The influence of cultivation conditions on the formation of psychoactive salvinorin A, salvinorin B, rosmarinic acid and caffeic acid in Coleus scutellarioides.

Coleus scutellarioides (L.) Benh. is a popular species in the world, known for its characteristic magnificent colourful leaves. The study has revealed that the contents of rosmarinic acid and caffeic acid are significantly higher in the plant tissues cultivated in vivo than when under in vitro conditions. The performed qualitative and quantitative analyses confirmed the presence (whose averaged content) of salvinorin A (6.65 µg/1 g of fresh plant) and salvinorin B (50.46 µg/1 g of fresh plant) in tissues of Coleus scutellarioides (L.) Benh. of 'Electric lime' variety. The greatest quantities of these compounds were recorded for plants cultivated in vitro on the MS medium enriched with NAA (naphthyl-1-acetic acid) at a concentration of 0.5 mg∙ dm-3. The research detected differences in the amounts of compounds between plants grown in vivo and those cultivated in vitro. Addition of plant growth regulators into the breeding medium under in vitro conditions was found affecting the amounts of compounds in plant tissues.

Coleus vettiveroides ethanolic root extract induces cytotoxicity by intrinsic apoptosis in HepG2 cells.

Hepatocellular carcinoma (HCC) contributes to more than 80% of all primary cancers globally and ranks fourth in cancer-related deaths, due to the lack of an effective, definite therapeutic drug. Coleus vettiveroides (CV) has been used in Indian traditional medicine to treat diabetes, liver ailments, skin diseases, leukoderma, and leprosy. This study investigates the anticancer effect of CV ethanolic root extract in HepG2 cells. HepG2 cells were treated with CV extract, and its cytotoxicity was analyzed by MTT assay. AO/EB staining, propidium iodide staining, DCFH-DA assay, phalloidine staining, flow cytometry, and qPCR studies were performed for ROS expression, apoptosis and cell cycle analysis. The phytochemical analysis confirmed the presence of quercetin and galangin in CV root extract. The results showed that CV inhibited the proliferation of HepG2 cells, with altered cellular and nuclear morphology. CV was also found to increase intracellular ROS levels and oxidative stress markers in HepG2 cells. CV significantly altered the actin microfilament distribution in HepG2 cells and caused cell cycle arrest at the sub G0 -G1 phase. CV also induced mitochondria-mediated apoptosis, as evidenced by increased expression of p53, Bax, cytochrome C, Apaf-1, PARP, caspase-3 and caspase-9, and downregulated Bcl-2 expression. Therefore, CV exerts its anticancer effect by inducing mitochondrial dysfunction, oxidative stress, cytoskeletal disorganization, cell cycle arrest, and mitochondria-mediated apoptosis, and it could be a potent therapeutic option for HCC.

Insights into the antimicrobial efficacy of Coleus aromaticus essential oil against food-borne microbes: Biochemical and molecular simulation approaches.

The study reported the antimicrobial efficacy of chemically characterized Coleus aromaticus essential oil (CEO) against food-borne bacteria, molds (Aspergillus flavus), aflatoxin B1 (AFB1) and explored its mechanism of action using biochemical and molecular simulation approaches. The chemical profile of CEO was explored by Gas chromatography-mass spectrometry (GC-MS) analysis, which revealed thymol (46.0%) as the major compound. The minimum inhibitory concentration values of CEO for bacterial species Escherichia coli, Salmonella enterica, Bacillus cereus, and Shigella flexneri was found to be 0.9 µl/ml, 0.7 µl/ml, 0.16 µl/ml, and 0.12 µl/ml respectively. The MIC value for A. flavus and AFB1 contamination was 0.6 µl/ml. The DPPH radical scavenging activity of CEO was recorded with IC50 0.32 µl/ml. Biochemical and computational approaches (docking and dynamics simulation) have been performed to explore the multi-faceted antimicrobial inhibitory effects of CEO at the molecular level, which shows the impairment in membrane functioning, leakage of cellular contents, release of 260-nm absorbing materials, antioxidative defense, carbon catabolism and vital genes (7AP3, Nor1, Omt1, and Vbs). The findings indicated that CEO could be used as natural antimicrobial agents against food-spoilage bacteria, A. flavus and AFB1 contamination to extend the shelf-life of food product and prevention of food-borne diseases.

