Steroidal Saponins from Water Eggplant (Fruits of Solanum torvum) Exhibit Anti-Epileptic Activity against Pentylenetetrazole-Induced Seizure Model in Zebrafish.

The fruits of Solanum torvum Swartz, a wild relative of eggplant, are consumed as a wild vegetable in tropical regions of Africa, Asia, and South America. In traditional Chinese medicine, it is believed to have anti-inflammatory and sedative effects. In the Philippines, water decoction is used to treat hyperactivity disorder. Twenty-two steroidal saponins were isolated and purified from the fruits grown in Yunnan, China, including six new compounds: torvosides U-Z (1-6). During drying and cooking, the saponins may undergo transformation, resulting in small amounts of sapogenins. These transformations can include dehydration of hydroxyl groups at position C22, formation of double bonds at position 20, 22 or 22, 23, and even formation of peroxide products. Saponin compounds torvoside X (4), torvoside Y (5), torvoside A (7), and (25S)-3-oxo-5α-spirostan-6α-yl-O-ß-d-xylopyranoside (20), which are glycosylated at C-6, showed certain anti-epileptic activity in a pentylenetetrazole-induced zebrafish seizure model. No antiproliferative activity was detected when tested on the cancer cell line HepG2, and no hepatotoxic effect was noted on normal liver cell line LO2.

Dietary supplementation with Weissella cibaria C-10 and Bacillus amyloliquefaciens T-5 enhance immunity against Aeromonas veronii infection in crucian carp (Carassiu auratus).

With the aim to discover novel lactic acid bacteria and Bacillus strains from fish as potential probiotics to replace antibiotics in aquaculture, the present study was conducted to isolate lactic acid bacteria and Bacillus from intestinal tract of healthy crucian carp (Carassiu auratus) and largemouth bass (Micropterus salmoides) and evaluate their resistance against Aeromonas veronii. Based on the evaluation of antibacterial activity and tolerance test, one strain of lactic acid bacteria (Weissella cibaria C-10) and one strain of Bacillus (Bacillus amyloliquefaciens T-5) with strong environmental stability were screened out. The safety evaluation showed that these two strains were non-toxic to crucian carp and were sensitive to most antibiotics. In vivo study, the crucian carps were fed a basal diet supplemented with W. cibaria C-10 (C-10), B. amyloliquefaciens T-5 (T-5) and W. cibaria C-10 + B. amyloliquefaciens T-5 (C-10+T-5), respectively, for 5 weeks. Then, various immune parameters were measured at 35 days of post-feeding. Results showed both probiotics could improve the activities of related immune enzymes, immune factors and non-specific immune antibodies in blood and organs (gill, gut, kidney, liver, and spleen) of crucian carp in varying degrees. Moreover, after 7 days of challenge experiment, the survival rates after challenged with A. veronii of W. cibaria C-10 (C-10), B. amyloliquefaciens T-5 (T-5) and W. cibaria C-10 + B. amyloliquefaciens T-5 (C-10+T-5) supplemented groups to the crucian carps were 20%, 33% and 22%, respectively. Overall, W. cibaria C-10 and B. amyloliquefaciens T-5 could be considered to be developed into microecological preparations for the alternatives of antibiotics in aquaculture.

Essential Oils from Spices Inhibit Cholinesterase Activity and Improve Behavioral Disorder in AlCl3 Induced Dementia.

The chemical compositions of essential oils (EOs) prepared from six spices including cinnamon, amomum tsao-ko, cardamom, amomum, black pepper and white pepper were analyzed by gas chromatography-mass spectrometry (GC/MS), which led to identify almost 200 volatile compounds. All EOs of spices showed cholinesterase inhibitory activity. Among them, pepper EO showed most potent acetylcholinesterase (AChE) inhibitory activity with IC50 values of 8.54 µg/mL (black pepper EO) and 5.02 µg/mL (white pepper EO). Molecular docking and in vitro validation suggested that 3-carene, α-pinene and ß-pinene with IC50 value of 1.73, 2.66, and 14.75 µg/mL, respectively, might be active constituents of spices oil in inhibiting AChE. Furthermore, amomum tsao-ko EO and amomum EO can improve behavioral disorder in dementia zebrafish induced by aluminum trichloride (AlCl3 ).

