Evaluation of Three-Dimensional Bioprinted Human Cartilage Powder Combined with Micronized Subcutaneous Adipose Tissues for the Repair of Osteochondral Defects in Beagle Dogs.

Cartilage lesions are difficult to repair due to low vascular distribution and may progress into osteoarthritis. Despite numerous attempts in the past, there is no proven method to regenerate hyaline cartilage. The purpose of this study was to investigate the ability to use a 3D printed biomatrix to repair a critical size femoral chondral defect using a canine weight-bearing model. The biomatrix was comprised of human costal-derived cartilage powder, micronized adipose tissue, and fibrin glue. Bilateral femoral condyle defects were treated on 12 mature beagles staged 12 weeks apart. Four groups, one control and three experimental, were used. Animals were euthanized at 32 weeks to collect samples. Significant differences between control and experimental groups were found in both regeneration pattern and tissue composition. In results, we observed that the experimental group with the treatment with cartilage powder and adipose tissue alleviated the inflammatory response. Moreover, it was found that the MOCART score was higher, and cartilage repair was more organized than in the other groups, suggesting that a combination of cartilage powder and adipose tissue has the potential to repair cartilage with a similarity to normal cartilage. Microscopically, there was a well-defined cartilage-like structure in which the mid junction below the surface layer was surrounded by a matrix composed of collagen type I, II, and proteoglycans. MRI examination revealed significant reduction of the inflammation level and progression of a cartilage-like growth in the experimental group. This canine study suggests a promising new surgical treatment for cartilage lesions.

Moscatilin Induces Apoptosis in Human Head and Neck Squamous Cell Carcinoma Cells via JNK Signaling Pathway.

Dendrobii Herba is an herbal medicine that uses the stems of Dendrobium species (Orchidacea). It has been traditionally used to treat fever, hydrodipsomania, stomach disorders, and amyotrophia. In our previous study, a bibenzyl compound, moscatilin, which is isolated from Dendrobii Herba, showed potent cytotoxicity against a FaDu human pharyngeal squamous carcinoma cell line. Prompted by this finding, we performed additional studies in FaDu cells to investigate the mechanism of action. Moscatilin induced FaDu cell death by using 5 µM of concentration and by mediating apoptosis, whereas cell proliferation following treatment with 1 µM of moscatilin was not suppressed to the same levels as by the anti-cancer agent, cisplatin. Apoptosis-related protein expression (cleaved caspase-8, cleaved caspase-7, cytochrome c, cleaved caspase-9, cleaved caspase-3, and poly (ADP-ribose) polymerase (PARP) was increased by treating with 5 µM of moscatilin. This suggests that moscatilin-mediated apoptosis is associated with the extrinsic and intrinsic apoptotic signaling pathways. In addition, moscatilin-induced apoptosis was mediated by the c-Jun N-terminal kinase (JNK) signaling pathway. Overall, this study identified additional biological activity of moscatilin derived from natural products and suggested its potential application as a chemotherapeutic agent for the management of head and neck squamous cell carcinoma.

Comparative Analysis of Phytochemical Composition of Gamma-Irradiated Mutant Cultivars of Chrysanthemum morifolium.

