Tuning of Poly-S-Nitrosated Human Serum Albumin as Superior Antitumor Nanomedicine

Macromolecules have been developed as carriers of low-molecular-weight drugs in drug delivery systems (DDS) to improve their pharmacokinetic profile or to promote their uptake in tumor tissue via enhanced permeability and retention (EPR) effects. We have previously demonstrated that poly-nitric oxide (NO) conjugated human serum albumin (Poly-SNO-HSA) has the potential to be a DDS carrier capable of accumulating NO in tumors. However, the stability of Poly-SNO-HSA in the circulation has to be improved, and its optimal molecular size for using the EPR effects has to be evaluated. In the present study, we performed two tuning methods for refining Poly-SNO-HSA, namely, pegylation and dimerization. We observed that pegylation enhanced the stability of Poly-SNO-HSA both in vitro and in vivo, and that dimerization of Poly-SNO-HSA enhanced the antitumor activity via more efficient delivery of NO in Colon 26 tumor-bearing mice. Intriguingly, dimerization resulted in a 10 times higher antitumor activity. These data suggest that pegylation and dimerization of Poly-SNO-HSA are very important tuners to optimize NO stability and accumulation, and thereby effect, in tumors. Thus, polyethylene glycol-Poly-SNO-HSA dimer seems to be a very appealing and safe NO carrier and thereby a strong candidate as an antitumor drug in future development of cancer therapeutics.     

Lactobacillus casei strain C1 attenuates vascular changes in spontaneously hypertensive rats

Hypertension can be caused by various factors while the predominant causes include increase in body fluid volume and resistance in the circulatory system that elevate the blood pressure. Consumption of probiotics has been proven to attenuate hypertension; however, the effect is much strain-dependent. In this study, a newly isolated Lactobacillus casei (Lb. casei) strain C1 was investigated for its antihypertensive properties in spontaneously hypertensive rats (SHR). Lactic acid bacteria (LAB) suspension of 11 log colony-forming unit (CFU) was given to SHR (SHR+LAB, n=8), and phosphate buffer saline (PBS) was given as a control in SHR (SHR, n=8) and in Wistar rats as sham (WIS, n=8). The treatment was given via oral gavage for 8 weeks. The results showed that the weekly systolic blood pressure (SBP), mean arterial pressure (MAP), diastolic blood pressure (DBP) and aortic reactivity function were remarkably improved after 8 weeks of bacterial administration in SHR+LAB. These effects were mostly attributed by restoration of wall tension and tensile stress following the bacterial treatment. Although not statistically significant, the level of malondialdehye (MDA) in SHR+LAB serum was found declining. Increased levels of glutathione (GSH) and nitric oxide (NO) in SHR+LAB serum suggested that the bacterium exerted vascular protection through antioxidative functions and relatively high NO level that induced vasodilation. Collectively, Lb. casei strain C1 is a promising alternative for hypertension improvement.     

The EPR effect: Unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect

The enhanced permeability and retention (EPR) effect is a unique phenomenon of solid tumors related to their anatomical and pathophysiological differences from normal tissues. For example, angiogenesis leads to high vascular density in solid tumors, large gaps exist between endothelial cells in tumor blood vessels, and tumor tissues show selective extravasation and retention of macromolecular drugs. This EPR effect served as a basis for development of macromolecular anticancer therapy. We demonstrated methods to enhance this effect artificially in clinical settings. Of great importance was increasing systolic blood pressure via slow angiotensin II infusion. Another strategy involved utilization of NO-releasing agents such as topical nitroglycerin, which releases nitrite. Nitrite is converted to NO more selectively in the tumor tissues, which leads to a significantly increased EPR effect and enhanced antitumor drug effects as well. This review discusses molecular mechanisms of factors related to the EPR effect, the unique anatomy of tumor vessels, limitations and techniques to avoid such limitations, augmenting tumor drug delivery, and experimental and clinical findings.     