Intelligent packaging based on chitosan/fucoidan incorporated with coleus grass (Plectranthus scutellarioides) leaves anthocyanins and its application in monitoring the spoilage of salmon (Salmo salar L.).

The innovation of this study was to develop a novel biodegradable intelligent packaging based on chitosan/fucoidan combined with different amounts (1, 3 and 5 wt% on chitosan basis) of coleus grass (Plectranthus scutellarioides) leaves anthocyanins (CGL) to monitor the spoilage of salmon (Salmo salar L.). The addition of fucoidan improved the barrier and mechanical properties of the chitosan films (CS) due to hydrogen bonds and intermolecular electrostatic interactions. Moreover, the addition of CGL not only improved the physical properties but also improved the biological activity of chitosan/fucoidan film (CF). The DPPH and ABTS radical scavenging activity of CF contained 5 wt% CGL was 1.83 and 1.75 times than CF, respectively. The inhibition zone size of CF films containing 5 wt% CGL (CF-5%CGL) was approximately 2.04 (Escherichia coli) and 2.16 (Staphylococcus aureus) times higher than that of CF. Moreover, CF-CGL displayed obvious color changes in different pH environments and is highly sensitive to ammonia gas. The CF-CGL has visible color changes during the monitoring of salmon spoilage and extended the shelf life of salmon. According to our findings, CF-CGL film might be employed as a possible intelligent packaging material for monitoring and preserving salmon in the future.

Isolation, cloning, and functional analysis of a putative constitutive promoter of E3 ubiquitin-protein ligase RF4 from Coleus amboinicus Lour.

A promoter is a region in the genome sequence located upstream of the transcription start site comprising cis acting elements that initiates and regulates the transcription of an associated genes and restriction endonucleases. As the need for genetically engineered plants has widened, the requirement to develop methods to optimize the control of transgene expression has also increased. Therefore, analyzing the functionality of the promoter is very important in understanding the target gene expression. The widespread use of viral constitutive promoters (cauliflower mosaic virus, CaMV35) has raised concerns about the safety and containment of transgene in the environment. Hence isolation and characterization of novel promoters using fast and efficient genetic engineering tools is the need of the hour. The present study, for the first time, describes the isolation and characterization of a novel constitutive promoter driving ubiquitin E3 ligase from the plant Coleus amboinicus, a perennial herb, of the Lamiaceae family. The functionality of the isolated promoter was demonstrated using the ß -glucuronidase as a reporter in tobacco var Petit havana. The development of blue color in the tobacco leaves indicated the presence of a functional promoter.

Antibacterial, antifungal, antidiabetic, and antioxidant activities potential of Coleus aromaticus synthesized titanium dioxide nanoparticles.

The nanoparticles based drug delivery and treatment related research has been increased significantly in the recent years. Hence, the antibacterial, antifungal, and antioxidant activity potential of pre synthesized and characterized Titanium dioxide nanoparticles (TiO2 NPs) were investigated in this study through respective standard protocols. Interestingly, the obtained results revealed that TiO2 NPs have concentration dependent antibacterial activity against bacterial pathogens such as E. coli, P.mirabilis, V. cholerae, P. aeruginosa, S. typhimurium, and S. aureus at 100 µg mL-1 concentration. Furthermore, these TiO2 NPs showed remarkable antifungal activity against aspergillosis causing fungal pathogens such as A. niger, A. fumigatus, A. nidulans, and A. flavus at 100 µg mL-1 concentration. α-glucosidase. This TiO2 NPs also effectively inhibit the α-amylase (17%) and α-Glucosidase (37%) enzyme activity at 100 µg mL-1 dosage. The DPPH assay revealed that TiO2 NPs effectively scavenge DPPH free radicals by up to 89% at 100 µg mL-1 concentration, which was comparable to butylated hydroxytoluene (96%). These results suggest that the plant-based TiO2 NPs have remarkable in-vitro antibacterial, antifungal, and antioxidant activity. These may be considered for additional in-vitro and in-vivo experiments to assess their potential biomedical applications.

Coleus Amboinicus Lour. Leaf Extract as an Antioxidant in Sepsis.

