Botanical Extracts and Compounds of Castanea Plants and Methods of Use: US20190125818A1 - The United States Patent Evaluation.

BACKGROUND: Bacterial infections are increasingly difficult to combat, which makes them a threat to public health on a global level. Staphylococcus aureus is considered one of the main causes of infections in hospitals, as it has a variety of virulence factors, as well as is able to produce bacterial biofilms, which, consequently, bring numerous damages to public health as a result of increased resistance to conventional antibiotics and a longer hospital stay. Therefore, the use of compounds extracted from medicinal plants is a potential pharmaceutically acceptable target, as they do not have toxicity and the potential to disrupt biofilms produced by Staphylococcus aureus already evidenced, thus revealing their relevance to our study. OBJECTIVE: The objective of this work was to perform a critical analysis of a patent with natural extracts against bacterial biofilms found in the United States Patent and Trademark Office (USPTO) database, to map the possible bioactive compounds that may serve as potential future antimicrobial drugs. METHODS: A technological survey was carried out to verify existing patents using natural extracts with anti-biofilm potential. For this, it was searched with the keywords: Botanical extracts AND biofilms; which were performed in the United States Patent and Trademark Office (USPTO) database. Thus, the selected patent used a non-aqueous extract partitioned and vacuum-contracted, subsequently lyophilized for assays with antimicrobial potential. Because of this, a patent was analyzed regarding its chemistry, and biological activity, followed by a critical analysis of the technology proposed in the invention. RESULTS: When using the keywords Botanical extracts AND biofilms in the USPTO, it was possible to find twenty-two inventions; however, only four patents in the USPTO were in agreement with the proposal of the natural extract having antimicrobial activity and an anti-biofilm potential, of which two belonged to the same applicant with similar proposals. The key point of this invention was to enable the compounds of the Castanea sativa plant and its methods of obtaining the extract to present a significant antimicrobial action associated or not with antibiotics, promoting the development of new therapies against bacterial infections capable of disrupting biofilms. The invention developed a methodology for extracting Castanea sativa, in which pentacyclic triterpene compounds were found mostly in its leaves. Whereas for the extraction, the crude methanol extracts called extracts 224 from the ground leaves were made by maceration, filtered, combined, concentrated under pressure in rotary evaporators, and lyophilized. After that, they were resuspended in water and partitioned in succession with hexane, ethyl acetate, and butanol. The most active refined partition was the 224C extract with the solvent ethyl acetate, which was subjected to further fractionation using silica column chromatography. Resulting in the most refined extract, which was 224C-F2, capable of acting directly on the quorum sensing of bacteria, mainly Staphylococcus aureus, blocking the translation of RNAIII, including a series of exotoxins. Regarding the antimicrobial capacity against Staphylococcus aureus, it presented Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of 1.56 µg/mL-1 and > 100 µg/mL -1, respectively. CONCLUSION: Given the analyzed patent, it was possible to verify the importance of alternatives to reduce the impact of bacterial biofilms, which causes damage to industries in general and to health. From this, the invention analyzed has a promising proposal with antimicrobial potential focusing on the great impact of bacterial biofilms. Therefore, natural extracts with antibiofilmic potential can help to minimize the economic losses caused to health due to these multidrug-resistant microorganisms with different virulence mechanisms.

Polymeric Nanoparticles for the Treatment of Prostate Cancer- Technological Prospecting and Critical Analysis.

BACKGROUND: Polymeric nanoparticles have a wide diversity, and due to their toxicity and biodegradability, they have been widely used in the health area. Its use allows stability of some compounds, targeted delivery, and increased half-life, in this context, making some treatment proposals more effective. Prostate cancer, in turn, is among the types of cancer with the highest mortality, and the lack of effective treatment causes several strategies to meet this need. OBJECTIVE: The objective of this work was to verify patents that use polymeric nanoparticles for the treatment of prostate cancer. METHODS: For that, specific keywords to direct the search were applied in Patent Scope. After obtaining the patents, one was selected for the development of critical analysis in relation to its chemistry and biology. RESULTS: A total of five patents were found and, of these, an invention that used PCL-PLGA-PEGCOOH polymeric nanoparticles with two natural compounds, resveratrol and celastrol, providing an alternative method to traditional monotherapies. CONCLUSION: The prospective analysis serves to direct us in relation to the technologies currently used in certain fields. Based on several cases of cancer and specifically the countless cases of prostate cancer, five recent patents were found using polymeric nanoparticles. However, only one brought a different aspect of all the treatments used lately. It brought an invention containing two natural compounds being carried with polymeric nanoparticles with promising results.

