Influence of Root Color and Tissue on Phytochemical Contents and Antioxidant Activities in Carrot Genotypes.

This study monitored changes in major carotenoids (lutein, ⍺-carotene, and ß-carotene), free sugars (fructose, glucose, and sucrose), ascorbic acid, vitamin E, phytosterols (campesterol, stigmasterol, and ß-sitosterol), fatty acid composition, total phenol content (TPC), total flavonoid content (TFC), total anthocyanin content, and antioxidant activities (AA); ferric-reducing antioxidant power (FRAP) and 2,2'-azino-bis (3-ethylbenzothiazoline-6sulfonic acid) [ABTS] assays, in the inner and outer root tissues of nine carrot genotypes with orange, white, and purple roots. The results showed a differential accumulation of bioactive compounds and antioxidant activities depending on root tissue and color. Carotenoids, free sugars, and total phytosterol contents were higher in genotypes with orange roots than in other genotypes. Ascorbic acid, TPC, TFC, total anthocyanin, and AA were highest in purple-colored carrots while vitamin E content was higher in white/purple carrots. Root color was highly related to the accumulation of individual carotenoids, vitamin E isomers, and total anthocyanin content most prominently among the analyzed bioactive compounds and AA. Free sugar and carotenoid contents were relatively higher in outer tissues than in inner tissues. Furthermore, ascorbic acid, TPC, TFC, and AA were statistically higher or similar in outer tissues when compared to inner tissues in all genotypes. In contrast, trends in vitamin E and phytosterol content were inconsistent between the inner and outer tissues, depending on the genotype. Although fatty acid composition was affected by both root color and tissue, the results were not significant. Thus, the phytochemical profile and content were highly dependent on root color and tissue in carrot genotypes. This may be useful in the food processing and pharmaceutical industries for the extraction of targeted bioactive compounds.

Apigenin induces apoptosis by regulating Akt and MAPK pathways in human melanoma cell A375SM.

Apigenin, an aromatic compound, exhibits antioxidant, anti­inflammatory and anti­viral effects. The present study aimed to investigate the effects of apigenin on cell proliferation and apoptosis of human melanoma cells A375P and A375SM. Therefore, melanoma cells were treated with apigenin to determine its anti­proliferative and survival effects, using wound healing and MTT assays. The results revealed that melanoma cell viability was decreased in a dose­dependent manner. Furthermore, chromatin condensation, indicating apoptosis, was significantly increased in a dose­dependent manner, as demonstrated by DAPI staining. In addition, increased apoptosis rate following treatment with apigenin was confirmed by Annexin V­propidium iodide staining. The changes in the expression levels of apoptosis­related proteins in A375P and A375SM melanoma cells were subsequently detected using western blot analysis. The results demonstrated that the protein expression levels of Bcl­2 were decreased, whereas those of Bax, cleaved poly ADP­ribose polymerase, cleaved caspase­9 and p53 were upregulated in a dose­dependent manner in apigenin­treated cells compared with those noted in untreated cells. In addition, in apigenin­treated A375P cells, phosphorylated (p)­p38 was upregulated and p­extracellular signal­regulated kinase (ERK), p­c­Jun N­terminal kinase (JNK) and p­protein kinase B (Akt) were downregulated. However, in A375SM cells, apigenin treatment increased p­ERK and p­JNK and decreased p­p38 and p­Akt protein expression levels. Subsequently, the inhibitory effect of apigenin on tumor growth was investigated in vivo. Tumor volume was significantly reduced in the 25 and 50 mg/kg apigenin­treated groups compared with the control group. Additionally, a TUNEL assay was performed to detect apoptotic cells. Immunohistochemical staining also revealed elevated p­ERK expression in the apigenin­treated group compared with the control group. Overall, the findings of the present study indicated that apigenin attenuated the growth of A375SM melanoma cells by inducing apoptosis via regulating the Akt and mitogen­activated protein kinase signaling pathways.

Profiling of Individual Desulfo-Glucosinolate Content in Cabbage Head (Brassica oleracea var. capitata) Germplasm.