Background: As broad-spectrum antibiotics can cause antimicrobial resistance in sepsis, there is the need for a complementary therapy to combat sepsis. Oxidative stress causes an increased severity and mortality in sepsis, whereas herbal medicines have been considered as an option due to its antioxidant potential. Coleus amboinicus Lour. has been documented for its therapeutic value due to the presence of flavonoid, an antioxidant compound. Objective: To study the effect of Coleus amboinicus Lour. leaf extract on total antioxidant capacity (TAC) and hepatic catalase (CAT) levels in septic rat model. Methods: Twenty-eight male Rattus norvegicus rats were divided into four groups: control (rats without sepsis induction and treatment), group 1 (septic rats treated with antibiotics), group 2 (septic rats treated with antibiotics and 250 mg/kg body weight of Coleus amboinicus Lour. leaf extract), and group 3 (septic rats treated with antibiotics and 500 mg/kg body weight of Coleus amboinicus Lour. leaf extract). The rats were sacrificed at the end of the eighth day of observation, and blood and liver tissues were gathered for examination. Results: Compared to the septic rat groups treated with only antibiotics, there was an increase in the TAC levels and CAT expression levels in septic rat groups given antibiotics and Coleus amboinicus Lour. leaf extract. However, the increase was not significant. Conclusion: Administering Coleus amboinicus Lour. leaf extract increases TAC levels and CAT expression levels in sepsis, decreasing oxidative stress. This will exert protective effects in the cells and therefore alleviate sepsis.

Ethanolic extract of Coleus aromaticus leaves impedes the proliferation and instigates apoptotic cell death in liver cancer HepG2 cells through repressing JAK/STAT cascade.

Liver cancer or hepatocellular carcinoma (HCC) has become a leading cause for cancer burden across the globe, and incidences have tripled since the last two decades. Poor diagnosis of primary liver cancer and limited treatment strategies aggravate the challenges. Researchers globally have shown a steep inclination toward the exploration of plant-based compounds for their nutraceutical and anticancer potential to fit into the role of novel chemotherapeutics. Coleus aromaticus is a well-known culinary herb that earlier has been reported for several medicinal attributes. The current investigation deals with exploring the anticancer potential of ethanolic leaf extract of C. aromaticus (CoL-EtOH) against hepatocellular carcinoma HepG2 cell line. The observations made it evident that CoL-EtOH extract impeded the viability of HepG2 at 400 µg/ml (p < .01). Additionally, the extract also succeeded in escalating ROS production (p < .01) which aided dissipation of mitochondrial membrane potential and disruption of nuclear morphology. CoL-EtOH further activated caspase-8, -9, and -3 which was reaffirmed by increase in apoptosis at 400 µg/ml (p < .01). Moreover, post treatment with CaLEt-OH extract significantly reduced the expression of JAK-1 & STAT-3 genes (p < .01) along with regulated expression of Mcl1, Bcl-2, cyclinD1, p21, and p27 within HepG2 cells. This evidence portrays the promising anticancer potential of CoL-EtOH projecting it as a novel chemotherapeutic agent against HCC. PRACTICAL APPLICATIONS: The herb Coleus aromaticus belonging to Lamiaceae family and Coleus genus is known by various names in different regions of the world and several language-specific vernacular names. The herb has been used in therapeutic and medicinal applications as well as in culinary preparations. Various attributes of the nutritional strength and functional characteristics of the leaves in terms of carotenoids, minerals, phenols, dietary fiber, and antioxidant activity have been reported by several researchers. Carvacrol and thymol are majorly found in the plant, while chlorogenic acid and rosmarinic acid etc. as the phenolic components. The herb has been used in therapeutic and medicinal implications as well as in culinary preparations.

Development of a multianalytical strategy for detection of frauds in Coleus forskohlii supplements.

A new multianalytical methodology based on gas chromatography (GC) and liquid chromatography (LC) coupled to mass spectrometry (MS) has been proposed to evaluate frauds affecting the composition of Coleus forskohlii root supplements (FKS). After optimization and validation of chromatographic methods, 24 FKS were analyzed. Forskolin, their main bioactive component, was only found in 50% of the FKS evaluated (in the 0.032-17.1% range), with 27% of these supplements showing concentrations of this bioactive lower than those declared in their labels. Application of this methodology also proved to be successful for the detection of frauds regarding the replacement of C. forskohlii by other vegetable sources (green tea, soy leaves and a plant of the Berberidaceae family) in 17% of supplements analyzed. A study on stability of forskolin under accelerated conditions allowed to rule out its degradation as responsible for the lack of this bioactive or other natural constituents in 25% of FKS evaluated. It can be concluded that the multianalytical methodology here developed is an advantageous alternative to address the wide diversity of frauds affecting these supplements.

