No-ozone cold plasma induces apoptosis in human neuroblastoma cell line via increased intracellular reactive oxygen species (ROS).

BACKGROUND: This study aimed to evaluate the effect of argon-based No-ozone Cold Plasma (NCP) on neuroblastoma cancer cell apoptosis. METHODS: Experiments were performed with SK-N-SH and HS 68. Cell cultures were treated with NCP for 1, 3, and 5 min. NCP was applied using three different strategies: direct NCP application to cell cultures, to only media, and to only cells. Evaluation of cell viability and the level of the reactive oxygen species (ROS) was performed. N-acetyl-L-cysteine (NAC) was also used to antagonize intracellular ROS. Cleaved caspase 3, PARP, aquaporin (AQP) 3 and 8 were detected. RESULTS: NCP induced a gradual decrease in the SK-N-SH cell viability. In contrast, the viability of HS 68 cells did not change. SK-N-SH cells viability was reduced the most when the only media-NCP application strategy was employed. Intracellular ROS levels were significantly increased with time. Cleaved caspase 3 and PARP were increased at 6 h after NCP application. SK-N-SH cells remained viable with NAC after NCP application. AQP 3 and 8 were over-expressed in SK-N-SH cells. CONCLUSION: These findings demonstrate the anti-cancer effect of NCP on neuroblastoma cells. NCP enhanced the selective apoptosis of neuroblastoma cells due to the increased intracellular ROS.

Antiviral Effects of Secondary Metabolites from Jatropha podagrica Leaves against the Pseudotyped Virus of SARS-CoV-2 Omicron.

Jatropha podagrica holds a longstanding place in traditional herbal medicine, primarily utilized for addressing skin infections, acting as antipyretics, diuretics, and purgatives. In this study, our primary objective was to investigate the secondary metabolites present in J. podagrica leaves, with the aim of pinpointing natural compounds exhibiting potential antiviral activities. Five secondary metabolites (1-5), including an auronol glycoside (1), two coumarins (2 and 3), a chromane (4) and a gallotannin (5), were isolated from J. podagrica leaves. Compound 1 presented as an amalgamation of unseparated mixtures, yet its intricate composition was adroitly unraveled through the strategic deployment of a chiral HPLC column. This tactic yielded the isolation of epimers (+)-1 and (-)-1, ascertained as unreported auronol glycosides. The structures of these novel compounds, (+)-1 and (-)-1, were elucidated to be (2S)-hovetrichoside C [(+)-1] and (2R)-hovetrichoside C [(-)-1] through NMR data and HR-ESIMS analyses, enzymatic hydrolysis, and comparison of optical rotation values. Cytotoxicity and antiviral effects were assessed for the isolated compounds ((+)-1, (-)-1 and 2-5), along with compound 1a (the aglycone of 1), in the A549 human alveolar basal epithelial cell line. Each compound demonstrated a cell viability of approximately 80% or higher, confirming their non-toxic nature. In the group of compounds, compounds 3-5 demonstrated antiviral effects based on RT-qPCR results, with individual enhancements ranging from approximately 28 to 38%. Remarkably, compound 4 exhibited the most substantial antiviral effect. Utilization of compound 4 to assess immune boosting and anti-inflammatory effects revealed increased levels of STING, RIG-I, NLRP3, and IL-10 along with a decrease in TNF-α and IL-6. Therefore, these findings underscore the potential of these active compounds 3-5 not only as therapeutic agents for SARS-CoV-2 but also as new contenders for upcoming pandemics.

N,N-Dimethyl-anthranilic Acid from Calvatia nipponica Mushroom Fruiting Bodies Induces Apoptotic Effects on MDA-MB-231 Human Breast Cancer Cells.

