Bioassay-Guided Fractionation and Biological Activity of Cardenolides from Streptocaulon juventas.

The discovery that Na/K-ATPase acts as a signal transducer led us to investigate the structural diversity of cardiotonic steroids and study their ligand effects. By applying Na/K-ATPase activity assay-guided fractionation, we isolated a total of 20 cardiotonic steroids from Streptocaulon juventas, including an undescribed juventasoside B (10: ) and 19 known cardiotonic steroids. Their structures have been elucidated. Using our platform of purified Na/K-ATPase and an LLC-PK1 cell model, we found that 10: , at a concentration that induces less than 10% Na/K-ATPase inhibition, can stimulate the Na/K-ATPase/Src receptor complex and selectively activate downstream pathways, ultimately altering prostate cancer cell growth. By assessing the ligand effect of the isolated cardiotonic steroids, we found that the regulation of cell viability by the isolated cardiotonic steroids was not associated with their inhibitory potencies against Na/K-ATPase activity but reflected their ligand-binding affinity to the Na/K-ATPase receptor. Based on this discovery, we identified a unique active cardiotonic steroid, digitoxigenin (1: ), and verified that it can protect LLC-PK1 cells from hypoxic injury, implicating its potential use in ischemia/reperfusion injury and inducing collagen synthesis in primary human dermal fibroblast cells, and implicating that compound 2: is the molecular basis of the wound healing activity of S. juventas.

Phenylethanoid glycosides of Callicarpa kwangtungensis Chun exert cardioprotective effect by weakening Na+-K+-ATPase/Src/ERK1/2 pathway and inhibiting apoptosis mediated by oxidative stress and inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: Callicarpa kwangtungensis Chun (C. kwangtungensis) is a very famous herbal medicine with the function of promoting blood circulation and removing blood stasis which is beneficial for cardiovascular disease (CVD). Phenylethanoid glycosides (PGs) are the major class of active ingredients in C. kwangtungensis and present significant anti-oxidative and anti-inflammatory property related to apoptosis. Therefore, this study aimed to investigate the effects of total phenylethanoid glycosides of C. kwangtungensis (CK-PGs) on isoproterenol (ISO) induced myocardial ischemic injury (MI) and the mechanisms related to the apoptosis mediated by oxidative damage and inflammation. METHODS: The myocardial ischemia animal model was established as subcutaneous injecting ISO. Echocardiography and biomarkers were employed to determine the degree of myocardial damage. Histopathological changes were observed by hematoxylin and eosin test. The TUNEL staining and activity of caspase-3 were measured to detect the level of apoptosis which is medicated by the oxidative damage detected by the level of MDA, GSH and ROS tested with the kit and the inflammation reflected by TNF-α. The activity of Na+-K+-ATPase (NKA) was detected by the commercial kits, whose expression was measured by immunohistochemistry analysis. At last, Western blot analysis was used to measure Na+-K+-ATPase/Src/ERK1/2 and Bax/Bcl-2 pathway. RESULTS: CK-PGs showed cardioprotective effect against ISO-induced myocardial ischemic injury evidenced by improving heart function and lowering myocardial injury markers. CK-PGs could inhibit the level of apoptosis as shown by the decrease of the TUNEL-positive cells, the activity of caspase-3 and increase of the expression of Bax. CK-PGs also reduced oxidative stress and inflammation to suppress apoptosis by decreasing the level of ROS, MDA, and increasing GSH activity and lowering the level of TNF-α. In addition, CK-PGs exerted the protection by increasing the activity and the expression of NKA. Meanwhile, Na+-K+-ATPase/Src/ERK1/2pathway was weakened for the inhibition of apoptosis. CONCLUSIONS: CK-PGs could protect cardiomyocytes from myocardial injury through suppressing Na+-K+-ATPase/Src/ERK1/2 pathway and inhibiting apoptosis mediated by oxidative stress and inflammation.

Pomegranate Juice and Extract Consumption Increases the Resistance to UVB-induced Erythema and Changes the Skin Microbiome in Healthy Women: a Randomized Controlled Trial.