Chitosan Oligomer and Zinc Oxide Nanoparticles for Treating Wastewaters: US20190134086 Patent Evaluation.

With the utilisation of algae, wastewater reuse is becoming a viable option for the energy industry, especially green energy. The growth of these algae in these wastewaters provides an alternative source for bioenergetics, however, the growth of other microorganisms can directly affect the production of bioenergy, requiring the removal and reduction of contaminants in these waters, in addition to being a source of contamination for workers. Therefore, the use of nanoparticles in bioremediation has been an alternative to mitigate the contamination of these wastewaters that have microorganisms capable of reducing the algae growth capacity. The objective of this work was to verify in the United States Patent and Trademarker office database (USPTO) patents that used chitosan nanoparticles as a form of wastewater treatment and to carry out the analysis of patent US20190134086, which addresses the use of zinc oxide nanoparticles associated with chitosan that was developed and used to evaluate their antibacterial activity against resistant microorganisms and biofilm producers present in wastewater. Escherichia coli, Enterococcus faecium, and/or Pseudomonas aeruginosa are the microorganisms involved in the evaluated invention, bacteria present in the gastrointestinal tract, of clinical and environmental importance. The synthesized nanoparticles are arranged as a pharmaceutically acceptable and toxic vehicle against resistant bacteria, thus being described as nanoremediators. Given the analyzed patent, it was possible to verify the importance of alternatives to reduce the impact that pollution, in general, has on the environment, in addition to the proposed technology serving to maintain the survival and development capacity of the algae that will be able to produce green energy, the nanoparticles with antibacterial potential can help indirectly reduce these pathogenic strains with resistance to several antibiotics in the environment.

Secondary Staphylococcus aureus intramammary colonization is reduced by non-aureus staphylococci exoproducts.

Non-aureus staphylococci (NAS) and Staphylococcus aureus are pathogens that cause bovine mastitis, a costly disease for dairy farmers, however; many NAS are considered part of the normal udder microbiota. It has been suggested that through a mechanism that remains to be elucidated, NAS intramammary colonization can prevent subsequent infection with other bacterial pathogens. This study shows that in a murine mastitis model, secondary Staph. aureus intramammary colonization is reduced by exoproducts from Staph. chromogenes and Staph. simulans, both NAS, while Streptococcus spp. exoproducts have much less ability to affect the course of the infection caused by S. aureus.

Peptide vaccines designed with the aid of immunoinformatic against Caseous Lymphadenitis promotes humoral and cellular response induction in mice.

Caseous Lymphadenitis (CLA) is a chronic disease that affects also small ruminants. CLA is caused by Corynebacterium pseudotuberculosis and is responsible for high economic losses due to the formation of superficial and visceral granulomas, the latter is considered as asymptomatic CLA causing high levels of dissemination. Several vaccination strategies, in which the use of synthetic peptides stands out. Thus, this work aimed to evaluate the protective potential of peptide vaccines designed to determine the immunodominant epitopes of CP40 against CLA in mice. The animals were divided into eight groups separated in controls (G1-PBS, G2-Saponin and G9-rCP40) and experimental (G3-pep1, G4- pep2, G5-pep3, G6-pep4, G7-pep5 and G8-pep6), these were vaccinated on days 0 and 15 by a subcutaneous route. 60 days after the first immunization, all animals were challenged with C. pseudotuberculosis. On days 0, 15, 60, and 120 after the first immunization, blood samples were taken to measure immunoglobulins. On the same day of the challenge, the splenocytes were isolated and assayed for the production of IL-2, IL-4, IL-6, IFN-γ, TNF-α, IL-17, and IL-10. After vaccinations, the animals were challenged and all of them were affected by the disease which led to their death. The G6 and G8 groups provided 10% protection and the G7 provided 20%. The G3 and G4 groups provided 30% and 40% protection respectively. The peptides showed the production of Total IgG antibodies and cytokines (IL-2, IL-4, IL-6, IFN-γ, and TNF-α), indicating a possible activation of the Th1 type response. However, groups G3, G5, G6, and G8 showed production of IL-17. None of the study groups showed IL-10 production. The immunogenicity of the peptides was not enough to protect these animals and it is believed that the use of adjuvants based on PAMPs may improve the immune response offered by these peptides.

