New Flavone C-Glycosides from Scleranthus perennis and Their Anti-Collagenase Activity

Three new flavone glycosides, one known flavone glycoside, and the phenolic derivative apiopaenonside were isolated and identified from the ethyl acetate fraction of the aerial parts of Scleranthus perennis. The planar structures were elucidated through extensive analysis of UV-Vis, IR, and 1H NMR and 13C NMR spectral data, including the 2D techniques COSY, HSQC, and HMBC, as well as ESI mass spectrometry. The isolated compounds were established as 5,7,3′-trihydroxy-4′-acetoxyflavone-8-C-β-d-xylopyranoside-2′′-O-glucoside (1), 5,7,3′-trihydroxy-4′-methoxyflavone-8-C-β-d-xylopyranoside-2′′-O-glucoside (2), 5,7-dihydroxy-3′-methoxy-4′-acetoxyflavone-8-C-β-d-xylopyranoside-2′′-O-glucoside (3), 5,7-dihydroxy-3′-methoxy-4′-acetoxyflavone-8-C-β-d-xylopyranoside-2′′-O-(4′′′-acetoxy)-glucoside (4), and apiopaenonside (5). Moreover, all isolated compounds were evaluated for anti-collagenase activity. All compounds exhibited moderate inhibitory activity with IC50 values ranging from 36.06 to 70.24 µM.

Screening Collagenase Activity in Bacterial Lysate for Directed Enzyme Applications

Collagenases are essential enzymes capable of digesting triple-helical collagen under physiological conditions. These enzymes play a key role in diverse physiological and pathophysiological processes. Collagenases are used for diverse biotechnological applications, and it is thus of major interest to identify new enzyme variants with improved characteristics such as expression yield, stability, or activity. The engineering of new enzyme variants often relies on either rational protein design or directed enzyme evolution. The latter includes screening of a large randomized or semirational genetic library, both of which require an assay that enables the identification of improved variants. Moreover, the assay should be tailored for microplates to allow the screening of hundreds or thousands of clones. Herein, we repurposed the previously reported fluorogenic assay using 3,4-dihydroxyphenylacetic acid for the quantitation of collagen, and applied it in the detection of bacterial collagenase activity in bacterial lysates. This enabled the screening of hundreds of E. coli colonies expressing an error-prone library of collagenase G from C. histolyticum, in 96-well deep-well plates, by measuring activity directly in lysates with collagen. As a proof-of-concept, a single variant exhibiting higher activity than the starting-point enzyme was expressed, purified, and characterized biochemically and computationally. This showed the feasibility of this method to support medium-high throughput screening based on direct evaluation of collagenase activity.

Tonsil-derived mesenchymal stem cells enhance allogeneic bone marrow engraftment via collagen IV degradation

Background Co-transplantation of bone marrow cells (BMCs) and mesenchymal stem cells (MSCs) is used as a strategy to improve the outcomes of bone marrow transplantation. Tonsil-derived MSCs (TMSCs) are a promising source of MSCs for co-transplantation. Previous studies have shown that TMSCs or conditioned media from TMSCs (TMSC-CM) enhance BMC engraftment. However, the factors in TMSCs that promote better engraftment have not yet been identified. Methods Mice were subjected to a myeloablative regimen of busulfan and cyclophosphamide, and the mRNA expression in the bone marrow was analyzed using an extracellular matrix (ECM) and adhesion molecule-targeted polymerase chain reaction (PCR) array. Nano-liquid chromatography with tandem mass spectrometry, real-time quantitative PCR, western blots, and enzyme-linked immunosorbent assays were used to compare the expression levels of metalloproteinase 3 (MMP3) in MSCs derived from various tissues, including the tonsils, bone marrow, adipose tissue, and umbilical cord. Recipient mice were conditioned with busulfan and cyclophosphamide, and BMCs, either as a sole population or with control or MMP3-knockdown TMSCs, were co-transplanted into these mice. The effects of TMSC-expressed MMP3 were investigated. Additionally, Enzchek collagenase and Transwell migration assays were used to confirm that the collagenase activity of TMSC-expressed MMP3 enhanced BMC migration. Results Mice subjected to the myeloablative regimen exhibited increased mRNA expression of collagen type IV alpha 1/2 (Col4a1 and Col4a2). Among the various extracellular matrix-modulating proteins secreted by TMSCs, MMP3 was expressed at higher levels in TMSCs than in other MSCs. Mice co-transplanted with BMCs and control TMSCs exhibited a higher survival rate, weight recovery, and bone marrow cellularity compared with mice co-transplanted with BMCs and MMP3-knockdown TMSCs. Control TMSC-CM possessed higher collagenase activity against collagen IV than MMP3-knockdown TMSC-CM. TMSC-CM also accelerated BMC migration by degrading collagen IV in vitro. Conclusions Collectively, these results indicate that TMSCs enhance BMC engraftment by the secretion of MMP3 for the modulation of the bone marrow extracellular matrix.