A hybrid bacterium with tumor-associated macrophage polarization for enhanced photothermal-immunotherapy

Remodeling the tumor microenvironment through reprogramming tumor-associated macrophages (TAMs) and increasing the immunogenicity of tumors via immunogenic cell death (ICD) have been emerging as promising anticancer immunotherapy strategies. However, the heterogeneous distribution of TAMs in tumor tissues and the heterogeneity of the tumor cells make the immune activation challenging. To overcome these dilemmas, a hybrid bacterium with tumor targeting and penetration, TAM polarization, and photothermal conversion capabilities is developed for improving antitumor immunotherapy in vivo. The hybrid bacteria (B.b@QDs) are prepared by loading Ag₂S quantum dots (QDs) on the Bifidobacterium bifidum (B.b) through electrostatic interactions. The hybrid bacteria with hypoxia targeting ability can effectively accumulate and penetrate the tumor tissues, enabling the B.b to fully contact with the TAMs and mediate their polarization toward M1 phenotype to reverse the immunosuppressive tumor microenvironment. It also enables to overcome the intratumoral heterogeneity and obtain abundant tumor-associated antigens by coupling tumor penetration of the B.b with photothermal effect of the QDs, resulting in an enhanced immune effect. This strategy that combines B.b-triggered TAM polarization and QD-induced ICD achieved a remarkable inhibition of tumor growth in orthotopic breast cancer.     

Screening of Lactobacillus casei strains for their ability to bind aflatoxin B1

It has been proposed that the consumption of lactic acid bacteria capable of binding or degrading foodborne carcinogens would reduce human exposure to these deleterious compounds. In the present study, the ability of eight strains of Lactobacillus casei to bind aflatoxin B₁ in aqueous solution was investigated. Additionally, the effect of addition of bile salts to the growth medium on aflatoxin B₁ binding was assessed. The eight strains tested were obtained from different ecological niches (cheese, corn silage, human feces, fermented beverage). The strains exhibited different degrees of aflatoxin binding; the strain with the highest AFB₁ binding was L. casei L30, which bound 49.2% of the available aflatoxin (4.6 μg/mL). In general, the human isolates bound the most aflatoxin B₁ and the cheese isolates the least. Stability of the bacterial–aflatoxin complex was assessed by repeated washings. Binding was to a limited degree (0.6–9.2% release) reversible; the L. casei 7R1–aflatoxin B₁ complex exhibited the greatest stability. L. casei L30, a human isolate, was the strain least sensitive to the inhibitory effects of bile salts. Exposure of the bacterial cells to bile significant increased aflatoxin B₁ binding and the differences between the strains was reduced.     

Enhancing cancer chemo-immunotherapy by biomimetic nanogel with tumor targeting capacity and rapid drug-releasing in tumor microenvironment

In the development of chemo-immunotherapy, many efforts have been focusing on designing suitable carriers to realize the co-delivery of chemotherapeutic and immunotherapeutic with different physicochemical properties and mechanisms of action. Besides, rapid drug release at the tumor site with minimal drug degradation is also essential to facilitate the antitumor effect in a short time. Here, we reported a cancer cell membrane-coated pH-responsive nanogel (NG@M) to co-deliver chemotherapeutic paclitaxel (PTX) and immunotherapeutic agent interleukin-2 (IL-2) under mild conditions for combinational treatment of triple-negative breast cancer. In the designed nanogels, the synthetic copolymer PDEA-co-HP-β-cyclodextrin-co-Pluronic F127 and charge reversible polymer dimethylmaleic anhydride-modified polyethyleneimine endowed nanogels with excellent drug-loading capacity and rapid responsive drug-releasing behavior under acidic tumor microenvironment. Benefited from tumor homologous targeting capacity, NG@M exhibited 4.59-fold higher accumulation at the homologous tumor site than heterologous cancer cell membrane-coated NG. Rapidly released PTX and IL-2 enhanced the maturation of dendritic cells and quickly activated the antitumor immune response in situ, followed by prompted infiltration of immune effector cells. By the combined chemo-immunotherapy, enhanced antitumor effect and efficient pulmonary metastasis inhibition were achieved with a prolonged median survival rate (39 days).     