Isolation, synthesis and absolute configuration of 4,5-dihydroxypiperines improving behavioral disorder in AlCl3-induced dementia.

A pair of stereoisomers of new 4,5-dihydroxypiperine was isolated from P. retrofractum and showed profound activity on AlCl3-induced dementia. In order to determine their absolute configurations and biological activities, all four possible stereoisomers of 4,5-dihydroxypiperine were synthesized from piperidine by Sharpless asymmetric dihydroxylation and Mitsunobu reaction. Their absolute configurations were established as (4R,5R) (1), (4S,5S) (2), (4S,5R) (3) and (4R,5S) (4) by NMR, optical rotation and CD spectra. It is note that only compound 4 improved behavioral disorder in AlCl3-induced dementia. Accordingly, the pair of stereoisomers isolated from P. retrofractum was determined to be (4S,5S) and (4R,5S)-isomers (2 and 4). The ratio of the epimers was present as 1:0.7 (4:2).

Modified Anterior Tibial Artery Perforator-Pedicled Propeller Flap for Soft-Tissue Coverage of the Ankle and Heel.

BACKGROUND: The ankle and heel are challenging regions to reconstruct functionally. Here, we explored the feasibility and clinical outcomes of a modified anterior tibial artery perforator-pedicled propeller flap for the repair of soft-tissue defects of the ankle and heel. PATIENTS AND METHODS: Between January 2013 and December 2015, 12 patients with soft-tissue defects of the ankle and/or heel underwent reconstructive surgery that included our flap technique. The flaps measured 20 × 8 cm to 7 × 4 cm. A hand-held Doppler was used to identify a proper constant perforator in the distal ankle. In each case, the base of the flap was well preserved. The flap was transposed (180° rotation) to reach and cover the defect. RESULTS: The average follow-up time was 13 months (10-28 months). We observed good texture matches and contour in all of the flaps. All patients could walk and wear normal footwear. All but one flap survived completely without complications. Partial loss was observed in one patient, and the necrotic region was healed with secondary intention. CONCLUSION: Our modified anterior tibial artery free-style perforator-pedicled propeller flap provides a novel option for functional ankle and heel reconstruction. LEVEL OF EVIDENCE: Level IV.

Calcimycin Suppresses S100A4 Expression and Inhibits the Stimulatory Effect of Transforming Growth Factor ß1 on Keloid Fibroblasts.

Recent researches have indicated that S100A4 participates in tissue fibrosis, whereas calcimycin inhibits this process as a novel S100A4 transcription inhibitor. However, the relationship and mechanisms between calcimycin and S100A4 in keloid fibroblasts (KFs) remain unknown. The present research was aimed to evaluate the effect of calcimycin on S100A4 expression and pathogenesis in KFs. Keloid fibroblasts were cultured and exposed to different concentrations of calcimycin in the absence or presence of transforming growth factor ß1 (TGF-ß1). The results showed that the expression of S100A4 was significantly increased in keloid derived fibroblasts compared with normal skin fibroblasts. Calcimycin depressed S100A4 in a concentration- and time-dependent manner. Moreover, calcimycin suppressed TGF-ß1-induced collagen type I, fibronectin, and α-smooth muscle actin expression and cell viability in cultured KFs. Furthermore, calcimycin modulated expression of TGF-ß/Smad target genes Smad7 and phosphorylation of TGF-ß1-induced Smad2/3. This research for the first time confirmed the presence of S100A4 in KFs. Calcimycin inhibits the expression of S100A4, as well as KF proliferation and migration and extracellular matrix (ECM) synthesis. Taken together, these results indicate that calcimycin might be a therapeutic candidate to keloid or other related fibrotic disorders.