Individual glucosinolates (GSLs) were assessed to select cabbage genotypes for a potential breeding program. One hundred forty-six cabbage genotypes from different origins were grown in an open field from March to June 2019; the cabbage heads were used for GSL analyses. Seven aliphatics [glucoiberin (GIB), progoitrin (PRO), epi-progoitrin (EPI), sinigrin (SIN), glucoraphanin (GRA), glucoerucin (GER) and gluconapin (GNA)], one aromatic [gluconasturtiin (GNS)] and four indolyl GSLs [glucobrassicin (GBS), 4-hydroxyglucobrassicin (4HGBS), 4-methoxyglucobrassicin (4MGBS), neoglucobrassicin (NGBS)] were found this study. Significant variation was observed in the individual GSL content and in each class of GSLs among the cabbage genotypes. Aliphatic GSLs were predominant (58.5%) among the total GSLs, followed by indolyl GSL (40.7%) and aromatic GSLs (0.8%), showing 46.4, 51.2 and 137.8% coefficients of variation, respectively. GIB, GBS and NGBS were the most common GSLs found in all genotypes. GBS was the most dominant GSL, with an average value of 3.91 µmol g-1 (0.79 to 13.14 µmol g-1). SIN, GIB, PRO and GRA were the other major GSLs, showing average values of 3.45, 1.50, 0.77 and 0.62 µmol g-1, respectively. The genotypes with relatively high contents of GBS, SIN, GIB and GRA warrant detailed studies for future breeding programs since the hydrolysis products of these GSLs have several anti-cancer properties.

Antitumor and Apoptosis-inducing Effects of Piperine on Human Melanoma Cells.

BACKGROUND/AIM: Piperine is a major pungent alkaloid present in black pepper (Piper nigrum L). This study investigated the potential anticancer effects of piperine on human melanoma cells and explored the potential pharmacological mechanisms in vitro and in vivo. MATERIALS AND METHODS: Studies were performed using the MTT assay, 4',6-diamidino-2-phenylindole (DAPI) staining, western blotting, a xenograft model, the terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and immunohistochemistry. RESULTS: Piperine inhibited the growth of melanoma cells. Several apoptotic events were observed following treatment, as revealed by DAPI staining. Piperine increased the expression of BCL2-associated X, apoptosis regulator (BAX), cleaved poly(ADP-ribose)polymerase, cleaved caspase-9, phospho-c-Jun N-terminal kinase and phospho-p38, and reduced that of B-cell lymphoma 2 (BCL2), X-chromosome-linked inhibitor of apoptosis, and phospho-extracellular signal-regulated protein kinase (ERK1/2) in a concentration-dependent manner. Treatment of mice for 4 weeks with piperine inhibited tumor growth without apparent toxicity. Piperine increased the expression of apoptotic cells and cleaved-caspase-3 protein and reduced the expression of phospho-ERK1/2 protein in melanoma tumors. CONCLUSION: Piperine suppressed the growth of human melanoma cells by the induction of apoptosis via the inhibition of tumor growth of human melanoma cells and tumor xenograft models.

Development of a rapid and sensitive electrochemical biosensor for detection of human norovirus via novel specific binding peptides.

Human noroviruses cause acute foodborne gastroenteritis outbreaks worldwide. In this study, a highly sensitive and selective electrochemical biosensor was fabricated for the detection of human norovirus using novel peptides as recognition elements. The electrochemical biosensor was fabricated by assembling of eight novel peptides separately on the gold electrode and investigated their efficiencies for sensing human noroviruses. Among eight peptides, NoroBP peptide coated onto the gold electrode exhibited a high binding affinity towards human noroviruses, resulting a progressive decrease in current signals with increasing concentration of human norovirus (0-105 copies/mL). As a result, NoroBP-nonFoul(FlexL)2-coated gold electrode acts an efficient electrochemical biosensor for highly selective detection of human norovirus with a detection limit of 1.7 copies/mL, which is 3-fold lower than the reported methods. The developed electrochemical biosensor was successfully applied to detect human norovirus prepared by standard procedure from oyster, which suggests that the developed biosensor can be used as a very sensitive and selective point-of-care bioanalytical platform for the detection of human norovirus in various food samples.

Pathogenesis of Human Norovirus Genogroup II Genotype 4 in Post-Weaning Gnotobiotic Pigs.