In situ apolipoprotein E-enriched corona guides dihydroartemisinin-decorating nanoparticles towards LDLr-mediated tumor-homing chemotherapy

The therapeutic efficiency of active targeting nanoparticulate drug delivery systems (nano-DDS) is highly compromised by the plasma proteins adsorption on nanoparticles (NPs) surface, which significantly hinders cell membrane receptors to recognize the designed ligands, and provokes the off-target toxicity and rapid clearance of NPs in vivo. Herein, we report a novel dihydroartemisinin (DHA)-decorating nano-DDS that in situ specifically recruits endogenous apolipoprotein E (apoE) on the NPs surface. The apoE-anchored corona is able to prolong PLGA-PEG2000-DHA (PPD) NPs circulation capability in blood, facilitate NPs accumulating in tumor cells by the passive enhanced permeability and retention (EPR) effect and low-density lipoprotein receptor (LDLr)-mediated target transport, and ultimately improve the in vivo antitumor activity. Our findings demonstrate that the strategy of in situ regulated apoE-enriched corona ensures NPs an efficient LDLr-mediated tumor-homing chemotherapy.     

Investigation of nepetolide as a novel lead compound: Antioxidant,antimicrobial, cytotoxic,anticancer, anti-inflammatory,analgesic activities and molecular docking evaluation

In the present study, we describe various pharmacological effects and computational analysis of nepetolide, a tricyclic clerodane-type diterpene, isolated from Nepeta suavis. Nepetolide concentration-dependently (1.0–1000?µg/mL) exhibited 1,1-diphenyl,2-picrylhydrazyl free radical scavenging activity with maximum effect of 87.01?±?1.85%, indicating its antioxidant potential, as shown by standard drug, ascorbic acid. It was moderately active against bacterial strain of Staphylococcus aureus. In brine shrimp’s lethality model, nepetolide potently showed cytotoxic effect, with LC₅₀ value of 8.7?µg/mL. When evaluated for antitumor activity in potato disc tumor assay, nepetolide exerted tumor inhibitory effect of 56.5?±?1.5% at maximum tested concentration of 1000?µg/mL. Nepetolide at 20?mg/kg reduced carrageenan-induced inflammation (P?<?.001 vs. saline group) in rat paw. Nepetolide dose-dependently (100–500?mg/kg) decreased acetic acid evoked writhes, as exhibited by diclofenac sodium. In-silico investigation of nepetolide was carried out against cyclooxygenase-2, epidermal growth factor receptor and lipoxygenase-2 targets. Virtual screening through Patchdock online docking server identified primarily hydrophobic interactions between ligand nepetolide and receptors proteins. Enhanced hydrogen bonding was predicted with Autodock showing 6–8 hydrogen bonds per target. These results indicate that nepetolide exhibits antioxidant, antibacterial, cytotoxic, anticancer, anti-inflammatory and analgesic activities and should be considered as a lead compound for developing drugs for the remedy of oxidative stress-induced disorders, microbial infections, cancers, inflammations and pain.     

Effect of low molecular lambda-carrageenan from Chondrus ocellatus on antitumor H-22 activity of 5-Fu.