Sesquiterpenoids from aged Artemisia argyi and their 3D-QSAR for anti-HBV activity.

Artemisia argyi Levl. Et Vant, commonly known as "Chinese Mugwort," has been utilized in traditional Chinese medicine and cuisine for centuries. Aged Chinese Mugwort has been uncovered to possess superior quality and safety, and its ethyl acetate extract has been found to exhibit anti-hepatitis B virus (HBV) activity. In this study, twenty-five sesquiterpenoids were isolated and characterized from three-year-aged A. argyi. Among them, 14 previously undescribed sesquiterpenoids (1-14), featuring double bond oxidation or ring opening. It is hypothesized that during the aging process, sesquiterpenes undergo oxidative transformation of their double bonds to form alcohols due to external factors and inherent properties. The anti-HBV activity and cytotoxicity of all compounds were assessed in vitro using HepG 2.2.15 cells, and their structure-activity relationships were analyzed through three-dimensional quantitative structure-activity relationship (3D-QASR) techniques. The α-methylene-γ-lactone sesquiterpenoid derivatives were discovered to have potent inhibitory activity against HBV. This research may broaden the potential applications of Chinese Mugwort and offer further guidance for its development and utilization as functional food or traditional Chinese medicine.

Pierisformotoxins A-D, polyesterified grayanane diterpenoids from Pieris formosa and their cAMP-decreasing activities.

Four highly acylated diterpenoids, designated as pierisformotoxins A-D (1-4, resp.), along with 26 known compounds, were isolated from the flowers of Pieris formosa. Among them, pierisformotoxins A and B (1 and 2, resp.) were new highly acylated grayanane diterpenoids, of which the five-membered ring A has undergone an oxidative cleavage between C(3) and C(4), followed by lactonization, to give rise to a five-membered lactone ring between C(3) and C(5), differing from the previously reported grayanane diterpenoids with a 5/7/6/5 ring system. Results of the cAMP-regulation-activity assay showed that pierisformotoxin C (3) at 10 µM (inhibitory ratio (IR): 10.1%) or 2 µM (9.8%), and pierisformotoxin B (2) at 50 µM (13.9%) significantly decreased the cAMP level in N1E-115 neuroblastoma cells (p<0.05).

Effect of heat and hypoxia stress on mitochondrion and energy metabolism in the gill of hard clam.

Aquatic animals suffer from heat and hypoxia stress more frequently due to global climate change and other anthropogenic activities. Heat and hypoxia stress can significantly affect mitochondrial function and energy metabolism. Here, the response and adaptation characteristics of mitochondria and energy metabolism in the gill of the hard clam Mercenaria mercenaria under heat (35 °C), hypoxia (0.2 mg/L), and heat plus hypoxia stress (35 °C, 0.2 mg/L) after 48 h exposure were investigated. Mitochondrial membrane potentials were depolarized under environmental stress. Mitochondrial fusion, fission and mitophagy played a key role in maintain mitochondrion function. The AMPK subunits showed different expression under environmental stress. Acceleration of enzyme activities (phosphofructokinase, pyruvate kinase and lactic dehydrogenase) and accumulation of anaerobic metabolites in glycolysis and TCA cycle implied that the anaerobic metabolism might play a key role in providing energy. Accumulation of amino acids might help to increase tolerance under heat and heat combined hypoxia stress. In addition, urea cycle played a key role in amino acid metabolism to prevent ammonia/nitrogen toxicity. This study improved our understanding of the mitochondrial and energy metabolism responses of marine bivalves exposed to environmental stress.

Compounds from Arnebia euchroma and their related anti-HCV and antibacterial activities.