In Silico Screening of Putative Corynebacterium pseudotuberculosis Antigens and Serological Diagnosis for Caseous Lymphadenitis in Sheep by Enzyme-Linked Immunosorbent Assay.

Corynebacterium pseudotuberculosis is the etiologic agent of Caseous Lymphadenitis (CLA), a disease leading to severe damage in sheep and goats farming due to the lack of serological diagnosis, treatment, and effective prophylaxis. In this context, several strategies in an attempt to discover new antigens to compose diagnosis assays or vaccines are fundamental. Therefore, this study aimed to use bioinformatics software to evaluate the critical chemical characteristics of unknown proteins of C. pseudotuberculosis by selecting them for heterologous expression in Escherichia coli. For this purpose, six protein sequences of ascorbate transporter subunit, UPF protein, MMPL family transporter, Ribonuclease, Iron ABC transporter domain-containing permease, and fimbrial subunit were obtained. In silico analyses were performed using amino acid sequences to access immunodominant epitopes and their antigenic and allergenic potential and physicochemical characterization. The expressed proteins were used as an antigen for serological diagnosis by ELISA. All proteins showed distinct immunodominant epitopes and potential antigenic characteristics. The only proteins expressed were PTS and Ribonuclease. In parallel, we expressed CP40 and all were used with ELISA antigen in 49 CLA positive sera and 26 CLA negative sera. The proteins alone showed 100% sensitivity and 96.2%, 92.3%, and 88.5% specificity for rPTS, rRibonuclease, and rCP40, respectively. When proteins were combined, they showed 100% sensitivity and 84.6%, 92.3%, 88.5%, and 92.3% specificity for rPTS/rCp40, rRibonuclease/rCP40, rPTS/rRibonuclease, and rPTS/rRibonuclease/rCP40, respectively. The results of this study show an excellent correlation of sensitivity and specificity with all proteins. None of the specificity values preclude the potential of rPTS, rRibonuclease, or rCP40 for use in ELISA diagnostic assays since the results of this work are superior to those of other studies on CLA diagnosis described in the literature.

Cymbopogon winterianus Essential Oil Attenuates Bleomycin-Induced Pulmonary Fibrosis in a Murine Model.

The essential oil of Cymbopogon winterianus (EOCW) is a natural product with antioxidant, anti-inflammatory, and antifibrotic properties. We studied the effect of EOCW in the progression of histological changes of pulmonary fibrosis (PF) in a rodent model. The oil was obtained by hydrodistillation and characterized using gas chromatography-mass spectrometry. Intratracheal instillation of bleomycin was performed in 30 rats to induce PF, while Sham animals were subjected to instillation of saline solution. The treatment was performed using daily oral administration of distilled water, EOCW at 50, 100, and 200 mg/kg, and deflazacort (DFC). After 28 days, hemogram and bronchoalveolar lavage fluid (BALF), tissue levels of malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) were assayed. Histological grading of PF, immunohistochemical expression of α-smooth muscle actin (α-SMA), and transforming growth factor-ß (TGF-ß) were also analyzed. The EOCW major compounds were found to be citronellal, geraniol, and citronellol. EOCW significantly reduced inflammation in BALF, reduced MDA levels, and increased SOD activity. EOCW attenuated histological grading of PF and reduced immunohistochemical expression of α-SMA and TGF-ß in a dose-dependent way, likely due to the reduction of oxidative stress, inflammation, and TGF-ß-induced myofibroblast differentiation.

Effects of exposure to triphenyltin (TPT) contaminant on sperm activity in adulthood of Calomys laucha exposed through breastfeeding.

Triphenyltin (TPT) is an organotin compound (OT), primarily used in agriculture and in the composition of antifouling paints for ships worldwide. Studies have showed its effects as an endocrine disrupter in several organisms by preventing enzymatic expression and causing reproductive toxicity. This study aimed to evaluate the effects of exposure to TPT, via breastfeeding, on reproductive physiology in the Calomys laucha species. The experimental design was compound of five groups, two controls and three with different doses of TPT. Moreover, females were exposed by gavage to the TPT for 20 days, from the 1st day postpartum to the 21st postnatal day (PND). Then, the pups were euthanized and the kinetics, organelles, and biochemistry of the sperm were evaluated. The results presented a reduction in total motility in the groups exposed to TPT. Regarding cellular organelles analysis, a loss in membrane integrity was evidenced; the functionality of mitochondria showed diminution followed by increased acrosome reaction. In conclusion, the TPT causes alteration of the reproductive parameters, decreasing the activity and sperm quality in individuals exposed in the breastfeeding phase.