λ-Carrageenan is a kind of sulfated galactan isolated from some red algae and reported to have more biological activities than other types, such as K-type [Güven KC, özsoy Y, Ulutin ON. Anticoagulant, fibrinolytic and antiaggregant activity of carrageenans and alginic acid. Bot Mar 1991; 34: 429–432]. The molecular weight (MW) of λ-carrageenan reaches up to 600–700 kDa, the high MW decreased their solubility and limited their bioactivities. At the same time, the production and applications of λ-carrageenan are limited for its some characteristics, such as the high viscosity, and the experiments of antitumor and immunomodulation also indicated that MW of polysaccharides had notably effect on the activities [Zhou G, Sun YP, Xin H, Zhang Y, Li Z, Xu Z. In vivo antitumor and immunomodulation activities of different molecular weight lambda-carrageenans from Chondrus ocellatus. Pharmacol Res 2004; 50(1) 47–53, Zhou G, Xin H, Sheng W, Sun Y, Li Z, Xu Z. In vivo growth-inhibition of S180 tumor by mixture of 5-Fu and low molecular λ-carrageenans from C. ocellatus. Pharmacol Res 2005; 51(2) 153–157].In this research, λ-carrageenan was extracted from C. ocellatus, an important economic alga in China. Then some small MW samples of 650, 240, 140, 15 and 9.3 kDa are obtained by the method of microwave degradation, respectively. The tumor inhibiting activities of the low MW λ-carrageenan and mixture of it and 5-Fu on transplanted H-22 tumor mice were investigated. The weight of immune organ, proliferation ratio of lymphocyte and concentration of Tumor Necrosis Factor-α (TNF-α) and histopathology of tumor from transplanted H-22 tumor mice were also determined. The results indicated that the degraded λ-carrageenan could add the antitumor activities of 5-Fu and improve the immunocompetence damaged by 5-Fu.     

Salmonella typhimurium strain SL7207 induces apoptosis and inhibits the growth of HepG2 hepatoma cells in vitro and in vivo

Salmonella typhimurium is probably most extensively studied tumor-targeting bacteria and SL7207 is one of its attenuated strains. SL7207 was first made for bacterial vaccine development and its therapeutic efficacy and safety for hepatocellular carcinoma has not been characterized. In this study, the inhibitory ability of SL7207-lux on human hepatoma HepG2 cells was tested in vitro and in vivo. A bacterial luminescent gene cluster (lux CDABE) was transfected into SL7207 to better monitor the invasion of the bacteria. The results show that SL7207-lux can rapidly enter HepG2 cells and localize in the cytoplasm. This invasion represses cell proliferation and induces apoptosis. In vivo real-time invasion studies showed that the bacteria gradually accumulate in the tumor. This enrichment was confirmed by anatomic observation at 5 days after inoculation. About 40% of tumor growth was inhibited by SL7207-lux at 34 days post-treatment without significant loss of body weight. The area of necrosis of tumor tissue was clearly increased in the treated group. Bacterial quantification showed that the number of colony-forming units per gram of bacteria within tumor tissue was approximately 1000-fold higher than that of liver and spleen. These data suggest that attenuated S. typhimurium strain SL7207 has potential for the treatment of cancers.     

Biocompatible Microemulsion Modifies the Tissue Distribution of Doxorubicin

The incorporation of doxorubicin (DOX) in a microemulsion (DOX-ME) has shown beneficial consequences by reducing the cardiotoxic effects of DOX. The aim of this study was to determine the distribution of DOX-ME in Ehrlich solid tumor (EST) and the heart, and compare it with that of free DOX. The distribution study was conducted with female Swiss mice with EST (n = 7 per group; 20–25 g). Animals received a single dose (10 mg/kg, i.p.) of DOX or DOX-ME 7 days after tumor inoculation. Fifteen minutes after administration, the animals were sacrificed, and the tumor and heart tissues were taken for immediate analysis by ultra-performance liquid chromatography. No difference was observed in DOX concentration in tumor tissue between DOX and DOX-ME administration. However, the most remarkable result in this study was the statistically significant reduction in DOX concentration in heart tissue of animals given DOX-ME. Mean DOX concentration in heart tissue was 0.92 ± 0.54 ng mg⁻¹ for DOX-ME and 1.85 ± 0.34 ng mg⁻¹ for free DOX. In conclusion, DOX-ME provides a better tissue distribution profile, with a lower drug concentration in heart tissue but still comparable tumor drug concentration, which indicates that antitumor activity would not be compromised. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:3297–3301, 2014     