The flowers of chrysanthemum species are used as a herbal tea and in traditional medicine. In addition, members of the genus have been selected to develop horticultural cultivars of diverse floral colors and capitulum forms. In this research, we investigated the phytochemical composition of eight gamma-irradiation mutant cultivars of Chrysanthemum morifolium and their original cultivars. The mutant chrysanthemum cultivars were generated by treatment with various doses of 60Co gamma irradiation of stem cuttings of three commercial chrysanthemum cultivars as follows: 'ARTI-Dark Chocolate' (50Gy), 'ARTI-Purple Lady' (30 Gy), and 'ARTI-Yellow Star' (50 Gy) derived from 'Noble Wine'; 'ARTI-Red Star' (50 Gy) and 'ARTI-Rising Sun' (30 Gy) from 'Pinky'; 'ARTI-Purple' (40 Gy) and 'ARTI-Queen' (30 Gy) from 'Argus'; and 'ARTI-Rollypop' (70 Gy) from 'Plaisir d'amour'. Quantitative analysis of flavonoids, phenolic acids, anthocyanins, and carotenoids in the flowers of the 12 chrysanthemum cultivars was performed using high performance liquid chromatography-diode array detector-electrospray ionization mass spectrometry (HPLC-DAD-ESIMS). Essential oils from the flowers of these cultivars were analyzed by gas chromatography-mass spectrometry (GC-MS). The mutant cultivars, 'ARTI-Dark Chocolate', 'ARTI-Purple Lady', 'ARTI-Purple', and 'ARTI-Queen' showed higher total amounts of flavonoid and phenolic acid compared with those of the respective original cultivars. The mutant cultivars, 'ARTI-Dark Chocolate', 'ARTI-Purple Lady' and 'ARTI-Purple', which produce purple to pink petals, contained more than two-times higher amounts of anthocyanins compared with those of their original cultivars. Of the mutant cultivars, 'ARTI-Yellow Star' in which petal color was changed to yellow, showed the greatest accumulation of carotenoids. Ninety-nine volatile compounds were detected, of which hydrocarbons and terpenoids were abundant in all cultivars analyzed. This is the first report that demonstrated the phytochemical analysis of novel chrysanthemum cultivars derived from C. morifolium hydrid using HPLC-DAD-ESIMS and GC-MS. These findings suggest that the selected mutant chrysanthemum cultivars show potential as a functional source of phytochemicals associated with the abundance of health-beneficial components, as well as good source for horticulture and pigment industries.

Phytochemical Composition and Antioxidant Activities of Two Different Color Chrysanthemum Flower Teas.

Chrysanthemum morifolium Ramat is a perennial flowering plant widely cultivated for use in a tea infusion and as a popular beverage. To identify and evaluate the tea infusion made with a γ-irradiated mutant chrysanthemum cultivar with dark purple petals (cv. ARTI-Dark Chocolate), its phytochemical composition and antioxidant activity were tested and compared with those of the commercially available chrysanthemum cultivar with yellow petals (cv. Gamguk) by HPLC-DAD-ESIMS, as well as DPPH and ABTS radical scavenging assays. The purple chrysanthemum tea contained anthocyanins and linarin, which were not detected in the yellow chrysanthemum tea and the content of chlorogenic acid, acacetin-7-O-ß-glucoside, and luteolin was higher compared with the yellow chrysanthemum tea. In contrast, the yellow chrysanthemum tea had higher luteolin-7-O-ß-glucoside, 3,5-dicaffeoylquinic acid, apigenin-7-O-ß-glucoside, and apigenin contents in comparison with the purple chrysanthemum tea. In addition, the content and antioxidant activity of the two chrysanthemum teas were investigated according to different water temperatures and infusing time. The yellow chrysanthemum tea did not show any significant differences according to infusing time and temperature, while the purple chrysanthemum tea was more influenced by the infusing time than water temperature, showing the highest total compound content in the infusing condition of 100 °C and 4 min. Moreover, the floral scent volatiles of the two chrysanthemum tea sources were analyzed using HS-SPME-GC-MS. In the DPPH radical scavenging assay, the purple chrysanthemum tea broadly showed greater antioxidant activity than did the yellow chrysanthemum tea, corresponding to the high content of anthocyanins known as the powerful antioxidant. Further, both chrysanthemum flower teas exhibited strong ABTS radical scavenging effects ranging from 76% to 61% under all infusing conditions. Therefore, the purple chrysanthemum cultivar, ARTI-Dark Chocolate, is worthy of breeding as a new tea cultivar.

Identification of Two New Phenanthrenes from Dendrobii Herba and Their Cytotoxicity towards Human Hypopharynx Squamous Carcinoma Cell (FaDu).