Three new hydroquinone terpenoids with benzogeijerene skeletons, euchroquinols A-C (1- 3), and a new monoterpenylbenzenoid, 9,17-epoxyarnebinol (4), along with five known compounds were isolated from the stem bark of ARNEBIA EUCHROMA. Shikonin (6) exhibited potent anti-HCV activity with a selective index of 43.56, and compounds 1, 6, and des-O-methyllasiodiplodin (7) showed anti-Staphylococcus aureus activity with MICs of 0.5, 0.125, and 0.125 mg/mL, respectively.

Two new grayanane diterpenoids from the flowers of Rhododendron molle.

A phytochemical investigation of the flowers of Rhododendron molle has yielded two new grayanane diterpenoids, rhodomolleins F and G (1, 2). Compounds 1 and 2 possessed an oxo-bridge between C-5 and C-9, and their structures were elucidated on the basis of interpretation of spectroscopic data, including 1D and 2D NMR techniques.

Genome-wide analysis of the hard clam mitogen-activated protein kinase kinase gene family and their transcriptional profiles under abiotic stress.

Mitogen-activated protein kinase kinase (MAPKK) was the hub component of the Mitogen-activated protein kinase (MAPK) signaling pathway and played an important role in the cellular response to environmental stress. In this study, we identified five MmMAPKK genes in hard clam Mercenaria mercenaria and found that all MmMAPKK genes contain a conserved protein kinase domain. The MmMAPKK genes derived from dispersed duplication were unevenly distributed in three chromosomes. Although the genome size was highly variable among different bivalve mollusks, the number of MAPKK genes was relatively stable. Phylogenetic analysis showed that bivalve MAPKK was divided into five clades, and amino acid sequences of MAPKK from the same clade consisted of similar conserved motifs. The syntenic analysis demonstrated that MmMAPKKs had the highest number of homologous gene pairs with Cyclina sinensis. MmMAPKKs were ubiquitously expressed in all examined tissues, and all MmMAPKK genes were highly expressed in the ovary. MmMAPKK genes showed stress-specific expression under envirionmental stress. MmMAPKK7 showed an upregulated in heat and heat plus hypoxia stress while MmMAPKK1 showed an upregulated in hypoxic stress groups. Dynamic changes of MmMAPKK7, MmMAPKK6 and MmMAPKK1 in hemocytes were observed in response to air exposure. MmMAPKK4 significantly downregulated after air exposure for five days. MmMAPKK7 and MmMAPKK6 might participate in adaptation to low salinity stress. Our results provided useful information about MAPKK and laid a foundation for further studies on MAPKK evolution in the bivalve.

Identification and characterization of olfactory receptor genes and olfactory perception in rapa whelk Rapana venosa (Valenciennes, 1846) during larval settlement and metamorphosis.

The rapa whelk Rapana venosa, an economically important marine fishery resource in China but a major invader all over the world, changes from a phytophagous to a carnivorous form following settlement and metamorphosis. However, the low settlement and metamorphosis rates (<1%) of larvae limit the abundance of R. venosa. This critical step (settlement and metamorphosis) remains poorly characterized but may be related to how larvae perceive the presence of shellfish, their new source of food. Here, we report that larvae may use olfactory perception to sense shellfish. Olfactory receptor (OR) genes are involved in odor sensing in animals. We identified a total of 463 OR genes, which could be grouped into nine clades based on phylogenetic analysis. When assessing the attraction of larvae at different developmental stages to oyster odor, R. venosa showed active settlement and metamorphosis behavior only at the J4 stage (competent larva, 1000-1500 µm shell length) and in the presence of shellfish odor at the same time. Expression of OR gene family members differed between stage 2 (four-spiral whorl stage) and stage 1 (single- to three-spiral whorl stage), indicating significant changes in the olfactory system during larval development. These findings broaden our understanding of olfactory perception, settlement, and metamorphosis in gastropods and can be used to improve R. venosa harvesting, as well as the sustainable development and utilization of this resource.

Metabolomics and biochemical assays reveal the metabolic responses to hypo-salinity stress and osmoregulatory role of cAMP-PKA pathway in Mercenaria mercenaria.