3-苯甲酰基-2H-1-苯并吡喃-2-酮衍生物的设计、合成及抗肿瘤活性

目的 设计合成3-苯甲酰基-2H-1-苯并吡喃-2-酮衍生物,并评价其抗肿瘤活性。方法 以取代苯乙酮为原料,首先与碳酸二乙酯经Claisen缩合得到相应的取代β-酮酸酯,再与取代水杨醛经Knoevenagel缩合,同时环合得到目标化合物。采用人急性早幼粒白血病细胞HL-60及人乳腺癌细胞T47D对部分目标化合物的抗肿瘤活性进行初步评价。结果 合成了18个目标化合物,其中13个未见文献报道,目标化合物的结构经核磁共振氢谱和红外光谱确证。化合物III₁₅对人乳腺癌细胞T47D的抑制活性较强,IC₅₀值为38 μmol.L^-1;化合物III₁、III₂、III₁₅对人急性早幼粒白血病细胞HL-60的抑制活性较好,IC₅₀值分别为37、36、16 μmol.L^-1。结论 3-苯甲酰基-2H-1-苯并吡喃-2-酮衍生物作为新型肿瘤抑制剂,其构效关系值得进一步研究。     

Dextran sulphate sodium colitis in C57BL/6J mice is alleviated by Lactococcus lactis and worsened by the neutralization of Tumor necrosis Factor α

TNFα has a well-established role in inflammatory bowel disease that affects the gastrointestinal tract and is usually manifested as Crohn's disease or ulcerative colitis. We have compared Lactococcus lactis NZ9000 displaying TNFα-binding affibody with control Lactococcus lactis and with anti-TNFα antibody infliximab for the treatment of mice with dextran sulphate sodium (DSS)-induced colitis. L. lactis NZ9000 alleviated the colitis severity one week after colitis induction with DSS, more effectively when administered in preventive fashion prior to, during and after DSS administration. TNFα-binding L. lactis was less effective than control L. lactis, particularly when TNFα-binding L. lactis was administered in preventive fashion. Similarly, an apparently detrimental effect of TNFα neutralization was observed in mice that were intraperitoneally administered anti-TNFα monoclonal antibody infliximab prior to colitis induction. The highest concentrations of tissue TNFα were observed in groups without DSS colitis that were treated either with TNFα-binding L. lactis or infliximab. To conclude, we have confirmed that L. lactis exerts a protective effect on DSS-induced colitis in mice. Contrary to expectations, but in line with some reports, the neutralization of TNFα aggravated disease symptoms in the acute phase of colitis and increased TNFα concentration in colon tissue of healthy mice. Nevertheless, we have demonstrated that oral administration of bacteria with surface displayed TNFα-binding affibody can interfere significantly with TNFα signaling and mimic the infliximab response in the given animal model of colitis.     

Phospholipid-stabilized mesoporous carbon nanospheres as versatile carriers for systemic delivery of amphiphobic SNX-2112 (a Hsp90 inhibitor) with enhanced antitumor effect

Systemic delivery of amphiphobic drugs (insoluble in both water and oil) represents a formidable challenge in drug delivery. This work aimed to engineer a functional mesoporous carbon material to efficiently load SNX-2112, an amphiphobic anticancer agent, and to evaluate its performance in tumor-targeting delivery. Hydrothermal reaction combined with high-temperature activation was used to fabricate glucose-based mesoporous carbon nanospheres (MCNs). SNX-2112-loaded MCNs stabilized by phospholipid (SN-PMCNs) were prepared by the absorption/solvent diffusion/high-pressure homogenization method. The obtained SN-PMCNs were 180 nm around in particle size, showing a high drug load (42.7%) and acceptable physical stability. SN-PMCNs demonstrated an enhanced in vitro antitumor effect and increased uptake into cancer cells in comparison with the formulation of SNX-2112 solution (SN-Sol). The in vivo antitumor effect and biodistribution in 4T1 xenograft tumor mice, a breast cancer model, were also significantly improved through SN-PMCNs. It was shown that specific clathrin-dependent and nonspecific caveolae-dependent endocytosis were involved in the cellular trafficking of SN-PMCNs. Glucose transporter-mediated transport, prolonged body residence time and improved biodistribution via EPR effect were the main mechanisms of enhanced antitumor effect. SN-PMCNs have presented excellent tumor targeting properties and should be a promising carrier to address the systemic delivery of SNX-2112.     