In vitro and animal studies have demonstrated that topical application and oral consumption of pomegranate reduces UVB-induced skin damage. We therefore investigated if oral pomegranate consumption will reduce photodamage from UVB irradiation and alter the composition of the skin microbiota in a randomized controlled, parallel, three-arm, open label study. Seventy-four female participants (30-45 years) with Fitzpatrick skin type II-IV were randomly assigned (1:1:1) to 1000 mg of pomegranate extract (PomX), 8 oz of pomegranate juice (PomJ) or placebo for 12 weeks. Minimal erythema dose (MED) and melanin index were determined using a cutometer (mexameter probe). Skin microbiota was determined using 16S rRNA sequencing. The MED was significantly increased in the PomX and PomJ group compared to placebo. There was no significant difference on phylum, but on family and genus level bacterial composition of skin samples collected at baseline and after 12 week intervention showed significant differences between PomJ, PomX and placebo. Members of the Methylobacteriaceae family contain pigments absorbing UV irradiation and might contribute to UVB skin protection. However, we were not able to establish a direct correlation between increased MED and bacterial abundance. In summary daily oral pomegranate consumption may lead to enhanced protection from UV photodamage.

Epicatechin-3-Gallate Signaling and Protection against Cardiac Ischemia/Reperfusion Injury.

At concentrations found in humans after ingestion of one to two cups of green tea, epicatechin-3-gallate (ECG) modulates Na/K-ATPase conformation and activity. Akin to ouabain, an archetypal Na/K-ATPase ligand of the cardiotonic steroid (CTS) family, ECG also activates protein kinase C epsilon type (PKCε) translocation and increases the force of contraction of the rat heart. This study evaluated whether, like ouabain, ECG also modulates Na/K-ATPase/Src receptor function and triggers pre- and postconditioning against ischemia/reperfusion (I/R) injury. In vitro, ECG activated the purified Na/K-ATPase/Src complex. In Langendorff-perfused rat hearts, submicromolar concentrations of ECG administered either before or after ischemia reduced infarct size by more than 40%, decreased lactate dehydrogenase release, and improved the recovery of cardiac function. ECG protection was blocked by PKCε inhibition and attenuated by mitochondrial KATP channel inhibition. In a unique mammalian cell system with depleted Na/K-ATPase α1 expression, ECG-induced PKCε activation persisted but protection against I/R was blunted. Taken together, these results reveal a Na/K-ATPase- and PKCε-dependent mechanism of protection by ECG that is also distinct from the mechanism of action of ouabain. These ECG properties likely contribute to the positive impact of green tea consumption on cardiovaascular health and warrant further investigation into the role of cardiac Na/K-ATPase signaling in the cardioprotective effect of green tea consumption. SIGNIFICANCE STATEMENT: Consumption of green tea, the richest dietary source of ECG, is associated with a reduced risk of cardiac mortality. Antioxidant effects of ECG and other tea polyphenols are well known, but reported for concentrations well above dietary levels. Therefore, the mechanism underlying the cardioprotective effect of green tea remains incompletely understood. This study provides experimental evidence that ECG concentrations commonly detected in humans after consumption of a cup of tea trigger the Na/K-ATPase/Src receptor in a cell-free system, activate a CTS-like signaling pathway, and provide PKCε-dependent protection against ischemia/reperfusion injury in rat hearts. Mechanistic studies in mammalian cells with targeted Na/K-ATPase depletion revealed that although Na/K-ATPase does not mediate ECG-induced PKCε activation, it is required for ECG-induced protection against ischemia/reperfusion injury.

Characterization, quantitation, similarity evaluation and combination with Na+,K+-ATPase of cardiac glycosides from Streblus asper.

Streblus asper Lour. (Moraceae) is a medicinal plant in Asian countries including India and Thailand, possessing activities of anti-tumor, anti-allergy, anti-parasitic and anti-bacterial. In this paper, characterization, quantitation and similarity evaluation of cardiac glycosides in different parts of S. asper were investigated by HPLC-Q-TOF-MS and chemometric methods. Then, the inhibition of Na+,K+-ATPase activity by the compounds isolated from S. asper was measured. Meanwhile, enzyme kinetics and molecular docking were determined to exhibit the combination modes between cardiac glycosides and Na+,K+-ATPase. As a result, twenty peaks of cardiac glycosides were assigned. Strophanthidin-3-O-α-l-rhamnopyranosyl-(1 → 4)-6-deoxy-ß-d-allopyranoside (1), glucostrebloside (2), strebloside (4) and mansonin (8) with a significant activity of inhibiting Na+,K+-ATPase (IC50 7.55-13.60 µM) were chosen for the determination of enzyme kinetics, exhibiting anticompetitive inhibitory characteristics towards Na+,K+-ATPase. Compound 4 could reasonably bind to the active sites of Na+,K+-ATPase, proved by molecular docking. Furthermore, the contents of the major compounds in four different parts of S. asper were extremely different, analyzed by chemometric methods, similarity analysis and principle compounds analysis. All these findings indicated that the contents of major compounds in different parts of S. asper were extremely different with a significant activity of inhibiting Na+,K+-ATPase, providing a reference for determination of effective part and administered dosage. The combination modes between cardiac glycosides and Na+,K+-ATPase were also revealed by enzyme kinetics and molecular docking, which provided a basis for further study of pharmacological activity.