Study on Chemotypic Variability of Coleus forskohlii Briq., Samples Collected from Different Phytogeographical Locations of India and Evaluation of Its Inhibitory Potential.

Coleus forskohlii Briq. is an important medicinal herb, endowed with a wide range of medicinal properties against the variety of ailments. Seven germplasm of C. forskohlii collected from different phyto-geographical locations and identification of elite chemotype was performed with the help of high performance thin layer chromatography. Data of soil analysis correlated with the bioactive compounds and inhibitory potential of the species. Quantification of forskolin and its isomer (iso-forskolin) content were done in all the collected samples of C. forskohlii, which revealed a wide range of variations, varying from 1.15-0.004% and 0.0091 to 0.1077% per dry weights basic, respectively. Variation in the bioactive content may be due to the soil nature and environmental factors. Soil analysis of collected samples demonstrated that there is significant variation in available NPK and micronutrient content and may be reasoned for existing chemotypic variability. In vitro biological activity (antioxidant and antidiabetic) analyses were performed, which reveals that germplasms have a high amount of forskolin and iso-forskolin, both show more activity. The aim of this study was to elucidate the effect of elicitors and precursors on the production of bioactive compounds and identification of best elite germplasm among the populations, to provide basic lead to the industry for commercial exploitability including its location-specific commercial cultivation.

Differences in diterpenoid diversity reveal new evidence for separating the genus Coleus from Plectranthus.

Covering: up to 2019The large and medicinally important tropical plant genus Plectranthus (Lamiaceae) was recently split into three separate genera on the basis of molecular and morphological evidence; Plectranthus sensu stricto, Coleus and Equilabium. We found striking differences between the diterpenoids which strongly support this taxonomic split. Coleus is characterised by abietanes oxygenated at C-14 such as royleanones, spirocoleons and acylhydroquinones, which could be useful chemotaxonomic markers to distinguish this genus from Plectranthus s.s. In contrast, the abietanes in Plectranthus s.s. lack C-14 oxygenation, but are often acylated with unusual acids. Equilabium species do not seem to produce diterpenoids. The structures of the nearly 240 abietanes so far reported from Coleus and Plectranthus and their distribution are presented. The aim of this Highlight is to provide an overview of the differences in diterpenoid diversity between these newly defined genera, which are relevant to predict which previously understudied species could hold untapped potential for their medicinal and other economic uses, and to underpin future research on how these plants have evolved to synthesise distinct abietane types.

Quantitative dissection of color patterning in the foliar ornamental coleus.

Coleus (Coleus scutellarioides) is a popular ornamental plant that exhibits a diverse array of foliar color patterns. New cultivars are currently hand selected by both amateur and experienced plant breeders. In this study, we reimagine breeding for color patterning using a quantitative color analysis framework. Despite impressive advances in high-throughput data collection and processing, complex color patterns remain challenging to extract from image datasets. Using a phenotyping approach called "ColourQuant," we extract and analyze pigmentation patterns from one of the largest coleus breeding populations in the world. Working with this massive dataset, we can analyze quantitative relationships between maternal plants and their progeny, identify features that underlie breeder-selections, and collect and compare public input on trait preferences. This study is one of the most comprehensive explorations into complex color patterning in plant biology and provides insights and tools for exploring the color pallet of the plant kingdom.

