Physicochemical Characterization, Antioxidant, and Proliferative Activity of Colombian Propolis Extracts: A Comparative Study.

Propolis extracts have been widely studied due to their popularity in traditional medicine, presenting incredible biodiversity. This study aimed to analyze propolis extracts' phytochemical, physicochemical, and biological activities from four different biogeographic zones of the Huila region (Colombia). The raw material samples were collected by the scraping method and the ethanolic extracts (EEPs) were obtained by cold maceration with ethanol (96%). The physicochemical and sensory characterization was carried out according to the protocols recommended by the Brazilian Ministry of Agriculture and the main components of the EEPs were identified by LC-HRMS analysis. The determination of total phenols and flavonoids was carried out using colorimetric techniques. The antioxidant activity, cytotoxicity, and cell cycle regulation analyses in L929 and HGnF cells were evaluated using DPPH, Alamar Blue, and 7-amino actinomycin D (7-AAD) assays. The propolis samples presented an average yield of 33.1%, humidity between 1.6 and 2.8%, melting point between 54 and 62 °C, ashes between 1.40 and 2.19%, and waxes of 6.6-17.9%, respectively. The sensory characteristics of all samples were heterogeneous, complying with the quality specifications established by international standards. The polyphenolic and total flavonoid content was representative in the samples from Quebradon (255.9 ± 9.2 mg GAE/g, 543.1 ± 8.4 mg QE/g) and Arcadia (543.1 ± 8.4 mg GAE/g, 32.5 ± 1.18 g QE/g) (p < 0.05) that correlated with high antioxidant activity (Quebradon: 37.2 ± 1.2 µmol/g, Arcadia: 38.19 ± 0.7 µmol/g). In the chemical composition analysis, 19 compounds were characterized as phenolic acids and flavonoids, the most representative being chrysoeriol-O-methyl-ether, ellagic acid, and 3,4-O-dimethylcaffeic acid. Regarding biological activity, Quebradon and Arcadia propolis presented low toxicity with IC50 of 2.83 ± 2.3 mg/mL and 4.28 ± 1.4 mg/mL in HGnF cells, respectively, and an arrest of the cell cycle in the G2/M phase of 71.6% and 50.8% compared to the control (11.9%) (p < 0.05). In general, the results of this study contribute to the identification of valid quality criteria to evaluate Colombian propolis, contributing to its study and chemical and biological characterization as a source of raw material for industrial and pharmaceutical use. In addition, Quebradon and Arcadia propolis can be important sources of bioactive molecules for the development of new drugs.

Down-regulation of human papillomavirus E6 oncogene and antiproliferative effect of Schisandra chinensis and Pueraria lobata natural extracts on Hela cell line.

ETHNOPHARMACOLOGICAL RELEVANCE: Cervical cancer is one of the most common malignancies in women that continues to be a public health problem worldwide. Human papillomavirus (HPV) infection is closely related as the causative agent of almost all cases of cervical cancer. Currently, there is no effective treatment for the persistence of HPV. Although vaccines have shown promising results in recent years, they are still a costly strategy for developing countries and have no therapeutic effect on existing infections, which is why the need arises to search for new strategies that can be used in treatment, suppressing oncogenic HPV and disease progression. Extracts of Schisandra Chinensis and Pueraria lobata have been used in traditional medicine, and it has been shown in recent years that some of their bioactive compounds have pharmacological, antioxidant, antitumor, apoptotic, and proliferation effects in HPV-positive cells. However, its mechanism of action has yet to be fully explored. AIM OF THE STUDY: The following study aimed to determine the chemical composition, antioxidant activity, and potential antiproliferative and viral oncogene effects of natural extracts of S. chinensis and P. lobata on HPV-18 positive cervical cancer cells. MATERIALS AND METHODS: The HPV-18-positive HeLa cells were treated for 24 and 48 h with the ethanolic extracts of S chinensis and P. lobata. Subsequently, cell viability was evaluated using the resazurin method, the effect on the cell cycle of the extracts (1.0, 10, and 100 µg/mL) was measured by flow cytometry, the gene of expression of the E6/E7, P53, BCL-2, and E2F-1 were determined by RT-PCR and the protein expression of p53, Ki-67, x|and Bcl-2 by immunohistochemistry. Additionally, the chemical characterization of the two extracts was carried out using LC-MS, and the total phenolics content (TPC), Total flavonoid content (TFC), and DPPH radical scavenging capacity were determined. Data were analyzed using the Mann-Whitney and Kruskal Wallis U test with GraphPad Prism 6 software. RESULTS: The natural extracts of Schisandra chinensis and Pueraria lobata induced down-regulation of E6 HPV oncogene (p<0.05) and a strong up-regulation of P53 (p<0.05), E2F-1 (p<0.05), and Bcl-2 (p<0.05) gene expression. Simultaneously, the natural extracts tend to increase the p53 protein levels and arrest the cell cycle of HeLa in the G1/S phase (p<0.05). Investigated extracts were characterized by the occurrence of bioactive lignans and isoflavones in S. chinensis and P. lobata, respectively. CONCLUSION: The extracts of S. chinensis and P. lobata within their chemical characterization mainly present lignan and isoflavone-type compounds, which are probably responsible for inhibiting the expression of the HPV E6 oncogene and inducing an increase in the expression of p53, Bcl -2 and E2F-1 producing cell cycle detection in S phase in HeLa cells. Therefore, these extracts are good candidates to continue studying their antiviral and antiproliferative potential in cells transformed by HPV.