Effects of Periploca forrestii Schltr on wound healing by Src meditated Mek/Erk and PI3K/Akt signals.

ETHNOPHARMACOLOGICAL RELEVANCE: Periploca forrestii Schltr. (PF) is a traditional folk medicine in China that has been used widely for treating rheumatoid arthritis and traumatic injuries for a long history. Previously, we have roughly demonstrated that the ethanol extract of PF possessed in vitro wound healing potential, and more in depth research deserves to be conducted. AIM OF THE STUDY: The present study is aiming to fully evaluate the wound healing activity of PF in vitro and in vivo, clarify the mechanism of actions and the primary constituents responsible for wound healing. MATERIALS AND METHODS: The total extract of Periploca forrestii Schltr. (EPF) and its fraction (65% ethanol fraction, EPFE65) were obtained and evaluated on in vitro wound healing properties using mouse dermal fibroblasts (L929). Cell proliferation was tested by MTT and EdU assay, confirmed by cell cycle analysis, cell migration was evaluated by scratch and transwell assay and collagen production was also determined. Then EPFE65 was tested on in vivo wound healing activity using the excision rat models. The wounded skin of rats was topically applied with 0.1% EPFE65 once daily for 6 days with hydrogel as the carrier and the recombinant bovine basic fibroblast growth factor hydrogel (rbFGF) as positive control. Histopathology of the wounded skin on day 6 and day 12 was studied via hematoxylin and eosin (HE) staining. The expression of phosphorylation of Src, Akt and Erk1/2 was determined after the treatment with EPFE65 by western blot. In order to figure out whether the activation of Src, Akt and Erk1/2 was directly in conjunction with wound healing process promoted by EPFE65, cell proliferation and migration were tested in the presence of three inhibitors of Src, Akt and Erk1/2. Finally, the chemical composition of the effective fraction EPFE65 was analyzed by HPLC-Q-TOF-MS/MS. RESULTS: In vitro experiments suggested that EPFE65 was comparable to EPF that had potent effect on promoting L929 fibroblasts proliferation, migration and increasing collagen production. 0.1% EPFE65 hydrogel also exhibited significant effect on promoting wound healing in rats. The wound closure was significantly faster in EPFE65 and positive rbFGF group than that in negative control group since the third day post wounding (p < 0.05). Specifically, on day10-12, the wounds in EPFE65 and rbFGF group were almost healed as the wound areas diminished into 13.3-5.3% and 7.7-4.0%, while the wound in control group was still apparent with 36.8-22.1% wound area. HE staining demonstrated that EPFE65 and rbFGF group could advance re-epithelialization in the early days and promote the transition of granulation tissue into complete dermis tissue with more skin appendages resembling those of normal skin in the last days. Western blot results suggested that the active fraction EPFE65 could increase the phosphorylation of Src, Akt and Erk1/2 in both dose-dependent and time-dependent manner, whereas Akt and Erk1/2 phosphorylation caused by EPFE65 could be abolished by Src inhibition. Inhibition experiments confirmed that the activation of Src, Akt and Erk1/2 were involved in cell proliferation and migration. All of these demonstrated that EPFE65 promoted wound healing at least in part via Src mediated Mek/Erk and PI3K/Akt signaling pathways. Analysis of chemical composition of EPFE65 revealed that cardiac glycosides were major components in EPFE65, among which periplocin showed effectiveness on promoting fibroblasts proliferation indicating that cardiac glycosides in EPFE65 maybe the active compounds responsible for wound healing. CONCLUSION: The present study confirmed that EPFE65, ethanol extract of Periploca forrestii Schltr. could accelerate wound healing in vitro and in vivo through Src meditated Mek/Erk and PI3K/Akt signaling pathways.

Periplocin promotes wound healing through the activation of Src/ERK and PI3K/Akt pathways mediated by Na/K-ATPase.