Isoforskolin, an adenylyl cyclase activator, attenuates cigarette smoke-induced COPD in rats.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by limited airflow due to pulmonary and alveolar abnormalities from exposure to cigarette smoke (CS). Current therapeutic drugs are limited and the development of novel treatments to prevent disease progression is challenging. Isoforskolin (ISOF) from the plant Coleus forskohlii is an effective activator of adenylyl cyclase (AC) isoforms. Previously we found ISOF could attenuate acute lung injury in animal models, while the effect of ISOF on COPD has not been elucidated. PURPOSE: In this study, we aimed to evaluate the efficacy of ISOF on COPD and reveal its potential mechanisms. METHODS: A rat model of COPD was established by long-term exposure to CS, then the rats were orally administered with ISOF (0.5, 1 and 2 mg/kg). The pulmonary function, lung morphology, inflammatory cells and cytokines in serum or bronchoalveolar lavage fluid (BALF) were evaluated. Transcriptomics, proteomics and network pharmacology analysis were utilized to identify potential mechanisms of ISOF. Droplet digital PCR was used to detect the mRNA expression of AC1-10 in donor lung tissues. AC activation was determined in recombinant human embryonic kidney 293 (HEK293) cells stably expressing human AC isoforms. In addition, ISOF caused trachea relaxation ex vivo were assessed in isolated trachea rings from guinea pigs. RESULTS: ISOF significantly ameliorated pathological damage of lung tissue and improved pulmonary function in COPD rats. ISOF treatment decreased the number of inflammatory cells in peripheral blood, and also the levels of pro-inflammatory cytokines in serum and BALF. Consistent with omics-based analyses, ISOF markedly downregulated the mTOR level in lung tissue. Flow cytometry analysis revealed that ISOF treatment reduced the ratio of Th17/Treg cells in peripheral blood. Furthermore, the expression levels of AC1 and AC2 are relatively higher than other AC isoforms in normal lung tissues, and ISOF could potently activate AC1 and AC2 in vitro and significantly relax isolated guinea pig trachea. CONCLUSION: Collectively, our studies suggest that ISOF exerts its anti-COPD effect by improving lung function, anti-inflammation and trachea relaxation, which may be related to AC activation, mTOR signaling and Th17/Treg balance.

Phytochemical Profile and Antioxidant Activities of Coleus amboinicus Lour. Cultivated in Indonesia and Poland.

Coleus amboinicus Lour., Lamiaceae, is a perennial herb that is native to Indonesia and also cultivated in Africa, Asia and Australia. The major phytochemicals responsible for its bioactivity are rosmarinic acid (RA) and its analogues, flavonoids and abietane diterpenoids. The possibility of cultivation in a colder climate would extend the use of this herb and provide new opportunities to herb growers and livestock farmers. Our study to compare feed value and phytochemical composition of C. amboinicus plants cultivated in its original region, Indonesia, and in Poland. The crude protein content was significantly higher in plants cultivated in Indonesia compared to those cultivated in Poland-21% and 13% of dry matter, respectively. The higher ADF contents were detected in C. amboinicus cultivated in Indonesia, 38-41%, in comparison to 34% in plants cultivated in Poland. The phytochemical composition was also significantly influenced by the cultivation location. Polish samples were higher in polyphenols (RA and its analogues), and also had 1.5-2-fold higher antioxidant potential, as measured by DPPH scavenging, phosphomolybdenum reduction and Fenton reaction driven lipid peroxidation. The Indonesian samples contained more diterpenoid compounds, such as dihydroxyroyleanone, and the sum of terpenoids was ca. 10 times higher than in samples from Poland (15.59-23.64 vs. 1.87 µg/g of extracts). In conclusion, C. amboinicus is suitable for cultivation in non-optimal climatic conditions but some nutritional properties and bioactivity are significantly affected.

Synthesis and characterization of TiO2 NPs by aqueous leaf extract of Coleus aromaticus and assess their antibacterial, larvicidal, and anticancer potential.