Breast cancer ranks among the most prevalent malignancies affecting women worldwide, and apoptosis-targeting drugs are attractive candidates for the treatment of cancer. In the current study, we investigated the in vitro cytotoxicity of the mushroom Calvatia nipponica in human breast cancer cells (MDA-MB-231), identified potential antitumor compounds through bioactivity-guided isolation, and elucidated the antitumor, pro-apoptotic molecular mechanisms of the identified bioactive compounds. C. nipponica is edible when young, and it has been used as a food source as well as a traditional medicine in wound dressings. However, only a limited number of studies have reported its chemical composition and biological activities. In the screening test, the methanol extract of C. nipponica fruiting bodies exhibited cytotoxicity against MDA-MB-231 cells. Bioactivity-guided fractionation of the methanol (MeOH) extract and chemical investigation of the active fractions resulted in the isolation of fourteen compounds (1-14), including six alkaloids (1-3, 5, 7, and 8), two phenolic compounds (4 and 6), one fatty acid (9), and five steroids (10-14). The structures of the isolated compounds were determined using NMR spectroscopic methods, liquid chromatography-mass spectrometry, and comparison of data with previously reported values. The isolated compounds (1-14) were tested for cytotoxicity against MDA-MB-231 cells, where compound 1, i.e., N,N-dimethyl-anthranilic acid, exhibited the most significant cytotoxicity against MDA-MB-231 cells, with an IC50 value of 90.28 ± 4.23 µM and apoptotic cell death of 56.01% ± 2.64% at 100 µM. Treatment with compound 1 resulted in an upregulation of protein levels, including cleaved caspase-8, cleaved poly (ADP-ribose) polymerase, Bcl-2-associated X protein (Bax), cleaved caspase-3, cleaved caspase-9, Bad, and Cytochrome c, but decreased the levels of B-cell lymphoma 2 (Bcl-2). Overall, these results indicate that N,N-dimethyl-anthranilic acid (1) may have anti-breast cancer activity and is probably involved in the induction of apoptosis mediated by extrinsic and intrinsic signaling pathways.

Enhanced Expression of p53 and Suppression of PI3K/Akt/mTOR by Three Red Sea Algal Extracts: Insights on Their Composition by LC-MS-Based Metabolic Profiling and Molecular Networking.

Brown algae comprise up to 2000 species with wide dissemination in temperate zones. A comprehensive untargeted metabolic profiling guided by molecular networking of three uninvestigated Red-Sea-derived brown algae, namely Sirophysalis trinodis, Polycladia myrica, and Turbinaria triquetra, led to the identification of over 115 metabolites categorized as glycerolipids, fatty acids, sterol lipids, sphingolipids, and phospholipids. The three algae exhibited low-to-moderate antioxidant capacity using DPPH and ABTS assays. Preliminary in vitro antiproliferative studies showed that the algal extracts displayed high cytotoxic activity against a panel of cancer cell lines. The most potent activity was recorded against MCF-7 with IC50 values of 51.37 ± 1.19, 63.44 ± 1.13, and 59.70 ± 1.22 µg/mL for S. trinodis, P. myrica, and T. triquetra, respectively. The cytotoxicity of the algae was selective to MCF-7 without showing notable effects on the proliferation of normal human WISH cells. Morphological studies revealed that the algae caused cell shrinkage, increased cellular debris, triggered detachment, cell rounding, and cytoplasmic condensation in MCF-7 cancer cells. Mechanistic investigations using flow cytometry, qPCR, and Western blot showed that the algae induced apoptosis, initiated cell cycle arrest in the sub-G0/G1 phase, and inhibited the proliferation of cancer cells via increasing mRNA and protein expression of p53, while reducing the expression of PI3K, Akt, and mTOR.

Methoxyflavones from Black Ginger (Kaempferia parviflora Wall. ex Baker) and their Inhibitory Effect on Melanogenesis in B16F10 Mouse Melanoma Cells.

Kaempferia parviflora Wall. ex Baker (Zingiberaceae), commonly known as Thai ginseng or black ginger, is a tropical medicinal plant in many regions. It has been traditionally used to treat various ailments, including ulcers, dysentery, gout, allergies, abscesses, and osteoarthritis. As part of our ongoing phytochemical study aimed at discovering bioactive natural products, we investigated potential bioactive methoxyflavones from K. parviflora rhizomes. Phytochemical analysis aided by liquid chromatography-mass spectrometry (LC-MS) led to the isolation of six methoxyflavones (1-6) from the n-hexane fraction of the methanolic extract of K. parviflora rhizomes. The isolated compounds were structurally determined to be 3,7-dimethoxy-5-hydroxyflavone (1), 5-hydroxy-7-methoxyflavone (2), 7,4'-dimethylapigenin (3), 3,5,7-trimethoxyflavone (4), 3,7,4'-trimethylkaempferol (5), and 5-hydroxy-3,7,3',4'-tetramethoxyflavone (6), based on NMR data and LC-MS analysis. All of the isolated compounds were evaluated for their anti-melanogenic activities. In the activity assay, 7,4'-dimethylapigenin (3) and 3,5,7-trimethoxyflavone (4) significantly inhibited tyrosinase activity and melanin content in IBMX-stimulated B16F10 cells. In addition, structure-activity relationship analysis revealed that the methoxy group at C-5 in methoxyflavones is key to their anti-melanogenic activity. This study experimentally demonstrated that K. parviflora rhizomes are rich in methoxyflavones and can be a valuable natural resource for anti-melanogenic compounds.

