different levels of phosphorylation of extracellular signal-regulated kinase1/2 and p38 MAPK. These findings demonstrate that the CBMEOs have antioxidative and anti-melanogenic activities in all the CBM harvesting stages, resulting in skin-whitening biological activities and that the levels of their component contents and bioactivities differ among the CBM harvesting stages. The CBMEOs may have the potential for use in cosmetics and alternative medicine.

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Seven genomic clones of tobacco (Nicotiana tabacum W38) cytosolic class I small heat shock proteins (sHSPs), probably representing all members in the class, were isolated and found to have 66 to 92% homology between their nucleotide sequences. Even though all seven sHSP genes showed heat shock-responsive accumulation of their transcripts and proteins, each member showed discrepancies in abundance and timing of expression upon high-temperature stress. This was mainly the result of transcriptional regulation during mild stress conditions and transcriptional and translational regulation during strong stress conditions. Open reading frames (ORFs) of these genomic clones were expressed in Escherichia coli and the sHSPs were purified from E. coli. The purified tobacco sHSPs rendered citrate synthase and luciferase soluble under high temperatures. At room temperature, non-denaturing pore exclusion polyacrylamide gel electrophoresis on three sHSPs demonstrated that the sHSPs spontaneously formed homo-oligomeric complexes of 200 ∼ 240 kDa. However, under elevated temperatures, hetero-oligomeric complexes between the sHSPs gradually prevailed. Atomic force microscopy showed that the hetero-oligomer of NtHSP18.2/NtHSP18.3 formed a stable oligomeric particle similar to that of the NtHSP18.2 homo-oligomer. These hetero-oligomers positively influenced the revival of thermally inactivated luciferase. Amino acid residues mainly in the N-terminus are suggested for the exchange of the component sHSPs and the formation of dominant hetero-oligomers under high temperatures.

TLHS1 is a class I low molecular weight heat-shock protein (LMW HSP) of tobacco (Nicotiana tabacum). For a functional study of TLHS1, a recombinant DNA coding for TLHS1 with a hexahistidine tag at the amino-terminus was constructed and expressed in Escherichia coli. An expressed fusion protein, H6TLHS1, was purified using a Ni2+ affinity column and a Sephacryl S400 HR column. A polyclonal antibody against H6TLHS1 was produced to follow the fate of H6TLHS1 in E. coli. The fusion protein in E. coli maintained its solubility at a temperature of up to 90 degrees C and most of the proteins in the E. coli cell lysate with H6TLHS1 were prevented from thermally induced aggregation at up to 90 degrees C. We compared the viability of E. coli cells expressing H6TLHS1 to the E. coli cells without H6TLHS1 at a temperature of 50 degrees C. After 8 h of high temperature treatment, E. coli cells with H6TLHS1 survived about three thousand times more than the bacterial cells without H6TLHS1. These results showed that a plant class I LMW HSP, TLHS1, can protect proteins of E. coli from heat denaturation, which could lead to a higher survival rate of the bacterial cells at high temperature.

Small heat-shock proteins (sHsps) are ubiquitous stress proteins with molecular chaperone activity. They share characteristic homology with the @-crystallin protein of the mammalian eye lens as well as being ATP-independent in their chaperone activity. We isolated a clone for a cytosolic class ! sHsp, NtHSP17.6, from Nicotiana tabacum, and analyzed its functional mode for such activity. Following its transformation into Escherichia coli and its over-expression, NtHSP17.6 was purified and examined in vitro. This purified NtHSP17.6 exhibited typical chaperone activity in a lightscattering test. It was enable to protect a model substrate, firefly luciferase, from heat-induced aggregation. Nondenaturing PAGE showed that NtHSP17.6 formed a dodecamer in its native conformation, and was bound to its substrate under heat stress. A labeling test with bis-ANS indicated that this binding might be linked to newly exposed hydrophobic sites of the NtHSP17.6 complexes during heat shock. Based on these data, we suggest that NtHSP17.6 is a molecular chaperone that functions as a dodecamer in a heat-induced manner.