Oral microbiome mediated inflammation, a potential inductor of vascular diseases: a comprehensive review.

The dysbiosis of the oral microbiome and vascular translocation of the periodontopathic microorganism to peripheral blood can cause local and systemic extra-oral inflammation. Microorganisms associated with the subgingival biofilm are readily translocated to the peripheral circulation, generating bacteremia and endotoxemia, increasing the inflammation in the vascular endothelium and resulting in endothelial dysfunction. This review aimed to demonstrate how the dysbiosis of the oral microbiome and the translocation of oral pathogen-induced inflammation to peripheral blood may be linked to cardiovascular diseases (CVDs). The dysbiosis of the oral microbiome can regulate blood pressure and activate endothelial dysfunction. Similarly, the passage of periodontal microorganisms into the peripheral circulation and their virulence factors have been associated with a vascular compartment with a great capacity to activate endothelial cells, monocytes, macrophages, and plaquettes and increase interleukin and chemokine secretion, as well as oxidative stress. This inflammatory process is related to atherosclerosis, hypertension, thrombosis, and stroke. Therefore, oral diseases could be involved in CVDs via inflammation. The preclinic and clinical evidence suggests that periodontal disease increases the proinflammatory markers associated with endothelial dysfunction. Likewise, the evidence from clinical studies of periodontal treatment in the long term evidenced the reduction of these markers and improved overall health in patients with CVDs.

Antiproliferative and Pro-Apoptotic Effects of a Phenolic-Rich Extract from Lycium barbarum Fruits on Human Papillomavirus (HPV) 16-Positive Head Cancer Cell Lines.

The in vitro antiproliferative activity of a phenolic-rich extract from Lycium barbarum fruits against head and neck HPV16 squamous cell carcinoma (OSCC) has been demonstrated, indicating for the first time that L. barbarum extract inhibits human papillomavirus (HPV) type 16 cell lines. Ethanol extract of L. barbarum was used for cell viability evaluation on SCC090, CAL27, and HGnF cell lines. After 24 and 48 h, the cell cycle effect of L. barbarum extract (at 1.0, 10, and 100 µg/mL) was measured via flow cytometry. In addition, the mRNA expression on E6/E7 and p53 via RT-PCR and the expression of p16, p53, Ki-67, and Bcl-2 via immunohistochemistry were also determined. Untreated cells, 20 µM cisplatin, and a Camellia sinensis-derived extract were used as negative and positive controls, respectively. We demonstrated that the studied L. barbarum extract resulted in G0/G1 arrest and S phase accumulation in SCC090 at 1.0 and 10 µg/mL. A reduction in mRNA levels of E6/E7 oncogenes (p < 0.05) with p53 overexpression was also observed through PCR, while immunohistochemical analyses indicated p16 overexpression (p > 0.05) and a decrease in p53 overexpression. The observed effects were associated with anticancer and immunomodulatory phenolics, such as flavonols/flavan-3-ols and tyramine-conjugated hydroxycinnamic acid amides, identified in the studied extract. These findings revealed that the phenolic-rich extract of L. barbarum fruits has promising properties to be considered further for developing new therapies against oral and oropharyngeal HPV lesions.

Repeated Porphyromonas gingivalis W83 exposure leads to release pro-inflammatory cytokynes and angiotensin II in coronary artery endothelial cells.