Estrogenic Activity of 4-Hydroxy-Benzoic Acid from Acer tegmentosum via Estrogen Receptor α-Dependent Signaling Pathways.

Acer tegmentosum, a deciduous tree belonging to Aceraceae, has been used in traditional oriental medicine for treating hepatic disorders, such as hepatitis, cirrhosis, and liver cancer. We evaluated the estrogen-like effects of A. tegmentosum using an estrogen receptor (ER)-positive breast cancer cell line, namely MCF-7, to identify potential phytoestrogens and found that an aqueous extract of A. tegmentosum promoted cell proliferation in MCF-7 cells. Five phenolic compounds (1-5) were separated and identified from the active fraction using bioassay-guided fractionation of crude A. tegmentosum extract and phytochemical analysis. The chemical structures of the compounds were characterized as vanillic acid (1), 4-hydroxy-benzoic acid (2), syringic acid (3), isoscopoletin (4), and (E)-ferulic acid (5) based on the analysis of their nuclear magnetic resonance spectra and liquid chromatography-mass spectrometry data. All five compounds were evaluated using an E-screen assay for their estrogen-like effects on MCF-7 cells. Among the tested compounds, only 4-hydroxy-benzoic acid (2) promoted the proliferation of MCF-7 cells, which was mitigated by the ER antagonist, ICI 182,780. The mechanism underlying the estrogen-like effect of 4-hydroxy-benzoic acid (2) was evaluated via western blotting analysis to determine the expression levels of extracellular signal-regulated kinase (ERK), phosphoinositide 3-kinase (PI3K), serine/threonine kinase (AKT), and ERα. Our results demonstrated that 4-hydroxy-benzoic acid (2) induced the increase in the protein expression levels of p-ERK, p-AKT, p-PI3K, and p-Erα, concentration dependently. Collectively, these experimental results suggest that 4-hydroxy-benzoic acid (2) is responsible for the estrogen-like effects of A. tegmentosum and may potentially aid in the control of estrogenic effects during menopause.

Bioactive Phytochemicals from Salix pseudolasiogyne Twigs: Anti-Adipogenic Effect of 2'-O-Acetylsalicortin in 3T3-L1 Cells.

Salix pseudolasiogyne (Salicaceae) is a willow tree and has been used as a medicinal herb in Korea to treat pain and fever. As a part of an ongoing study to identify bioactive natural products, potential anti-adipogenic compounds were investigated using the ethanol (EtOH) extract of S. pseudolasiogyne twigs. Phytochemical investigation of the EtOH extracts using liquid chromatography-mass spectrometry (LC/MS) led to the separation of two compounds, oregonin (1) and 2'-O-acetylsalicortin (2). The structures of the isolates were identified using nuclear magnetic resonance spectroscopy and LC/MS analysis. To the best of our knowledge, it is the first report identifying oregonin (1) in twigs of S. pseudolasiogyne. Here, we found that the isolated compounds, oregonin (1) and 2'-O-acetylsalicortin (2), showed anti-adipogenic effects during 3T3-L1 cell differentiation. Notably, 2'-O-acetylsalicortin (2), at a concentration of 50 µM, significantly suppressed lipid accumulation. Moreover, the mRNA and protein levels of lipogenic and adipogenic transcription factors were reduced in 2'-O-acetylsalicortin (2)-treated 3T3-L1 cells. Taken together, these results indicate that 2'-O-acetylsalicortin (2), isolated from S. pseudolasiogyne twigs, has the potential to be applied as a therapeutic agent to effectively control adipocyte differentiation, a critical stage in the progression of obesity.

Effect of No-ozone Cold Plasma on Regeneration after Crushed Mental Nerve Injury in rats.