Immune-Informatic Analysis and Design of Peptide Vaccine From Multi-epitopes Against Corynebacterium pseudotuberculosis.

Caseous lymphadenitis (CLA) is a disease caused by Corynebacterium pseudotuberculosis bacteria that affects sheep and goats. The absence of a serologic diagnose is a factor that contributes for the disease dissemination, and due to the formation of granuloma, the treatment is very expensive. Therefore, prophylaxis is the approach with best cost-benefit relation; however, it still lacks an effective vaccine. In this sense, this work seeks to apply bioinformatic tools to design an effective vaccine against CLA, using CP40 protein as standard for the design of immunodominant epitopes, from which a total of 6 sequences were obtained, varying from 10 to 16 amino acid residues. The evaluation of different properties of the vaccines showed that the vaccine is a potent and nonallergenic antigen remaining stable in a wide range of temperatures. The initial tertiary structure of the vaccine was then predicted and a model selected. Later, the process of CP40 protein and TLR2 receptor binding was performed, presenting interaction with this receptor, which plays an important role in the activation of the immune response.

Recombinant CP40 from Corynebacterium pseudotuberculosis confers protection in mice after challenge with a virulent strain.

BACKGROUND: Caseous Lymphadenitis (CLA) is a contagious, infectious, chronic disease caused by Corynebacterium pseudotuberculosis, which affects mainly sheep and goats. The clinical prevalence of CLA in Brazil is 30%, resulting in decreased milk production, weight loss, and unusable meat and leather. Prophylaxis is based on vaccination; however, current vaccinations do not offer effective protection against the infection, which makes the development of a new vaccine essential to control this disease. EXPERIMENTAL APPROACH: Here, we developed a recombinant vaccine based on CP40 protein (rCP40) combined with an adjuvant (Freund's complete adjuvant or saponin) and evaluated its efficacy in a murine model of CLA. Female BALB/c mice were used in an immunization assay. KEY RESULTS: rCP40 induced high levels of IgG2a and IgG2b antibodies. After challenge with a virulent strain of C. pseudotuberculosis C57 (10(4)CFU/mL), the levels of IgG2a and IgG2b were sustained, indicating a Th1 response. The groups immunized with rCP40 protein (GES and GEF groups) showed 100% protection and was statistically significant in the GES and GEF groups (p<0.037 and p<0.0952, respectively). CONCLUSIONS: The results indicated the recombinant protein CP40 induced an specific immune response in mice that was able to afford protection after challenge, regardless the adjuvant used in the formulation.

Corynebacterium pseudotuberculosis cp09 mutant and cp40 recombinant protein partially protect mice against caseous lymphadenitis.

BACKGROUND: Caseous lymphadenitis (CLA) is an infectious disease that affects small ruminants and is caused by Corynebacterium pseudotuberculosis. This disease is responsible for high economic losses due to condemnation and trim of infected carcasses, decreased leather and wool yield, loss of sales of breeding stock and deaths from internal involvement. Treatment is costly and ineffective; the most cost-effective strategy is timely immunisation. Various vaccine strategies have been tested, and recombinant vaccines are a promising alternative. Thus, in this study, different vaccine formulations using a recombinant protein (rCP40) and the CP09 live recombinant strain were evaluated. Five groups of 10 mice each were immunised with saline (G1), rCP40 (G2), CP09 (G3), a combination of CP09 and rCP40 (G4) and a heterologous prime-boost strategy (G5). Mice received two immunisations within 15 days. On day 30 after primary immunisation, all groups were challenged with a C. pseudotuberculosis virulent strain. Mice were monitored and mortality was recorded for 30 days after challenge. RESULTS: The G2, G4 and G5 groups showed high levels of IgG1 and IgG2a; G2 presented significant IgG2a production after virulent challenge in the absence of IgG1 and IgG3 induction. Thirty days after challenge, the mice survival rates were 20 (G1), 90 (G2), 50 (G3), 70 (G4) and 60% (G5). CONCLUSIONS: rCP40 is a promising target in the development of vaccines against caseous lymphadenitis.