Hypo-salinity events frequently occur in marine ecosystem due to persistent rainfall and freshwater inflow, reducing the cytosol osmolarity and triggering cellular stress responses in aquatic organisms. Euryhaline bivalves have developed sophisticated regulatory mechanisms to adapt to salinity fluctuations over a long period of evolution. In this study, we performed multiple biochemical assays, widely targeted metabolomics, and gene expression analysis to investigate the comprehensive metabolic responses to hypo-salinity stress and osmoregulation mechanisms in hard clam Mercenaria mercenaria, which is a euryhaline bivalve species widely cultured in China. During hypo-salinity stress, increased vacuoles appeared in gill filaments. The Na+ and Cl- concentrations in gills significantly decreased because of the up-regulation of Na+/K+-ATPase (NKA) activity. The cAMP content dramatically decreased at 5 d post hypo-salinity stress. Meanwhile, the gene expression levels of adenylate cyclase, proteinkinase A, and sodium and calcium channel proteins were evidently down-regulated, suggesting that cAMP-PKA pathway was inhibited to prevent ambient inorganic ions from entering the gill cells. Antioxidant metabolites, such as serine and Tyr-containing dipeptides, were significantly up-regulated to resist oxidative stress. Glycerolipid metabolism was strengthened to stabilize membrane structure when hypo-salinity stress was prolonged to 5 days. At 1 d post hypo-salinity stress, an increase in alanine and lactate contents marked the initiation of anaerobic metabolism. Acylcarnitines accumulation indicated that fatty acids ß-oxidation was promoted to provide energy for osmoregulation. The potential biomarkers of hypo-salinity stress were identified in hard clams. This study provides novel insights into the metabolic regulatory mechanisms to hypo-salinity stress in euryhaline bivalves.

Lyonin A, a new 9,10-Secograyanotoxin from Lyonia ovalifolia.

Phytochemical studies on the branches and leaves of Lyonia ovalifolia yielded a new grayanane diterpenoid, lyonin A (1), together with two known compounds, secorhodomollolides A and D (2 and 3, resp.). The structure of 1 was elucidated by combination of 1D- and 2D-NMR, and MS analyses. Compound 1 turned out to be a new, highly O-acylated grayanane diterpenoid, of which ring B has undergone an oxidative cleavage between C(9) and C(10), yielding a system differing from the previously reported grayanane type with a 5/7/6/5 ring system. Results of the cAMP regulation activity assay showed that compounds 2 and 3 at 50 µM induced a significantly decreased cAMP level in N1E-115 neuroblastoma cells (p<0.001), indicating neuropharmacological potential.

[Effects of hypoxia on iron metabolism of rat L6 skeletal muscle cells].

The present study was aimed to investigate the effects of hypoxia on iron metabolism of skeletal muscle. Rat L6 skeletal muscle cells were randomly divided into three groups which were exposed to hypoxia (1% O(2)) for 0, 12, 24 h, respectively. Iron isotope tracing method was used to determine iron uptake and release. Iron content of labile iron pool (LIP) was investigated by flow cytometry, and the expressions of transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1), hypoxia-inducible transcription factor-1 (HIF-1) in L6 cells were observed by Western blot. The results showed that, compared with 0-hour hypoxia group, 12-hour hypoxia group exhibited significantly increased iron uptake and LIP (P < 0.05), as well as decreased iron release (P < 0.01). Not only iron uptake and release, but also LIP in 24-hour hypoxia group were significantly decreased, compared with those in 0- and 12-hour hypoxia groups (P < 0.01). The expressions of HIF-1, DMT1 (IRE), DMT1 (non-IRE) and TfR1 in 12-hour hypoxia group were significantly increased compared with those in 0-hour hypoxia group (P < 0.01). On the contrary, the expressions of DMT1 (IRE), DMT1 (non-IRE) and FPN1 in 24-hour hypoxia group were significantly decreased compared with those in the other two groups. However, TfR1 expression in 24-hour hypoxia was higher than those in 0- and 12-hour hypoxia groups (P < 0.05 or P < 0.01). These results suggest hypoxia plays an important role in iron metabolism of skeletal muscle cells. Moderate hypoxia can increase iron uptake and decrease iron release, resulting in higher LIP, but a prolonged hypoxia induces a disordered iron metabolism of skeletal cells.