Two new phenanthrenes, (1R,2R)-1,7-hydroxy-2,8-methoxy-2,3-dihydrophenanthrene-4(1H)-one (1) and 2,7-dihydroxy-phenanthrene-1,4-dione (2), were isolated from the ethyl acetate-soluble fraction of Dendrobii Herba, together with seven known phenanthrenes (3-9), two bibenzyls (10-12), and a lignan (13). Structures of 1 and 2 were elucidated by analyzing one-dimensional (1D) and two-dimensional (2D)-NMR and High-resolution electrospray ionization mass spectra (HR-ESI-MS) data. The absolute configuration of compound 1 was confirmed by the circular dichroism (CD) spectroscopic method. In cytotoxicity assay using FaDu human hypopharynx squamous carcinoma cell line, compounds 3-6, 8, 10, and 12 showed activities, with IC50 values that ranged from 2.55 to 17.70 µM.

Isoegomaketone Alleviates the Development of Collagen Antibody-Induced Arthritis in Male Balb/c Mice.

In this study, we attempted to identify and assess effects of isoegomaketone (IK) isolated from Perilla frutescens var. crispa on the development of rheumatoid arthritis (RA). RA was induced in male Balb/c mice by collagen antibody injection. Experimental animals were randomly divided into five groups: normal, collagen antibody-induced arthritis (CAIA), CAIA + IK (5 mg/kg/day), CAIA + IK (10 mg/kg/day), and CAIA + apigenin (16 mg/kg/day) and respective treatments were administered via oral gavage once per day for four days. Mice treated with IK (10 mg/kg/day) developed less severe arthritis than the control CAIA mice. Arthritic score, paw volume, and paw thickness were less significant compared to the control CAIA mice at day seven (73%, 15%, and 14% lower, respectively). Furthermore, histopathological examination of ankle for inflammation showed that infiltration of inflammatory cells and edema formation were reduced by IK treatment. Similarly, neutrophil to lymphocyte ratio (NLR) in whole blood was lower in mice treated with IK (10 mg/kg/day) by 85% when compared to CAIA mice. Taken together, treatment with IK delays the onset of the arthritis and alleviates the manifestations of arthritis in CAIA mice.

Comparison of the Anti-Inflammatory Activities of Supercritical Carbon Dioxide versus Ethanol Extracts from Leaves of Perilla frutescens Britt. Radiation Mutant.

In this study, we aimed to compare supercritical carbon dioxide extraction and ethanol extraction for isoegomaketone (IK) content in perilla leaf extracts and to identify the optimal method. We measured the IK concentration using HPLC and inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells from the extracts. The IK concentration was 10-fold higher in perilla leaf extracts by supercritical carbon dioxide extraction (SFE) compared with that in perilla leaf extracts by ethanol extraction (EE). When the extracts were treated in LPS-induced RAW 264.7 cells at 25 µg/mL, the SFE inhibited the expression of inflammatory mediators such as nitric oxide (NO), monocyte chemoattractant protein-1 (MCP-1), interleutkin-6 (IL-6), interferon-ß (IFN-ß), and inducible nitric oxide synthase (iNOS) to a much greater extent compared with EE. Taken together, supercritical carbon dioxide extraction is considered the optimal process for obtaining high IK content and anti-inflammatory activities in leaf extracts from the P. frutescens Britt. radiation mutant.

A New Monoterpene from the Leaves of a Radiation Mutant Cultivar of Perilla frutescens var. crispa with Inhibitory Activity on LPS-Induced NO Production.