The role of Porphyromonas gingivalis (P. gingivalis) or its virulence factors, including lipopolysaccharide (LPS) not only has been related with periodontitis but also with endothelial dysfunction, a key mechanism involved in the genesis of atherosclerosis and hypertension that involving systemic inflammatory markers as angiotensin II (Ang II) and cytokines. This study compares the effect of repeated and unique exposures of P. gingivalis W83 LPS and live bacteria on the production and expression of inflammatory mediators and vasoconstrictor molecules with Ang II. Human coronary artery endothelial cells (HCAEC) were stimulated with purified LPS of P. gingivalis (1.0, 3.5 or 7.0 µg/mL) or serial dilutions of live bacteria (MOI 1: 100 - 1:0,1) at a single or repeated exposure for a time of 24 h. mRNA expression levels of AGTR1, AGTR2, IL-8, IL-1ß and MCP-1 were determined by RT-qPCR, and IL-6, MCP-1, IL-8, IL-1ß and GM-CSF levels were measured by flow cytometry, ELISA determined Ang II levels. Live bacteria in a single dose increased mRNA levels of AGTR1, and repeated doses increased mRNA levels of IL-8 and IL-1ß (p < 0.05). Repeated exposure of live-P. gingivalis induced significant production IL-6, MCP-1 and GM-CSF (p < 0.05). Moreover, these MCP-1, IL-6 and GM-CSF levels were greater than in cells treated with single exposure (p < 0.05), The expression of AGTR1 and production of Ang II induced by live-P. gingivalis W83 showed a vasomotor effect of whole bacteria in HCAEC more than LPS. In conclusion, the findings of this study suggest that repeated exposure of P. gingivalis in HCAEC induces the activation of proinflammatory and vasoconstrictor molecules that lead to endothelial dysfunction being a key mechanism of the onset and progression of arterial hypertension and atherosclerosis.

Croton schiedeanus Schltd prevents experimental hypertension in rats induced by nitric oxide deficit

Croton schiedeanus Schltd (N.V.: "almizclillo") is a plant used in traditional medicine as an antihypertensive in Colombia. It contains flavonoid, diterpenoid and fenilbutanoid metabolites that have vasodilatation effects linked to the NO/cGMP pathway. This work aimed to assess the capacity of a 96% EtOH extract to prevent the hypertension induced by nitric oxide (NO) deficiency in rats. The NO synthase inhibitor L-NAME (10 mg/kg/d, i.p) was administered during five weeks to three groups of rats (6-7 animals): C. Schiedeanus (200 mg/kg/d, p.o), enalapril (reference, 10 mg/kg/d, p.o) and vehicle (control: olive oil 1 ml/kg/d, p.o). In addition, the blank group received only vehicle. The arterial blood pressure (BP) and heart rate (HR) were measured daily for six weeks. After sacrificing the animals, the aortic rings were isolated, contraction was triggered with phenylephrine (PE 10-6 M) and relaxant responses were achieved with cumulative concentrations of acetylcholine (ACh, 10-10 - 10-4 M). L-NAME increased the systolic arterial pressure in the control group, attaining mean values of 131 mm Hg at week 5, whereas the C. schiedeanus, enalapril and blank groups maintained blood pressure under 100 mm Hg. The capacity of PE to contract aortic rings was greater in the C. schiedeanus, enalapril and blank groups than in the control group (2157, 2005, 1910 and 1646 mg, respectively). The pEC50 values for ACh were as follows: C. Schiedeanus (6.89) >enalapril (6.39) > blank (5.68) > control (5.09). These results give support to C. Schiedeanus as a natural antihypertensive source.

Croton schiedeanus Schltd (NV: "almizclillo") é utilizado na medicina tradicional da Colômbia para o tratamento da hipertensão arterial. Outras pesquisas demonstraram que a planta tem metabólitos como os flavonoides, os diterpenoides e os fenilbutanoides, os quais têm comprovados efeitos vasodilatadores vinculados com a via NO/GMPc. O objetivo deste estudo foi avaliar a capacidade do extrato de Croton schiedeanus Schltd em EtOH a 96% na prevenção da hipertensão induzida pela deficiência de óxido nítrico (NO), em ratos. O inibidor da NO sintetase L-NAME (10 mg/kg/d, ip) foi administrado durante cinco semanas em três grupos de ratos (6-7 animais): C. schiedeanus (200 mg/kg/d, v.o.), enalapril (referência, 10 mg/kg/d, v.o.) e o veículo (controle: azeite de oliva 1 mL/kg/d, v.o.). O grupo branco recebeu somente o veículo. A pressão sanguínea (BP) e a frequência cardíaca (FC) foram medidas diariamente em um período de seis semanas. Após o sacrifício, os anéis aórticos foram isolados e contraídos, utilizando fenilefrina (PE 10-6 M) e as respostas para a relaxação foram obtidas com doses acumulativas de acetilcolina (ACh, 10-10-10-4 M). Os resultados demonstraram que o L-NAME provocou incremento significativo da pressão nos ratos do grupo controle, obtendo-se valores médios de 131 mm Hg na quinta semana. No entanto, os grupos C. schiedeanus, enalapril e branco mantiveram a pressão arterial aos níveis médios iniciais 100 mm Hg. A capacidade da PE para fazer a contração dos anéis da aorta foi maior nos grupos C. schiedeanus, enalapril e branco do que no grupo controle (2157, 2005, 1910 and 1646 mg, respectivamente). Os valores de pCE50 de ACh foram os seguintes: C. schiedeanus (6,89) > enalapril (6,39) > branco (5,68) > controle (5,09). Pode-se afirmar que estes resultados dão suporte à C. schiedeanus como fonte natural anti-hipertensiva.