C21, C22 pregnane glycosides and cytotoxic C27 spriostanol steroids from Asparagus cochinchinesis.

A preliminary chemical investigation on 70% MeOH extract of the roots of Asparagus cochinchinensis resulted in the isolation of nine steroids. These isolates comprised of four new C21 (1-4) and one new pregnane (5) glycosides, and four known C27 (6-9) spirostanol steroids. Their structures were identified via analysis of the spectroscopic data and the results of hydrolytic cleavage. The cytotoxic activities of the compounds were tested toward the human tumor cell line Hela (cervical cancer), and compounds 7 and 8 displayed moderate activity with IC50 values of 35.5 and 39.6 µM, respectively.

New grayanol diterpenoid and new phenolic glucoside from the flowers of Pieris formosa.

A new grayanol diterpenoid, grayanotoxin XXII (1), and a new phenolic glucoside, benzyl 2-hydroxy-4-O-[beta-xylopyranosyl(1'' --> 6')-beta-glucopyranosyl]-benzoate (2), were isolated from the flowers of Pieris formosa. Their structures were determined on the basis of spectroscopic analysis and chemical methods.

Steaming Changes the Composition of Saponins of Panax notoginseng (Burk.) F.H. Chen That Function in Treatment of Hyperlipidemia and Obesity.

Saponins of Panax notoginseng (Burk.) F.H. Chen have been classified as a type of composition in functional foods for numerous diseases. However, its mild effects and other characteristics limited clinical applications in diseases. Inspired by "nine steaming and nine processing" of P. notoginseng in traditional Chinese medicine, we developed a "steaming"-mimic protocol, which significantly changed the composition of saponins of P. notoginseng from the original, R1, Rg1, Re, Rb1, and Rd (raw-PNS), to the products after steaming, 20S/R-Rh1, Rk3, Rh4, 20S/R-Rg3, Rk1, and Rg5 (N-PNS). Surprisingly, N-PNS demonstrated promising activities in improving hyperlipidemia and reducing body weight and weight of white adipose tissue and the inhibition of adipogenesis in obese mice. In accordance with the results in vivo, N-PNS remarkably blunted adipogenesis at the early stage of differentiation dose-dependently in vitro. Moreover, we demonstrated that the activity may involve the adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway by promoting phosphorylation of AMPKT172 and downregulating its downstream factors: sterol regulatory element binding protein 1c, stearoyl-CoA desaturase 1, and fatty acid synthase. Taken together, the steaming-induced eight compositions of saponins showed a very promising function in improving hyperlipidemia and obesity both in vivo and in vitro, providing fundamental evidence for future study and application in treatment of hyperlipidemia, obesity, and other lipid-related metabolic syndromes.

Isolation of two new prenylflavonols from Epimedium brevicornum and their effects on cytokine production in vitro.

In an attempt to search for bioactive natural products, two new prenylflavonols, 2''-hydroxy-3''-en-anhydroicaritin (1) and 2''-hydroxy-beta-anhydroicaritin (2), were isolated from the Chinese medicinal herb Epimedium brevicornum. Their structures were determined by 1D and 2D NMR spectroscopic analysis. The effects of compounds 1 and 2 on cytokine production in vitro were investigated. Compound 1 could significantly downregulate tumor necrosis factor-alpha (TNF-alpha) production and increase interleukin-10 (IL-10) production. These results suggested that compound 1 may have anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated mouse macrophages. In addition, the biogenetic relationships among compounds 1 and 2 are discussed.