The leaves of Perilla frutescens var. crispa (Lamiaceae)-known as 'Jureum-soyeop' or 'Cha-jo-ki' in Korean, 'ZI SU YE' in Chinese, and 'Shiso' in Japan-has been used as a medicinal herb. Recent gamma irradiated mutation breeding on P. frutescens var. crispa in our research group resulted in the development of a new perilla cultivar, P. frutescens var. crispa (cv. Antisperill; PFCA), which has a higher content of isoegomaketone. The leaves of PFCA were extracted by supercritical carbon dioxide (SC-CO2) extraction, and phytochemical investigation on this extract led to the isolation and identification of a new compound, 9-hydroxy-isoegomaketone [(2E)-1-(3-furanyl)-4-hydroxy-4-methyl-2-penten-1-one; 1]. Compound 1 exhibited inhibitory activity on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated RAW264.7 cells with an IC50 value of 14.4 µM. The compounds in the SC-CO2 extracts of the radiation mutant cultivar and the original plant were quantified by high-performance liquid chromatography with diode array detection.

Characterization of the complete chloroplast genome of Amsonia elliptica (Apocynaceae).

Amsonia elliptica (Apocynaceae), endangered species in Korea, is a perennial herb that is economically important as traditional medicine and used as ornamentals. Natural populations of this species are facing extinction due to small population size and isolated distribution. Here, we report the complete chloroplast (cp) genome of A. elliptica using Illumina HiSeq sequencing and its phylogenetic position in subfamily Rauvolfioideae based on 20 Apocynaceae cp genomes. The cp genome of A. elliptica was 154,242 bp in length with a pair of inverted repeats of 25,711 bp, separated by large single-copy and small single-copy regions of 85,382 bp and 17,438 bp, respectively. Our phylogenomic analyses revealed that A. elliptica was closely related to Rhazya stricta in Rauvolfioideae (Apocynaceae).

Content of Two Major Steroidal Glycoalkaloids in Tomato (Solanum lycopersicum cv. Micro-Tom) Mutant Lines at Different Ripening Stages.

Esculeoside A and tomatine are two major steroidal alkaloids in tomato fruit (Solanum lycopersicum) that exhibit anti-inflammatory, anticancer, and anti-hyperlipidemia activities. Tomatine contained in immature tomato fruit is converted to esculeoside A as the fruit matures. To develop new tomato varieties based on the content analysis of functional secondary metabolites, 184 mutant lines were generated from the original cultivar (S. lycopersicum cv. Micro-Tom) by radiation breeding. Ultra-performance liquid chromatography coupled with evaporative light scattering detector was used to identify the mutant lines with good traits by analyzing tomatine and esculeoside A content. Compared with the original cultivar, candidates for highly functional cultivars with high esculeoside A content were identified in the mature fruit of the mutant lines. The mutant lines with low and high tomatine content at an immature stage were selected as edible cultivars due to toxicity reduction and as a source of tomatine with various pharmacological activities, respectively. During the process of ripening from green to red tomatoes, the rate of conversion of tomatine to esculeoside A was high in the green tomatoes with a low tomatine content, whereas green tomatoes with a high tomatine content exhibited a low conversion rate. Using methanol extracts prepared from unripe and ripe fruits of the original cultivar and its mutant lines and two major compounds, we examined their cytotoxicity against FaDu human hypopharynx squamous carcinoma cells. Only tomatine exhibited cytotoxicity with an IC50 value of 5.589 µM, whereas the other samples did not exhibit cytotoxicity. Therefore, radiation breeding represents a useful tool for developing new cultivars with high quality, and metabolite analysis is applicable for the rapid and objective selection of potential mutant lines.

Isoegomaketone from Perilla frutescens (L.) Britt Stimulates MAPK/ERK Pathway in Human Keratinocyte to Promote Skin Wound Healing.