BACKGROUND: Periploca forrestii(PF) is mainly utilized for treatment of arthritis and traumatic injury historically. We had previously demonstrated that a fraction rich in cardiotonic steroids isolated from PF had the potential to facilitate wound healing. However, the exact material basis and mechanism of action responsible for wound healing is still unclear. Periplocin(PP) is the highest level of cardiotonic steroid included in PF. The present study aims to evaluate the efficacy of periplocin on wound healing systematically in vitro and in vivo. MATERIALS AND METHODS: The L929 proliferation was determined by both MTT and EdU assay. Cell migration was tested by both scratch and transwell assay. The total amount of soluble collagen was assessed using a Sircol Collagen Assay Kit. The wound healing activity was evaluated in vivo using the excision rat models. Histopathology of the wounded skin on day 9 was studied via hematoxylin and eosin staining (HE) for general morphological observations and masson's trichrome staining for collagen deposition, respectively. The alteration in Src/ERK and PI3K/Akt pathways mediated by Na/K-ATPase was determined by western blot after the treatment with periplocin. The interaction between Na/K-ATPase and Src was tested by immunoprecipitation and immunostaining analysis. RESULTS: The results revealed that periplocin could significantly boost proliferation, migration and stimulate collagen production in fibroblast L929 cells, which is dependent on activation of Src/ERK and PI3K/Akt pathways mediated by Na/K-ATPase, and thus promoting wound healing. Indeed, inhibition of Na/K-ATPase/Src complex receptor by Src specific inhibitor or knocking down the Na/K-ATPase expression would abolish the subsequent activation of Src/ERK and PI3K/Akt pathways and attenuate periplocin-induced beneficial effects on wound healing. Additionally, the wound healing activity is also confirmed in a rat excisional wound model as evidenced by increased rate of wound closure, reepithelization, formation of granulation tissue and collagen accumulation. CONCLUSIONS: Collectively, we lay the rationale for traditional usage for traumatic injury, suggesting that periplocin and periploca forrestii is a promising candidate for management of chronic wounds.

Identification of poliumoside metabolites in rat feces by high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.

Poliumoside is one of the major phenylethanoid glycosides (PhGs) isolated from Callicarpae Caulis et Folium (CCF) which is a traditional Chinese medicine used for hemostasis in clinic. In this study, high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (HPLC/Q-TOF-MS) was applied to investigate the metabolites of poliumoside in rat feces after oral administration. A total of 66 metabolites were confirmed or tentatively identified. Poliumoside could be partly transformed into its positional isomer isopoliumoside in vivo, and poliumoside was easily hydrolyzed and metabolized into degradation products. The parent compound and its degradation products could further undergo extensive phase I and phase II metabolism. The results indicated that hydrolysis, hydroxylation, acetylation, sulfation, hydration, reduction, dehydrogenation and dimethylation were the major metabolic pathways of poliumoside. The major metabolic soft spots of poliumoside and the fragmentation patterns of the metabolites were also proposed. This study provided valuable information regarding the metabolites of poliumoside in rats.

Studies on the flavone glycosides from Fructus Kochiae.

A series of flavone glycosides were isolated from Fructus Kochiae for the first time, including two new flavone glycosides. The structures were established by interpretation of their spectroscopic data. Two new flavone glycosides are quercetin 3-O-ß-d-apiofuranosyl-(1 â†’ 2)-ß-d-galactopyranosyl-7-O-ß-d-glucopyranoside (1) and quercetin 3-O-α-l-rhamnopyranosyl-(1 â†’ 6)-ß-d-galactopyranosyl-7-O-ß-d-sophoroside (2). The others are quercetin 7-O-ß-d-glucopyranoside (3), quercetin 3-O-ß-d-apiofuranosyl-(1 â†’ 2)-ß-d-galactopyranoside (4), quercetin 3-O-ß-d-galactopyranosyl-7-O-ß-d-glucopyranoside (5), and quercetin 7-O-ß-d-sophoroside (6).

Characterization and controlled release aloe extract of collagen protein modified cotton fiber.

For exploiting the novel multifunctional cotton fibers, a collagen protein modified cotton fiber (CPMCF) was prepared by the oxidation of cotton fiber with sodium periodate solution and subsequent crosslinking reaction with an aqueous solution of collagen protein in acetic acid. Infrared spectra and X-ray photoelectron spectrometry (XPS) analysis of the CPMCF illuminated that the C=N double bond was formed through the imine reaction of the aldehyde group on oxidized cotton fiber with the amino group of collagen protein. X-ray diffractograms indicated that the crystallinity of the oxidized cotton fiber increased from 65.6 to 69.3% after collagen protein treatment. Scanning electron microscopy photographs displayed that the collagen protein combined on the surface of oxidized cotton fiber. The resulting optimum conditions to prepare the CPMCF achieved the sufficient aldehyde groups in oxidized cotton fiber and the collagen protein content on CPMCF, whereas the mechanical strength of the oxidized cotton fiber had no significant change. Meanwhile, a model experiment for the controlled release of aloe anthraquinone extract on CPMCF showed a satisfactory result compared with those release of the original cotton fiber, demonstrated potential application of the synthetic collagen protein-cotton fiber as a carrier for the sustained release of drugs.