Mini Review: Chemical Characterization and Evaluation of Antiviral and Antibacterial Activity of Extracts from Graptopetalum paraguayense E. Walther (Crassulaceae)

Graptopetalum paraguayense E. Walther (GP) is a traditional Chinese herbal medicine belonging to the Crassulaceae family As herbal medicine, GP possesses several biological/pharmacological perspectives. It was found that GP aqueous extracts exhibit anti-inflammatory and hepatoprotective, antioxidant, anti-hypertensive and antihyperglycemic, immunomodulatory, antineoplastic, antiviral and antibacterial activities. The GP contains in the high range levels phenolic compounds and anthocyanins. Despite the intense study of the plant, little data exist regarding its chemical composition. It was found that the GP aqueous extract has no cytotoxic effect on RD and Lep cells. The extract effectively inhibited herpes simplex virus (HSV) replication in dose-dependent manner moderate activity as well as Gram-positive bacterial strains

Improvement of the Antimicrobial Activity of Oregano Oil by Encapsulation in Chitosan—Alginate Nanoparticles

Oregano oil (OrO) possesses well-pronounced antimicrobial properties but its application is limited due to low water solubility and possible instability. The aim of this study was to evaluate the possibility to incorporate OrO in an aqueous dispersion of chitosan—alginate nanoparticles and how this will affect its antimicrobial activity. The encapsulation of OrO was performed by emulsification and consequent electrostatic gelation of both polysaccharides. OrO-loaded nanoparticles (OrO-NP) have small size (320 nm) and negative charge (−25 mV). The data from FTIR spectroscopy and XRD analyses reveal successful encapsulation of the oil into the nanoparticles. The results of thermogravimetry suggest improved thermal stability of the encapsulated oil. The minimal inhibitory concentrations of OrO-NP determined on a panel of Gram-positive and Gram-negative pathogens (ISO 20776-1:2006) are 4–32-fold lower than those of OrO. OrO-NP inhibit the respiratory activity of the bacteria (MTT assay) to a lower extent than OrO; however, the minimal bactericidal concentrations still remain significantly lower. OrO-NP exhibit significantly lower in vitro cytotoxicity than pure OrO on the HaCaT cell line as determined by ISO 10993-5:2009. The irritation test (ISO 10993-10) shows no signs of irritation or edema on the application site. In conclusion, the nanodelivery system of oregano oil possesses strong antimicrobial activity and is promising for development of food additives

Biopolymeric Nanogel as a Drug Delivery System for Doxorubicin—Improved Drug Stability and Enhanced Antineoplastic Activity in Skin Cancer Cells

In this study, doxorubicin was loaded in a chitosan–albumin nanogel with the aim of improving its stability and exploring the potential of the system in the treatment of skin cancer. Infrared spectroscopy and X-ray diffraction confirmed the encapsulation of the drug. Transmission electron microscopy revealed the spherical shape of the nanogel particles. The drug-loaded nanogel was characterized with a small diameter of 29 nm, narrow polydispersity (0.223) and positive zeta potential (+34 mV). The exposure of encapsulated doxorubicin to light (including UV irradiation and daylight) did not provoke any degradation, whereas the nonencapsulated drug was significantly degraded. In vitro studies on keratinocytes (HaCaT) and epidermoid squamous skin carcinoma cells (A-431) disclosed that the encapsulated doxorubicin was more cytotoxic on both cell lines than the pure drug was. More importantly, the cytotoxic concentration of encapsulated doxorubicin in carcinoma cells was approximately two times lower than that in keratinocytes, indicating that it would not affect them. Thus, the loading of doxorubicin into the developed chitosan–albumin nanogel definitely stabilized the drug against photodegradation and increased its antineoplastic effect on the skin cancer cell line

New Insights in Routine Procedure for Mathematical Evaluation of in vitro Cytotoxicity Data from Cancer Cell Lines

In oncopharmacology, the common procedure to evaluate median-effect concentrations (IC50) on experimental data is based on the use of well-established kinetic models representing inhibition effects of drugs on human cancer cell lines. Several widespread software programs, such as GraphPad Prism and CompuSyn offer possibilities for calculation of IC50 through the model of Chou. In recent study, we analyzed the results from those two software programs and compared them with the non-linear programming procedure written by us in the MAPLE symbolic software. The last evaluated IC50 more precisely and the correlation coefficient R value was better in all trails. We demonstrated the efficiency of non-linear programming procedures in examples of two cancer cell lines treated with three different drugs. The response surface analysis showed the potential of the applied kinetic model. As a result, we were able to define better the IC50 values and to use them in planning further experiments in human cancer cell lines related to single drug influence and drug-drug interference