Skin wound healing is essential for recovery from injury, and delayed or impaired wound healing is a severe therapeutic challenge. Keratinocytes, a major component of the epidermis, play crucial roles in reepithelialization during wound healing including cell proliferation. Recent studies have shown that compounds from natural products have candidates for healing skin injury. Isoegomaketone (IK), isolated from leaves of Perilla frutescens var. crispa (Lamiaceae), has various bioactivities. However, the effect of IK on cutaneous wound healing processes has not been studied yet. In this study, we demonstrated that IK exhibits therapeutic wound healing effects using the human keratinocyte cell line HaCaT. Notably, IK promoted cell proliferation and migration in a dose-dependent manner in vitro, and treatment with 10 µM IK upregulated these processes by approximately 1.5-fold after 24 h compared with the control. IK induced the activation of the MAPK/ERK pathway and cell cycle progression to the S and G2/M phases. Thus, this study demonstrates IK as a potential candidate to upregulate wound healing that may provide therapeutic benefits to patients with delayed wound healing.

9­Hydroxy­isoegomaketone inhibits LPS­induced NO and inflammatory cytokine production in RAW264.7 cells.

Isoegomaketone (IK) is a known component of Perilla frutescens that reportedly exhibits anti­inflammatory, anti­cancer and anti­allergic properties. A novel compound known as 9­HIK has been isolated from the extract of a radiation mutant P. frutescens var. crispa using supercritical carbon dioxide. In the present study, 9­HIK induced heme oxygenase­1 (HO­1) mRNA expression in RAW264.7 cells, with maximal levels observed 4 h after 9­HIK treatment. In addition, 9­HIK inhibited the mRNA and protein expression of pro­inflammatory mediators, such as IL­6 and interferon­ß, as well as the production of nitric oxide (NO) in lipopolysaccharide­stimulated RAW264.7 cells. Furthermore, N­acetyl­L­cysteine, a reactive oxygen species scavenger, inhibited NO production and HO­1 mRNA expression levels through the nuclear factor erythroid 2­related factor 2 pathway. Overall, 9­HIK displayed anti­inflammatory properties in LPS­induced RAW264.7 cells via direct suppression of inflammatory mediators and HO­1 induction.

Chemical and Biological Profiles of Dendrobium in Two Different Species, Their Hybrid, and Gamma-Irradiated Mutant Lines of the Hybrid Based on LC-QToF MS and Cytotoxicity Analysis.

The Dendrobium species (Orchidaceae) has been cultivated as an ornamental plant as well as used in traditional medicines. In this study, the chemical profiles of Dendrobii Herba, used as herbal medicine, Dendrobium in two different species, their hybrid, and the gamma-irradiated mutant lines of the hybrid, were systematically investigated via ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QToF MS). Among the numerous peaks detected, 17 peaks were unambiguously identified. Gigantol (1), (1R,2R)-1,7-hydroxy-2,8-methoxy-2,3-dihydrophenanthrene-4(1H)-one (2), tristin (3), (-)-syringaresinol (4), lusianthridin (5), 2,7-dihydroxy-phenanthrene-1,4-dione (6), densiflorol B (7), denthyrsinin (8), moscatilin (9), lusianthridin dimer (10), batatasin III (11), ephemeranthol A (12), thunalbene (13), dehydroorchinol (14), dendrobine (15), shihunine (16), and 1,5,7-trimethoxy-2-phenanthrenol (17), were detected in Dendrobii Herba, while 1, 2, and 16 were detected in D. candidum, 1, 11, and 16 in D. nobile, and 1, 2, and 16 in the hybrid, D. nobile × candidum. The methanol extract taken of them was also examined for cytotoxicity against FaDu human hypopharynx squamous carcinoma cells, where Dendrobii Herba showed the greatest cytotoxicity. In the untargeted metabolite analysis of 436 mutant lines of the hybrid, using UPLC-QToF MS and cytotoxicity measurements combined with multivariate analysis, two tentative flavonoids (M1 and M2) were evaluated as key markers among the analyzed metabolites, contributing to the distinction between active and inactive mutant lines.

Preparative Separation of Three Monoterpenes from Perilla frutescens var. crispa Using Centrifugal Partition Chromatography.