Phenolic Compounds from Leaves and Flowers of Hibiscus roseus: Potential Skin Cosmetic Applications of an Under-Investigated Species

The use of plant extracts in skin-care cosmetics is a modern trend due to their richness in polyphenols that act as anti-aging molecules. Hibiscus roseus is a perennial species naturalized in Italy, with beautiful soft pink flowers; its phenolic composition and biological activities have not been studied yet. The aim of this study was to characterize and quantify the phenolics and to evaluate the antioxidant, sun protection factor (SPF), and anti-collagenase activities of the ethanolic extracts of H. roseus leaves (HL) and flowers (HF). p-Coumaric, chlorogenic, and trans-ferulic acids derivatives as well as quercetin and kaempferol flavonoids were the main phenolic compounds detected. Catechin, epicatechin, kaempferol-3-O-rutinoside, kaempferol-3-O-glucoside, kaempferol-7-O-glucoside, tiliroside, oenin, and peonidin-3-O-glucoside were detected only in HF, while phloridzin was exclusive from HL, which also showed greater amounts of hydroxycinnamic acid derivatives. HF was richer in flavonoids and total phenolics, also exhibiting greater antioxidant capacity. The SPF and anti-collagenase activity of both extracts were similar and comparable to those of synthetic standards. The overall results demonstrate that H. roseus extracts are promising sources of bioactive phenolic compounds that could be potentially applied as anti-aging agents in skin-care cosmetics.

Obtusifolin, an Anthraquinone Extracted from Senna obtusifolia (L.) H.S.Irwin & Barneby, Reduces Inflammation in a Mouse Osteoarthritis Model

Osteoarthritis (OA) is an age-related degenerative disease that causes cartilage dysfunction and inflammation. Obtusifolin, an anthraquinone extracted from Senna obtusifolia (L.) H.S.Irwin & Barneby seeds, has anti-inflammatory functions; it could be used as a drug component to relieve OA symptoms. In this study, we investigated the effects of obtusifolin on OA inflammation. In vitro, interleukin (IL)-1β (1 ng/mL)-treated mouse chondrocytes were co-treated with obtusifolin at different concentrations. The expression of matrix metalloproteinase (Mmp) 3, Mmp13, cyclooxygenase 2 (Cox2), and signaling proteins was measured by polymerase chain reaction and Western blotting; collagenase activity and the PGE2 level were also determined. In vivo, OA-induced C57BL/6 mice were administered obtusifolin, and their cartilage was stained with Safranin O to observe damage. Obtusifolin inhibited Mmp3, Mmp13, and Cox2 expression to levels similar to or more than those after treatment with celecoxib. Additionally, obtusifolin decreased collagenase activity and the PGE2 level. Furthermore, obtusifolin regulated OA via the NF-κB signaling pathway. In surgically induced OA mouse models, the cartilage destruction decreased when obtusifolin was administered orally. Taken together, our results show that obtusifolin effectively reduces cartilage damage via the regulation of MMPs and Cox2 expression. Hence, we suggest that obtusifolin could be a component of another OA symptom reliever.

In Vitro Anti-Collagenase Activity and Total Phenolic Content Of Five Selected Herbs: A Review

Etlingera elatior, Persicaria odorata, Centella asiatica, Senna alata and Phyllanthus emblica were reviewed for their in vitro anti-collagenase activity and its total phenolic contents. Plant herbs play a vital role to promote the production of collagen which is the component of the skin. Different solvents during plant extraction and various plant parts were used to determine anti-collagenase properties and total phenolic content. Generally, anti-collagenase assay and Folin-Ciocalteu method were used to determine the anti-collagenase activity and total phenolic content, respectively. The extracts of these herbs have the potential of inhibiting collagenase activity and they possess total phenolic content. The total phenolic was found to be important elements in collagenase inhibition activity other than flavonoid. The selected herbs are popular in Malaysia and this review can be a useful reference or information for future application in pharmaceutical, food and cosmetics fields. Keywords: Anti-collagenase activity, total phenolic contents; plant Extraction; flavonoids

Collagenase and Melanogenesis Inhibitory Effects of Perilla Frutescens Pomace Extract and Its Efficacy in Topical Cosmetic Formulations

In previous studies, Perilla frutescens pomace was shown to contain bioactive phenolic compounds and good anti-oxidative activity. However, reports about collagenase activity and melanogenesis inhibitory effects of P. frutescens pomace are limited. This study aimed to evaluate the bioactivity of P. frutescens pomace extract and incorporate the extract into a cosmetic formulation for evaluating its effects on collagenase and melanogenesis inhibition on human skin. The P. frutescens seeds after an oil pressing process were extracted with ethanol (70% v/v) in order to examine the remaining phytochemical compounds, the bioactivity in pomace perilla, and its efficacy as a skincare product. In this study, total phenolic and total flavonoid contents of P. frutescens seed extract (PFSE) were determined using spectrophotometry. The free radical scavenging activity was determined with 2, 2′-diphenyl-1-picrylhydrazyl (DPPH) radicals and 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) ABTS radicals. Additionally, the effects on collagenase activity, melanin content, and alpha-Melanocyte stimulating hormone (α-MSH) on the viability of cultured B16F10 melanoma cells were investigated. Skin irritation and efficacy of PFSE cream for skin elasticity and skin color were also clinically evaluated. The total phenolic content with gallic acid equivalents (GAE) value and total flavonoids content with catechin equivalents (CE) value were, respectively, 92.79 ± 1.19 and 56.02 ± 2.83 mg/g. Furthermore, PFSE significantly inhibited the collagenase activity (p < 0.001) at the concentration of 400 µg/mL (82 ± 3.23%). These results clearly demonstrated the anti-melanogenic effects on B16F10 cells without causing any cytotoxicity or death. Although there was a slight improvement in skin elasticity in the 4th week compared to the previous week, the 4th week melanin content of the skin significantly decreased from the beginning (p < 0.05) without any irritations. In conclusion, PFSE could be cosmetically considered as a key ingredient that effectively lessens the effects of skin aging and skin hyperpigmentation disorders.