ANALYTICAL STUDY AND ANTIMICROBIAL ACTIVITY OF ALPHA-DEFENSIN 2 DISSOLVED IN PHARMACOPOEIA BUFFERS WITH DIFFERENT pH

Human neutrophil peptides (HNPs, alpha-defensins) are a group of six defensins being considered as new antimicrobial drugs because of their multifunctional efficiency against bacteria, viruses and fungi. Regardless of the unique biological properties alpha-defensins are unstable compounds and their activity depends on many physical and chemical factors as well as on the kind of the used cultivation media. This leads to research difficulties and obstructions in their therapeutic application. The purpose of this study was to determine antibacterial activity of alpha-defensin 2 dissolved in pharmacopeia buffers and in parallel to develop selective and accurate analytical tests for identification and assay of alpha-defensin 2 in the course of study. The antibacterial effect of alpha-defensin 2 was determined against the strain Escherichia coli (ATCC 25922). It was found that 10 mg/l of Human neutrophil peptide-2 (HNP-2, alpha-defensin 2) dissolved in pH 9.0 buffer caused 90% inhibition of the bacterial respiratory activity. This buffer was considered as a suitable environment for deploying the antibacterial activity of the alpha-defensin. А selective MS analysis method for identification of alpha-defensin 2 in sample mixtures was developed. Also HPLC method with alternative selectivity was elaborated for identification and assay of mixtures containing alpha-defensin 2 and aminoacids in Mueller Hinton broth. The procedure includes development of system suitability test determination

Reduced Expression of the Retinoblastoma Protein Shows That the Related Signaling Pathway Is Essential for Mediating the Antineoplastic Activity of Erufosine

<div><p>Erufosine is a new antineoplastic agent of the group of alkylphosphocholines, which interferes with signal transduction and induces apoptosis in various leukemic and tumor cell lines. The present study was designed to examine for the first time the mechanism of resistance to erufosine in malignant cells with permanently reduced expression of the retinoblastoma (Rb) protein. Bearing in mind the high number of malignancies with reduced level of this tumor-suppressor, this investigation was deemed important for using erufosine, alone or in combination, in patients with compromised RB1 gene expression. For this purpose, clones of the leukemic T-cell line SKW-3 were used, which had been engineered to constantly express differently low Rb levels. The alkylphosphocholine induced apoptosis, stimulated the expression of the cyclin dependent kinase inhibitor p27^Kip1 and inhibited the synthesis of cyclin D3, thereby causing a G₂ phase cell cycle arrest and death of cells with wild type Rb expression. In contrast, Rb-deficiency impeded the changes induced by eru-fosine in the expression of these proteins and abrogated the induction of G₂ arrest, which was correlated with reduced antiproliferative and anticlonogenic activities of the compound. In conclusion, analysis of our results showed for the first time that the Rb signaling pathway is essential for mediating the antineoplastic activity of erufosine and its efficacy in patients with malignant diseases may be predicted by determining the Rb status.</p></div

Cytotoxicity and clonogenic activity of erufosine in Rb-deficient cells.

<p>The figure depicts the survival (A) or clonogenicity (B) of SKW-3 cell clones with 99% (shRNA1) or 83% (shRNA 2) stable Rb-knockdown after treatment with 2, 4, 8, 16 or 32 µM erufosine for 48 h. A significant difference versus the respective nonsense control is marked by an asterisk (Student’s t-test; p<0.05). Bars denote standard deviation. The table under the graphs gives the IC₅₀ values of erufosine after 48 h of treatment as well as the respective 95% confidence intervals.</p

Erufosine-induced apoptosis is inhibited by Rb-loss.

<p>The expression of proteins is shown, which are involved in induction of the intrinsic apoptotic pathway, respectively, for nonsense or antisense-transduced cell clones with 99% or 83% Rb-knockdown after exposure to erufosine (16 µM, 48 h). The values under the protein bands denote their intensity compared to the untreated control and are calculated after densitometric analysis with the Quantity One 4.6.6 Program (Bio-Rad).</p

Rb-loss inhibits the erufosine-induced G2 cell cycle arrest.

<p>The flow cytometry histograms present the distribution of nonsense- or antisense transduced SKW-3 cell clones with 99% (shRNA 1) and 83% (shRNA 2) Rb-knockdown in G1-, S- and G2-phases of the cell cycle before and after exposure to 16 µM erufosine for 24 and 48 h. The percentage of the cell fractions is calculated with the ModFit LT software and given in the table below the graphs.</p

Efficacy of the Rb-knockdown.

<p>(A) Fluorescence imaging of SKW-3 cells at 72 h after viral transduction with three different pLL 3.7-constructs (nonsense shRNA – NSO, antisense shRNAs–21 bp, 1 or 27 bp, 2) and Cell Sorting using the eGFP signal. (B) The efficacy of the Rb-knockdown estimated by Western blot before and after selection. The Rb-knockdown on protein level was calculated as percentage of the respective nonsense-control cells after densitometric analysis of the protein bands using the Quantity One 4.6.6 Program (Bio-Rad Laboratories).</p