The frequent applications of synthetic chemical insecticides and drugs create resistance among insects and microbes, creating a new threat to human and environmental welfare. This investigation focused on evaluating the possibilities of fabricating and characterizing the titanium dioxide nanoparticles (TiO2 NPs) from titanium dioxide (TiO2) through the aqueous leaf extract of Coleus aromaticus. Their biological applications were studied against the larvae of Aedes aegypti human pathogenic bacteria, and cancer cell line. The results revealed that the aqueous leaf extract had the metal reducing proficiency to produce nanoparticles from TiO2. The synthesized TiO2 NPs were initially confirmed by visible color changes and Ultraviolet-Visible Spectrophotometer analysis that showed a predominant peak at 332 nm. Furthermore, the nanocrystals, structural alignment, functional groups and elemental compositions were studied by following standard operating protocol in XRD (X-ray Powder Diffraction), FTIR (Fourier Transform Infrared Spectroscopy), TEM (Transmission Electron Microscopy), and EDX (Energy-Dispersive X-ray Spectroscopy) techniques, respectively. The results attained from these techniques confirmed that the plant mediated and fabricated particles were in the nanoscale range (12-33 nm) with a hexagonal shape. The synthesized TiO2 NPs had an outstanding (1000 µg mL-1) larvicidal activity against the four stages of instars larvae of Ae. aegypti at 1000 µg mL-1. It also had an excellent antibacterial potential against E. faecalis (33 mm), followed by S. boydii (30 mm) at 30 mg L-1 concentration. The green fabricated TiO2 NPs had a fabulous (92.37%) cytotoxic activity on the HeLa cell line at 100 µg mL-1 dosage within one day of exposure. The entire results concluded that the C. aromaticus mediated TiO2 NPs have excellent biological applications and thus, could be considered for the welfare of human beings.

Constitutive and facultative crassulacean acid metabolism (CAM) in Cuban oregano, Coleus amboinicus (Lamiaceae).

Plants exhibiting the water-conserving crassulacean acid metabolism (CAM) photosynthetic pathway provide some of the most intriguing examples of photosynthetic diversity and plasticity. Here, a largely unnoticed facet of CAM-plant photosynthesis is highlighted: the co-occurrence of ontogenetically controlled constitutive and environmentally controlled facultative CAM in a species. Both forms of CAM are displayed in leaves of Coleus amboinicus Lour. (Lamiaceae), a semi-succulent perennial plant with oregano-like flavour that is native to southern and eastern Africa and naturalised elsewhere in the tropics. Under well-watered conditions, leaves assimilate CO2 predominantly by the C3 pathway. They also display low levels of CO2 uptake at night accompanied by small nocturnal increases in leaf tissue acidity. This indicates the presence of weakly expressed constitutive CAM. CAM expression is strongly enhanced in response to drought stress. The drought-enhanced component of CAM is reversible upon rewatering and thus considered to be facultative. In contrast to C. amboinicus, the thin-leaved closely related Coleus scutellarioides (L.) Benth. exhibits net CO2 fixation solely in the light via the C3 pathway, both under well-watered and drought conditions. However, low levels of nocturnal acidification detected in leaves and stems indicate that the CAM cycle is present. The highly speciose mint family, which contains few known CAM-exhibiting species and is composed predominantly of C3 species, appears to be an excellent group of plants for studying the evolutionary origins of CAM and for determining the position of facultative CAM along the C3-full CAM trajectory.

Anti-MRSA activity of abietane diterpenes from Coleus blumei Benth.

Two heretofore uncharacterised abietane diterpenes, sincoetsin C (1) and 3-hydroxyspirocoleon 7-O-ß-D-glucoside (4), were isolated from a methanolic extract of Coleus blumei Benth. (Lamiaceae), along with the known compounds, scutellarioidone A (2) and spirocoleon 7-O-ß-D-glucoside (3) using chromatographic techniques. Their structures were determined by 1D and 2D nuclear magnetic resonance including HSQC, HMBC, COSY and NOESY experiments, mass spectrometry (HR-MS) and other spectroscopic methods (UV, IR). Their antibacterial activity against the reference strain of methicillin-resistant Staphylococcus aureus subsp. aureus CCM 4750 (MRSA) was evaluated using optical absorption to obtain quantitative information on their growth. All isolated compounds displayed anti-MRSA 4750 activity at the concentration of 512 µg/mL. Sincoetsin C (1) was the abietane diterpene most active against MRSA 4750, with a minimum inhibitory concentration of 128 µg/mL.

Anticancer Potential of Calli Versus Seedling Extracts Derived from Rosmarinus officinalis and Coleus hybridus.