The Methanol Extract of Azadirachta indica A. Juss Leaf Protects Mice Against Lethal Endotoxemia and Sepsis

In the present study, the inhibitory effect of neem leaf extract (NLE) on lipopolysaccaride (LPS)-induced nitric oxide (NO) and tumor necrosis factor-α (TNF-α) production was examined both in vitro and in vivo. In vitro study revealed that NLE treatment (100 μg/ml) inhibits LPS (100 ng/ml)-induced NO production by 96% and TNF-α production by 32%. The reduction in NO production is probably conferred by the complete suppression of inducible nitric oxide synthase (iNOS) expression. Interestingly, in vivo NLE significantly improved the survival rate of mice in an experimental sepsis model. Administration of NLE (100 mg/kg) 24 h before LPS treatment (20 mg/kg) improved the survival rate of mice by 60%. The inhibition of plasma NO and TNF-α production by NLE is likely to account for the improved survival of mice. Our results suggest that NLE may present a promising avenue in the development of therapeutic agents for the treatment of inflammatory diseases.     

Antibacterial,antitumor and hemolytic activities of α‐helical antibiotic peptide,P18 and its analogs

Abstract: The α‐helical antibiotic peptide (P18: KWKLFKKIPKFLHLAKKF‐NH₂) designed from the cecropin A(1–8)–magainin 2 (1?12) hybrid displayed strong bactericidal and tumoricidal activity without inducing hemolysis. The effect of the Pro⁹ residue at central position of P18 on cell selectivity was investigated by Pro⁹ → Leu or Pro⁹ → Ser substitution. Either substitution markedly reduced the antibacterial activity of P18 and increased hemolysis, although it did not significantly affect cytotoxicity against human transformed tumor and normal fibroblast cells. These results suggest that a proline kink in α‐helical antibiotic peptide P18 serves as a hinge region to facilitate ion channel formation on bacterial cell membranes and thus plays an important role in providing high selectivity against bacterial cells. Furthermore, to investigate the structure?antibiotic activity relationships of P18, a series of N‐ or C‐terminal deletion and substitution analogs of P18 were synthesized. The C‐terminal region of P18 was related to its antibiotic activity and α‐helical conformation on lipid membranes rather than N‐terminal one. Higher α‐helicity of the peptides was involved in the hemolytic and antitumor activity rather than antibacterial activity. Except for [L⁹]‐P18 and [S⁹]‐P18, all the designed peptides containing a Pro residue showed potent antibacterial activity, although they did not induce a cytolytic effect against human erythrocyte and normal fibroblast cells at the concentration required to kill bacteria. In particular, P18 and some analogs (N‐1, N‐2, N‐3, N‐3L and N‐4L) with potent bactericidal and tumoricidal activity and little or no normal cell toxicity may serve as an attractive candidate for the development of novel anti‐infective or antitumor agents.     

Regulation of proliferation of mouse bone marrow-derived mast cells by activated fibroblasts

Nitric oxide (NO) is synthesized by various cells involved in inflammatory reactions and may then act on mast cells. In the present work, we attempted to clarify the role of this molecule on the proliferation of mouse bone marrow derived-mast cells (BMMC). Swiss 3T3 fibroblasts produced nitrite (NO₂) and nitrate (NO₃) upon treatment with interferon γ (IFN-γ). This formation was dependent ofL-arginine and could be inhibited by theL-arginine analogue N^G-monomethyl-L-arginine (N ^GMMA). The effect of IFN-γ was drastically increased by cotreatment with tumor necrosis factor γ (TNF-γ). BMMC were maintained in vitro for as long as 30 days when cocultured with Swiss 3T3 fibroblasts. Coculture withN ^GMMA, significantly increased the number of BMMC. These results indicate that NO involves the inhibition of proliferation of BMMC when cocultured with Swiss 3T3 fibroblasts.     