Norovirus is the most common cause of acute gastroenteritis. Its pathogenesis is poorly understood owing to the difficulty of establishing viral infection in animal models. Here, post-weaning gnotobiotic pigs were infected with human norovirus genogroup II genotype 4 (HuNoV GII.4) to investigate the pathogenesis and replication of the virus. Three groups of four pigs were infected with 1 × 105, 1 × 106, or 1 × 107 genomic equivalent (GE) copies of HuNoV GII.4. Four pigs were used as negative controls. Blood and rectal swab samples were collected after viral infection, and gross legions were examined after necropsy. Diarrhea was induced in 25% and 75% of pigs infected with 1 × 106 and 1 × 107 GE copies, respectively. Viral shedding was detected in 50%, 75%, and 50% of pigs infected with 1 × 105, 1 × 106, and 1 × 107 GE copies, respectively. Viremia was detected in 25% of pigs infected with either 1 × 106 or 1 × 107 GE copies. When gross lesions of gastroenteritis were investigated, the ileum walls of the infected pigs were thinner than those of the controls. Villi atrophy and inflammatory cell infiltration were identified in the ileum of each infected pig. Viral capsid was identified in the jejunum, ileum, colon, spleen, and mesenteric lymph node. Virus replication was newly verified in the spleen and mesenteric lymph nodes by detection of negative-sense viral RNA. In conclusion, HuNoV GII.4 could induce acute gastroenteritis and replicate in the extraintestinal lymphoid tissues in post-weaning gnotobiotic pigs. Therefore, such pigs would be a suitable animal model for studying the pathogenesis and replication of HuNoV.

Anti­inflammatory effect of quercetin and galangin in LPS­stimulated RAW264.7 macrophages and DNCB­induced atopic dermatitis animal models.

Flavonols are compounds that have been shown to possess potent anti­inflammatory effects in cellular and animal models of inflammation. In the present study, the anti­inflammatory effects and mechanisms of two natural flavonols, quercetin and galangin, in lipopolysaccharide (LPS)­stimulated RAW264.7 macrophages were investigated. It was identified that quercetin and galangin markedly reduced the production of nitric oxide (NO), inducible NO synthase and interleukin­6, and the nuclear translocation of nuclear factor­κB (NF­κB). In addition, LPS­induced activation of extracellular signal­regulated kinase 1/2 (Erk1/2) and c­Jun N­terminal kinase (JNK) was suppressed by quercetin and galangin. Taken together, these data implied that NF­κB, Erk1/2 and JNK may be potential molecular targets of quercetin and galangin in an LPS­induced inflammatory response. Subsequently, the effects of oral administration of quercetin or galangin, either alone or in combination, in a 2,4­dinitrochlorobenzene­induced atopic dermatitis (AD) mouse model were investigated. As a result, measurements of ear thickness and the levels of serum immunoglobulin E, and histological analysis revealed that the two flavonols led to a decrease in inflammation, whereas, in combination, they were even more effective. These results suggested that quercetin and galangin may be promising therapeutic agents for AD. Additionally, their combination may be a novel therapeutic strategy for the prevention of AD.

Anticancer effects of Ixeris dentata (Thunb. ex Thunb.) nakai extract on human melanoma cells A375P and A375SM.

ETHNOPHARMACOLOGICAL RELEVANCE: The plant species Taraxacum coreanum (TC), Youngia sonchifolia (YS), and Ixeris dentata (ID) belong to the family Compositae and are used for medicinal purposes in traditional medicine. However, the anticancer effects of TC, YS, and ID extracts and the underlying molecular mechanisms in melanoma cells have not been elucidated. AIM OF THE STUDY: To investigate the potential anticancer effects of TC, YS, and ID extracts on human melanoma cells and explore the potential pharmacological mechanisms in vitro and in vivo. MATERIALS AND METHODS: In this comparative study, we investigated the effects of TC, YS, and ID extracts on cell proliferation in human melanoma A375P and A375SM cells using MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays. Apoptotic cells were detected by 4',6-diamidino-2-phenylinodole (DAPI) staining. We also investigated whether the growth-inhibitory effects were associated with the induction of apoptosis and whether the mechanisms of cell death were the result of signaling molecules such as p53, Bax, Bcl-2, caspase-9, Poly-ADP ribose polymerase (PARP), and Erk (Extracellular signal-regulated protein kinase) 1/2. The in vivo antitumor effects were evaluated by measuring the tumor volume and weight and performing Terminal deoxynucleotidyl transferase (TdT) dUTP Nick End Labeling (TUNEL) assay and immunohistochemistry (IHC) in tumor xenograft models. RESULTS: TC, YS, and ID extracts effectively inhibited the growth of A375P and A375SM cells. In addition, several apoptotic events were observed following treatment, including DNA fragmentation and chromatin condensation by DAPI staining. The extracts increased p53, Bax, cleaved-caspase-9 and cleaved-PARP expression, whereas the expression of Bcl-2 was decreased in both cell lines. Furthermore, ID extract significantly inhibited the activation of Erk1/2 in both cell lines. Among the three extracts, ID had the strongest apoptotic effects. The administration of ID extract to mice inhibited tumor growth without any toxicity following 4 weeks of treatment. This extract increased the expression of apoptotic cells and p53 protein and decreased phospho-Erk1/2 protein. CONCLUSION: TC, YS, and ID extracts suppress the growth of human melanoma cells through apoptosis. Among these extracts, ID has the strongest anticancer and apoptotic effects. It induces apoptosis through the inhibition of Erk1/2 in A375P and A375SM human melanoma cells and in tumor xenograft models and may be a potential chemotherapeutic agent against melanoma.