Three monoterpenes, namely, 9-hydroxy isoegomaketone (1), isoegomaketone (2), and perilla ketone (3), were successfully separated from the supercritical carbon dioxide (SC-CO2) extract of the leaves of Perilla frutescens var. crispa (cv. Antisperill; Lamiaceae) by centrifugal partition chromatography (CPC). To obtain large quantities of these materials required for studies on their mechanism of action and in vivo effectiveness in inflammation, we used CPC because of its high loading capacity and reproducibility to purify the three compounds. Compound 1 (2.60 mg, 96.7% purity at 254 nm) was purified from 500 mg of the SC-CO2 extract of P. frutescens var. crispa (cv. Antisperill), using a two-phase solvent system comprising n-hexane/ethyl acetate/ethanol/water (5:5:5:5 v/v) in a descending mode. As compounds 2 (56.1 mg, 97.6% purity at 254 nm) and 3 (78.6 mg, 96.1% purity at 254 nm) are highly volatile and difficult to recover from an aqueous mobile phase after purification during the drying process, they were obtained from the same amount of the processed extract in an ascending mode using the upper organic phase as the mobile phase (n-hexane/ethyl acetate/ethanol/water, 8:2:8:2 v/v). The structures of compounds 1-3 were confirmed by 1H- and 13C-NMR analysis. Thus, based on our findings, we recommend centrifugal partition chromatography as a powerful technique for purifying the active principal compounds 1 and 2 from the leaves of P. frutescens var. crispa.

In Vitro and In Silico Insights into sEH Inhibitors with Amide-Scaffold from the Leaves of Capsicum chinense Jacq.

Two compounds termed 1 and 2 were isolated from the leaves of Capsicum chinense using column chromatography. Their structures were identified as amide scaffolds by analyzing spectroscopic signals. Compounds 1 and 2 have been confirmed to be competitive soluble epoxide hydrolase (sEH) inhibitors that suppress the catalytic reaction of sEH in a dose-dependent manner in vitro. Molecular docking was used for analyzing two binding clusters of ligand and receptor. The results confirmed that the key amino acids interacting with the ligand were Asp335, Tyr383, and Gln384. On the basis of molecular dynamics, inhibitors 1 and 2 were noted to interact at a distance of 3.5 Šfrom Asp335, Tyr383, Leu408 and Tyr466, and Asp335, Tyr383, and Tyr466, respectively. These results highlight the potential of N-trans-coumaroyltyramine (1) and N-trans-feruloyltyramine (2) as sEH inhibitors.

Quantification of Antioxidant Phenolic Compounds in a New Chrysanthemum Cultivar by High-Performance Liquid Chromatography with Diode Array Detection and Electrospray Ionization Mass Spectrometry.

The flowers of Chrysanthemum morifolium Ramat. have been used as an herbal tea and in traditional medicine, and the plant has been developed to produce horticultural cultivars of various colors and shapes. In this study, a new chrysanthemum cultivar with dark purple petals (C. morifolium cv. ARTI-Dark Chocolate; ADC) was developed by radiation-induced mutation breeding of its original cultivar with purple striped white petals (C. morifolium cv. Noble Wine, NW). The phenolic profile and antioxidant property of ADC were investigated and compared with NW and the commercially available medicinal herb, C. morifolium with yellow petals (CM), in order to find a scientific support to produce a new source of natural antioxidant. Flavonoid and phenolic acid profiles of the ethanol extracts of the three flowers were analyzed by high-performance liquid chromatography-diode array detector-electrospray ionization mass spectrometry (HPLC-DAD-ESIMS), while antioxidant properties were evaluated using the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging assay. Among the tested flowers, ADC possessed the strongest antioxidant capacity and the highest phenolic contents. Flavonoids (acacetin, apigenin, luteolin, acacetin-7-O-ß-glucoside, apigenin-7-O-ß-glucoside, luteolin-7-O-ß-glucoside, and linarin) and phenolic acids (chlorogenic acid and mixture of 1,4-, 1,5-, and 3,5-dicaffeoylquinic acids) were identified and quantified.