A novel method to load topotecan into liposomes driven by a transmembrane NH4EDTA gradient

Antitumor drugs not only cause cytocidal effect on cancer cells, but also damage on normal healthy tissues, resulting in side effects. Liposome encapsulation can result in reduced systematic distribution due to the enhanced permeability and retention (EPR) effect, accompanied by drug accumulation in liver, spleen, and other immune organs, which can cause damage to those organs. It has been demonstrated that EDTA, frequently used as a chelator, possesses a synergistic antitumor effect. Indeed, our previous study showed that EDTA could reduce the toxicity of anthracyclines to the heart and immune organs. In this study, we intended to encapsulate topotecan within liposome adopting transmembrane NH₄EDTA gradient in order to increase the antitumor activity and decrease the toxicity against normal immune organs. Regarding the encapsulation efficiency of topotecan liposomes, both the pH value of the buffer and the cholesterol content showed significant effects on encapsulation and drug retention. Liposome encapsulation dramatically increased the antitumor activity of topotecan compared to free drug (p < 0.05), while similar efficacy was obtained from liposomes prepared by a NH₄EDTA gradient or a (NH₄)₂SO₄ gradient (tumor inhibition ratios were 85.6% and 84.1%, respectively). However, a significant decrease in toxicity against the immune organs was found in liposomes prepared by a NH₄EDTA gradient compared to those prepared by a (NH₄)₂SO₄ gradient. These results suggest the superiority of the proposed gradient for topotecan encapsulation in decreasing its toxicity on immune systems.     

Evaluation of the viability of Lactobacillus spp. after the production of different solid dosage forms

The work aims to provide evidence on the viability of Lactobacillus spp. and a spore form of Bacillus subtilis from nonprocessed bacteria to coated dosage forms (i.e., mini-tablets, pellets, and their coated forms). Lactobacillus spp. were cultivated overnight in MRS broth (10⁹ cfu/mL) and B. subtilis spores were produced on plate count agar (10⁷ cfu/mL) for 2 weeks. Bacteria and spores were freeze-dried in skim milk enriched with glycerol. The cakes were further processed into tablets (2.5 mm diameter) by direct compression with or without microcrystalline cellulose and inulin. Pellets (1–1.4 mm diameter) were produced by extrusion–spheronization of bacterial and spore suspensions with microcrystalline cellulose, lactose, inulin, and skim milk. Both tablets and pellets were film coated. The properties of the dosage forms, particularly the bacterial viability, were evaluated immediately after production and throughout storage for 6 months at 4°C. The study has shown that for an adequate stabilization of the bacteria a protective matrix (e.g., skim milk) and cryoprotectors (e.g., glycerol) must be present at early stages of bacterial de-hydration. Tabletting had a less deleterious effect (<2 log units) on bacteria when compared to pelletization (in some cases 3 log units). Enteric coating (15%, w/w) of either tablets or pellets did not affect the viability of the bacteria. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3329–3339, 2009     

Chemical composition and antibacterial activity of the essential oils from the medicinal plant Mentha cervina L. grown in Portugal

Mentha cervina is a medicinal plant traditionally used in Portugal in folk medicine, in different gastric disorders and inflammations of the respiratory tract. In order to validate those traditional uses, M. cervina essential oils (EOs) were characterized by GC and GC–MS and their antimicrobial activity was tested against 23 bacterial strains (including multiresistant strains). The EOs were dominated by the monoterpenes pulegone (52–75%), isomenthone (8–24%), limonene (4–6%), and menthone (1–2%). The antibacterial activity of these EOs was compared to that of the main components standards. The most effective antibacterial activity was expressed by the EOs against the Gram-negative bacteria, Escherichia coli and Acinetobacter baumanni, with MIC values of 1?mg/ml. The EOs complex mixtures were more active than the individual aromatic components supporting the hypothesis that the EOs antibacterial activity is a function of the synergistic effect of their different aromatic components. These results show the potential role of M. cervina EOs as antibacterial agents and validate the traditional use of this plant.