Expresión de sustancia P en la articulación temporomandibular / Substance P expression in the temporomandibular joint

Antecedentes: neuropéptidos como la sustancia P se relacionan con el desarrollo y progresión de enfermedad degenerativa. Estudios previos mostraron su papel en la respuesta vascular y nociceptiva en artritis, y su papel modulador en hperalgesia y dolor de tipo artrítico, comprobando su presencia en líquido sinovial de la articulación temporomandibular. Propósito: evaluar la presencia y contenido de sustancia P en tejido retrodiscal hiperplásico de la articulación temporomandibular en pacientes con enfermedad degenerativa articular, mediante radioinmunoensayo. Métodos: 7 pacientes mujeres (13 articulaciones) premenopáusicas, no embarazadas ni lactando por un año, fueron diagnosticadas con enfermedad articular (osteoartrosis). Se registraron niveles de dolor con escala visual análoga, donde 0 es ausencia de dolor y 16 dolor agudo; se clasificó la degeneración ósea entre leve, moderada y severa, de acuerdo con hallazgos de resonancia magnética nuclear. Las pacientes fueron sometidas a cirugía abierta de la articulación temporomandibular, donde se tomaron las muestras. Los especímenes se colocaron en bloques plásticos con medio congelante y se almacenaron a -70§C hasta la extracción del neuropéptido por radioinmunoensayo. Resultados: se estableció una relación directamente proporcional entre grade degeneración ósea y expresión de sustancia P, y entre clasificación de osteoartrosis con escala visual análoga. Los hallazgos muestran correlación definitiva entre niveles de dolor y expresión de sustancia P. Conclusiones: la sustancia P se expresa en el tejido retrodiscal de la articulación temporomandibular en humanos con enfermedad degenerativa articular, relacionándose directamente con niveles de osteoartrosis y particularmente con niveles de dolor

Expresión de neuropéptidos en la articulación temporomandibular, 1: GGRP y articulación temporomandibular / Neuropeptides expression in the temporomandibular joint, 1: CGRP and temporomandibular joint

Antecedentes: neuropéptidos como el péptido relacionado con el gen de la calcitonina (CGRP), sustancia P (SP) y neurokinina A (NKA) se relacionan con desarrollo y progresión de enfermedad degenerativa articular. Estudios previos mostraron su rol en respueta vascular y nociceptiva en artritis y su papel modulardor en hiperalgesia y dolor de tipo artrítico, comprobando su presencia en líquido sinovial de articulación temporomandibular. Propósito: evaluar presencia y contenido de neuropéptidos en tejidos retrodiscal hiperplásico de la articulación temporomandibular en pacientes con enfermedad degenerativa articular mediante radioinmunoensayo. Métodos: ocho pacientes de sexo femenino (15 articulaciones), premenopáusicas, no embarazadas ni lactando por un año, fueron diagnosticadas con enfermedad articular degenerativa (osteoartrosis). Se registraron niveles de dolor en escala visual análoga, donde 0 es ausencia de dolor y 16 dolor agudo; se clasificó la degeneración ósea entre leve, moderada y severa, de acuerdo con hallazgos de resonanacia nuclear magnética. Las pacientes fueron sometidas a cirugía abierta de ATM donde se tomaron las muestras. Los especímenes se colocaron en bloques plásticos con medio congelante y se almacenaron a -700C hasta la extracción de los neuropéptidos por radioinmunoensayo con el estuche específico para cada uno. Resultados: se estableció una relacion directamente proporcional entre grado de degeneración ósea y expresión de CGRP, y entre clasificación de osteoartrosis con escala visual análoga. Los hallazgos mostraron correlación definitiva entre niveles de dolor y expresión del péptido relacionado con el gen de la calcitonina. Conclusiones: CGRP, SP y NKA, si se expresan en tejido retrodiscal de ATM en humanos con enfermedad degenerativa articular, relacionándose CGRP directamente con niveles de osteoartrosis y dolor