Antitumor actions of baicalein and wogonin in HT-29 human colorectal cancer cells.

The purpose of this study was to determine the effects of baicalein and wogonin, which are compounds derived from the Chinese herb Scutellaria baicalensis, in suppressing the viability of HT-29 human colon cancer cells. Following treatment with baicalein or wogonin, several apoptotic events were observed, including DNA fragmentation, chromatin condensation and increased cell cycle arrest in the G1 phase. Baicalein and wogonin decreased Bcl-2 expression, whereas the expression of Bax was increased in a dose-dependent manner compared with the control. Furthermore, the induction of apoptosis was accompanied by an inactivation of phosphatidylinositol 3-kinase (PI3K)/Akt in a dose-dependent manner. The administration of baicalein to mice resulted in the inhibition of the growth of HT-29 xenografts without any toxicity following 5 weeks of treatment. The results indicated that baicalein induced apoptosis via Akt activation in a p53-dependent manner in the HT-29 colon cancer cells and that it may serve as a chemopreventive or therapeutic agent for HT-29 colon cancer.

Overexpression of the pineapple fruit bromelain gene (BAA) in transgenic Chinese cabbage (Brassica rapa) results in enhanced resistance to bacterial soft rot

Bromelain is a crude protein extract obtained from pineapple stems, which comprises a variety of proteolytic enzymes. It exhibits potential therapeutic activities against trauma, inflammation, autoimmune diseases and malignant disorders. In this study, we cloned BAA1 (the gene encoding fruit bromelain) into a plant expression vector that was then used to transform Brassica rapa and overexpress BAA1 under the control of the cauliflower mosaic virus (CaMV) 35S promoter. We demonstrate that constitutive overexpression of BAA1 in B. rapa confers enhanced resistance to the soft rot pathogen Pectobacterium carotovorum ssp. carotovorum. These results suggest that it could be utilized for protecting plants from attack by bacterial pathogens.

Abiotic-stress induces demethylation and transcriptional activation of a gene encoding a glycerophosphodiesterase-like protein in tobacco plants.

To examine the relationship between gene expression and DNA methylation, transcriptionally activated genes were screened in hypomethylated transgenic tobacco plants expressing an anti-DNA methyltransferase sequence. Among 16 genes initially identified, one clone was found to encode a glycerophosphodiesterase-like protein (NtGPDL), earlier reported to be responsive to aluminium stress. When detached leaves from wild type tobacco plants were treated with aluminium, NtGPDL transcripts were induced within 6 h, and corresponding genomic loci were demethylated at CCGG sites within 1 h. Direct bisulfite methylation mapping revealed that CG sites in coding regions were selectively demethylated, and that promoter regions were totally unmethylated regardless of the stress. Salt and low temperature treatments also induced similar demethylation patterns. Such effects could be attributable to oxidative stress, since reactive oxygen species generated by paraquat efficiently induced the same pattern of demethylation at coding regions. Pathogen infection induced neither transcripts nor genomic demethylation. These results suggested a close correlation between methylation and expression of NtGPDL upon abiotic stresses with a cause-effect relationship. Since DNA methylation is linked to histone modification, it is conceivable that demethylation at coding regions might induce alteration of chromatin structure, thereby enhancing transcription. We propose that environmental responses of plants are partly mediated through active alteration of the DNA methylation status.