Background: This experimental research aimed to determine whether No-ozone Cold Plasma (NCP) has regenerative effect on crushed injured sensory nerves in a rat model (Wistar A) and to evaluate whether NCP can be used as an alternative treatment method for sensory nerve injury in the oral-maxillofacial region. Methods: A total of 10 Wistar A rats were used for this experiment. They were divided into three groups according to whether the mental nerve of the left mandible was injured and NCP was applied or not: group 1 (n=3) (non-mental nerve damage, non-MD) - the left mental nerve was exposed and non-damaged; group 2 (n=3) (mental nerve damage, MD) - the left mental nerve was exposed and damaged, NCP was not applied; and group 3 (n=4) (mental nerve damage and NCP, MD-NCP) - the left mental nerve was exposed and damaged, NCP was applied with regular intervals (three times a week). Results: For the behavior analysis, von Frey test was used. Furthermore, the nerve tissues were examined with hematoxylin and eosin (H&E) staining, and the extent of neurorecovery was evaluated with the immunofluorescence staining of certain markers. The behavioral analysis showed that the function recovery sensory nerve was faster in group 3 (MD-NCP). In the histomorphologic and immunofluorescence analyses, the expression of the factors involved in neurorecovery was much higher in group 3 than in group 2 (MD). Conclusions: The expeditious recovery of sensory nerve function as well as the higher expression of the factors indicating nerve function recovery in the NCP-treated group suggest that NCP has a positive effect on regeneration after sensory nerve crushing injury. Therefore, in the case of sensory impairment of the oral-maxillofacial region, no-ozone cold plasma can be applied for therapeutic effect.

Non-thermal plasma directly accelerates neuronal proliferation by stimulating axon formation.

Among the various methods, Non Thermal Plasma (NTP) has been recently introduced and is being studied to recover the damaged nerve. In the recent years, several studies have suggested that NTP accelerates nerve cell regeneration, but the mechanism remains unknown. This study evaluated the effect of NTP on neuronal proliferation in SH-SY5Y (Human neuroblastoma cells) cells differentiated by retinoic acid (RA) and investigated the mechanism by which NTP promotes cell proliferation. We analyzed the morphology of differentiated SH-SY5Y cells, and performed western blot analysis and reverse transcription polymerase chain reaction (RT-PCR). Immunofluorescence analysis was performed in an in vivo study by categorizing Wistar A rats into three groups: non-nerve damage (Non-ND), nerve damage (ND), and nerve damage + NTP treatment (ND + NTP). The cell morphology analysis revealed that the number of cells increased and axonal elongation progressed after NTP treatment. In addition, western blots indicated that tau expression increased significantly after NTP treatment. The RT-PCR results revealed that the expression of tau, wnt3a, and ß-catenin increased after NTP treatment. The in vivo immunofluorescence assay showed that NTP increased the markers for tau and S100B while regulating the over-expression of MAP2 and GAP43. NTP treatment accelerated cell proliferation and regeneration of damaged neurons in differentiated SH-SY5Y cells. These results establish the fact of NTP as a noninvasive and effective treatment for nerve injury.

Phytochemical Investigation of Marker Compounds from Indigenous Korean Salix Species and Their Antimicrobial Effects.

Salix species, including willow trees, are distributed in the temperate regions of Asian countries, including South Korea. Willow trees are used to treat pain and inflammatory diseases. Due to the medicinal properties of willow trees, pharmacological studies of other Salix spp. have gained attention; however, only a few studies have investigated the phytochemicals of these species. As part of our ongoing natural product research to identify bioactive phytochemicals and elucidate their chemical structures from natural resources, we investigated the marker compounds from indigenous Korean Salix species, namely, Salix triandra, S. chaenomeloides, S. gracilistyla, S. koriyanagi, S. koreensis, S. pseudolasiogyne, S. caprea, and S. rorida. The ethanolic extract of each Salix sp. was investigated using high-performance liquid chromatography combined with thin-layer chromatography and liquid chromatography−mass spectrometry-based analysis, and marker compounds of each Salix sp. were isolated. The chemical structures of the marker compounds (1−8), 3-(4-hydroxyphenyl)propyl ß-D-glucopyranoside (1), 2-O-acetylsalicin (2), 1-O-p-coumaroyl glucoside (3), picein (4), isograndidentatin B (5), 2'-O-acetylsalicortin (6), dihydromyricetin (7), and salicin (8) were elucidated via nuclear magnetic resonance spectroscopy and high-resolution liquid chromatography−mass spectrometry using ultrahigh-performance liquid chromatography coupled with a G6545B Q-TOF MS system with a dual electrospray ionization source. The identified marker compounds 1−8 were examined for their antimicrobial effects against plant pathogenic fungi and bacteria. Dihydromyricetin (7) exhibited antibacterial activity against Staphylococcus aureus, inducing 32.4% inhibition at a final concentration of 125 µg/mL with an MIC50 value of 250 µg/mL. Overall, this study isolated the marker compounds of S. triandra, S. chaenomeloides, S. gracilistyla, S. koriyanagi, S. koreensis, S. pseudolasiogyne, S. caprea, and S. rorida and identified the anti-Staphylococcus aureus bacterial compound dihydromyricetin.