BACKGROUND: In Saudi Arabia, the incidence and mortality rates of breast cancer are high. Although current treatments are effective, breast cancer cells develop resistance to these treatments. Numerous studies have demonstrated that active compounds in plant extracts, such as the phenolic compound Rosmarinic Acid (RA), exert anti-cancer effects. OBJECTIVE: We investigated the anticancer properties of methanolic crude extracts of seedlings and calli of Rosmarinus officinalis and Coleus hybridus, two Lamiaceae species. METHODS: MCF-7 human breast cancer cells were treated with methanolic crude extracts obtained from plant calli and seedlings generated in vitro, and cell proliferation was evaluated. Transcriptional profiling of the seedling and callus tissues was also conducted. RESULTS: The mRNA expression levels of RA genes were higher in C. hybridus seedlings than in R. officinalis seedlings, as well as in C. hybridus calli than in R. officinalis calli, except for TAT and C4H. In addition, seedling and callus extracts of both R. officinalis and C. hybridus showed anti-proliferative effects against MCF-7 cells after 24 or 48 h of treatment. DISCUSSION: At a low concentration of 10 µg/mL, C. hybridus calli and seedling extracts showed the most significant anti-proliferative effects after 24 and 48 h of exposure (p < 0.01); controls (doxorubicin) also showed significant inhibition, but lesser than that observed with C. hybridus (p < 0.05). Results with R. officinalis callus and seedling extracts did not significantly differ from those with untreated cells. CONCLUSION: Methanolic extracts of R. officinalis and C. hybridus are potentially valuable options for breast cancer treatment.

High-Throughput Sequencing Analysis of Small RNAs Derived from Coleus Blumei Viroids.

Characterization of viroid-derived small RNAs (vd-sRNAs) is important to understand viroid-host interactions; however, vd-sRNAs belonging to the genus Coleviroid are yet to be identified and characterized. Herein, we used coleus plants singly infected with coleus blumei viroid (CbVd)-1, -5, or -6 and doubly infected with CbVd-1 and -5 to identify and analyze their vd-sRNAs. We found sense and antisense vd-sRNAs for CbVd-1, -5 and -6, and 22-nt vd-sRNAs were the most abundant; moreover, the 5'-terminal nucleotides (nts) of CbVd-1, -5, and -6 were biased toward U and C, and sRNAs derived from these three viroids were unevenly distributed along their genomes. We also noted that CbVd-5 and -6 share a fragment that forms the right half of the rod-like secondary structure of these viroids, which implied that they generated almost the same type of vd-sRNAs. This finding indicated that vd-sRNA biogenesis is mainly determined by the primary sequence of their substrates. More importantly, we found two complementary vd-sRNAs (22 nt) that were generated from the central conserved region (CCR) of these three viroids, suggesting an important role of CCR in vd-sRNA biogenesis. In conclusion, our results provide novel insight into the biogenesis of vd-sRNAs and the biological roles of CCR.

Modification of the first constant domain of heavy chain enabled effective folding of functional anti-forskolin antigen-binding fragment for sensitive quantitative analysis.

The assembly between heavy and light chains is a critical step of immunoglobulin (Ig) and fragment antigen-binding (Fab) antibody expression and of their binding activity. The genes encoding Fab were obtained from hybridoma cells secreting monoclonal antibody (MAb, IgG2b) against adenylate cyclase activator forskolin (FOR). The subclass of the first constant domain of heavy chain (CH 1) of IgG2b was modified to IgG1 via overlap extension polymerase chain reaction and expressed via Escherichia coli bacterial system. Since both Fabs (IgG2b and IgG1) were expressed as inclusion bodies, functional analysis was performed after in vitro refolding via stepwise dialysis. The result indicated that the folding efficiency between VH -CH 1 and VL -CL was improved by the CH 1 modification from IgG2b to IgG1 subclass, although their specificity for FOR was not altered. Effective folding of IgG1 was also observed when they were expressed in the hemolymph of silkworm larvae using the Bombyx mori nuclear polyhedrosis virus bacmid system. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was then developed for the determination of FOR using effectively prepared Fab IgG1. The sensitivity of FOR determination was in the range of 3.91-62.5 ng/mL with less than 9% relative standard deviation, implying the sensitive and reliable analysis of developed icELISA. In addition, high accuracy of the icELISA was supported by the results of spiked-and-recovery tests, ranging from 100.2 to 102.3%. Therefore, Fab could be utilized reliably for icELISA instead of the more expensive MAb. Collectively, this approach improved productivity of Fab and reduced the cost of antibody production.