Non-thermal plasma application enhances the recovery of transected sciatic nerves in rats.

This experimental research aimed to investigate the effects of non-thermal plasma on nerve regeneration after transected nerve damage using the sciatic nerve in Wistar albino (A) rats. The experiments were performed on 27 Wistar A rats. The rats underwent surgery for right sciatic nerve exposure and were divided into three groups (each group, n = 9) according to sciatic nerve transected injury (SNTI) and non-thermal plasma application: a non-nerve damage (non-ND) group, a only nerve damage without non-thermal plasma application (ND) group, and a nerve damage with non-thermal plasma application (ND + NTP) group. Subsequent to SNTI and immediate suture, non-thermal plasma was administered three times per week for eight weeks. Evaluation for functional recovery was performed using the static sciatic index measured over the full treatment period of eight weeks. The sciatic nerve specimens were obtained after euthanasia and third day from the last non-thermal plasma application. The sciatic nerve tissues were subjected to histological analysis. Behavior analysis presented that the ND + NTP group showed improved static sciatic index compared with the nerve damage group. Histopathological findings demonstrated that the ND + NTP group had more dense Schwann cells and well-established continuity of nerve fibers, greater than the nerve damage group. Immunohistochemistry showed that the ND + NTP group had increased levels of markers for microtubule-associated protein 2 (MAP2), tau, S100 calcium-binding protein B, and neurofilament-200 and regulated the overexpression of CD68 and MAP2. These results indicated that non-thermal plasma enhanced the motor function and restored the neuronal structure by accelerating myelination and axonal regeneration. Additionally, non-thermal plasma was confirmed to have a positive effect on the recovery of SNTI in rats.

Non-thermal plasma accelerates the healing process of peripheral nerve crush injury in rats.

The objective of this study was to evaluate the effect of non-thermal plasma (NTP) on the healing process of peripheral nerve crush injuries, which can occur during dental implant procedures. For this, a rat model of sciatic nerve crush injury (SNCI) was adopted. The rats were divided into three groups: non-nerve damage (non-ND), nerve damage (ND), and ND+NTP group. To evaluate the sciatic nerve (SN) function, the static sciatic index was calculated, and the muscle and SN tissues were subjected to a histologic analysis. The results showed that NTP effectively accelerated the healing process of SNCI in rats. In contrast to the ND group, which showed approximately 60% recovery in the SN function, the NTP-treated rats showed complete recovery. Histologically, the NTP treatments not only accelerated the muscle healing, but also reduced the edema-like phenotype of the damaged SN tissues. In the ND group, the SN tissues had an accumulation of CD68-positive macrophages, partially destroyed axonal fibers and myelinated Schwann cells. Conversely, in the ND+NTP group, the macrophage accumulation was reduced and an overall regeneration of the damaged axon fibers and the myelin sheath was accomplished. The results of this study indicate that NTP can be used for healing of injured peripheral nerves.

Whitlockite Granules on Bone Regeneration in Defect of Rat Calvaria.

Whitlockite (WH; Ca18Mg2(HPO4)2(PO4)12) is a calcium phosphate based ceramic that contains magnesium ions. As the second most abundant mineral in living bone, WH occupies 25-35 wt % of the inorganic portion of human bone. Compared to hydroxyapatite (HAp, Ca10(PO4)6(OH)2), WH possesses better mechanical properties, faster resorbability, and promotion behavior on the osteogenesis. In this article, we introduced a fabrication method of interconnected porous WH granules through vacuum filtration, followed by sintering treatment based on the thermal stability of WH synthesized using the tri-solvent system. This study presents a histological, radiological, and immunohistochemical evaluation of the bone healing potential of these WH granules in a 5 mm diameter calvarial bone defect in rats. The histological evaluation shows no inflammation or foreign body reaction in the WH group. The WH group displays newly formed bone at the same thickness as the original bone. On the contrary, bone formation is not observed in the nontreated (NT) group. Besides, immunohistochemistry (IHC) confirmed that WH granules promoted bone regeneration with the significantly higher expression of bone morphogenetic proteins-2 (BMP-2), alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) compared to the NT group without the addition of exogenous cells or growth factors. These results suggest that WH has excellent potential for application in bone tissue regeneration.