ABSTRACT
Evaluate the effect of 12 wks of concurrent training (CT) in the extracellular matrix (ECM) of subcutaneous adipose tissue (SAT) in people living with HIV/AIDS (PLWHA). In the non-randomized clinical trial, 19 participants, 11 healthy (HIV-) and 18 PLWHA under the use of highly active antiretroviral therapy (HAART) for at least 1 year (HIV+). All participants engaged in a moderate-intensity CT program for 12 weeks, 3 times a week. Before and after CT, aerobic and strength performance were assessed, as well as anthropometric and biochemical blood profiles. In addition, SAT biopsies were performed for histologic and morphometric analyses. Statistical analysis was carried out with R Studio, using descriptive and inferential analysis, ANOVA test, and mixed-effect model (P < 0.05). HIV+ showed higher levels of very-low-density lipoproteins and triglycerides and lower levels of high-density lipoproteins at baseline than HIV- (P < 0.05). All groups showed improved aerobic and strength performances (P < 0.05). Both groups showed reduced adipocyte sizes after CT (P < 0.05). Lastly, HIV+ presented smaller adipocytes and higher elastic fiber deposition at baseline and decreased after training only in HIV+, similar to the HIV group. Thus, CT in PLWHA promoted a decrease in the size heterogeneity of adipocytes and elastic fiber deposition, remodeling the ECM, and improving the SAT fibrosis profile. Brazilian Clinical Trials Registry (ensaiosclinicos.gov.br - UTN: U1111-1214-3022). Novelty: Adipose tissue fibrosis is improved by training in people living with HIV. Concurrent training remodels adipose tissue extracellular matrix.
Subject(s)
Exercise/physiology , Extracellular Matrix/metabolism , HIV Infections/metabolism , HIV Infections/pathology , Subcutaneous Fat/metabolism , Adipocytes/pathology , Adult , Antiretroviral Therapy, Highly Active/adverse effects , Collagen/metabolism , Elastic Tissue/pathology , HIV Infections/drug therapy , Humans , Lipoproteins, HDL/blood , Lipoproteins, LDL/blood , Middle Aged , Muscle Strength/physiology , Physical Conditioning, Human/methods , Physical Conditioning, Human/physiology , Real-Time Polymerase Chain Reaction , Triglycerides/bloodABSTRACT
The sugarcane cystatin (CaneCPI-5) was recently cloned and showed strong binding force to dental enamel and protection against initial erosion. However, evaluations on its safety and efficacy in a situation closer to the clinical condition are necessary. In the present study we analyzed 1) the cytotoxicity of CaneCPI-5 on human gingival fibroblasts (HGFs); 2) the ability of CaneCPI-5 to reduce enamel erosion and erosion+abrasion in situ. In part 1, HGFs were treated with CaneCPI-5 (0.025, 0.05, 0.1, 0.5 or 1.0 mg/mL) or no treatment (control). The cytotoxicity was assessed after 60 s and 24 h by mitochondrial activity (MTT), confocal microscopy, and hematoxylin/eosin staining. In part 2, 15 volunteers participated in a double-blind crossover protocol consisting of 3 phases, according to the following treatments: 1) 0.1 mg/mL CaneCPI-5; 2) SnCl2/NaF/AmF (Elmex; positive control); 3) water (negative control). The volunteers wore an appliance containing 4 bovine enamel specimens for 5 d. Each day, the specimens were individually treated with 50 µL of the tested solutions per 60 s and then subjected to erosive challenges (0.1% citric acid, pH 2.5, for 90 s, 4 times per day). After the first and last erosive challenge each day, 2 samples were abraded (toothbrushing, 15 s). Enamel wear was measured by contact profilometry. One or two-way analysis of variance (ANOVA)/Tukey's or Sidak's tests (P < 0.05) were applied. Regardless of the concentration and the experimental time, CaneCPI-5 did not decrease the cell viability compared to the negative control (P < 0.05). Erosion+abrasion led to significantly greater wear compared to erosion only. For both conditions, the lowest wear was found for SnCl2 and CaneCPI-5, which did not differ significantly from each other, but showed significant protection when compared to the negative control. In conclusion, CaneCPI-5 is safe on HGFs and reduces enamel erosive wear to the same extent as a commercial solution used to control erosive tooth wear (ETW).
Subject(s)
Cystatins , Tooth Abrasion , Tooth Erosion , Tooth Wear , Animals , Cattle , Cross-Over Studies , Dental Enamel , Humans , Tooth Erosion/chemically induced , Tooth Erosion/prevention & control , ToothbrushingABSTRACT
Objetivo: O objetivo do presente estudo foi verificar os níveis de condição, composição corporal e pressão arterial de policiais rodoviários do estado do Paraná - Brasil. Método: Fizeram parte da amostra 52 oficiais do sexo masculino (idade: 38,3 ± 6,3 anos, massa corporal: 89,6 ± 18,4 kg) de diferentes patentes. Foram realizadas diversas mensurações para obtenção do índice de massa corporal (IMC), circunferência de cintura (CC), relação cintura/quadril (RCQ), composição corporal por meio da espessura de dobras cutâneas, potência aeróbia estimada indiretamente em teste de esforço ergométrico, resistência muscular localizada (RML) de membros superiores e abdominal e os níveis pressóricos foram aferidos por método auscultatório. Resultados: Considerando as variáveis analisadas, os policiais rodoviários apresentaram IMC de 28,6 ± 4,8 kg/m2, risco cardiovascular elevado (95,4 ± 10,8 cm) para CC e alto (0,92 ± 0,05) para RCQ. O percentual de gordura corporal apresentou-se acima dos valores recomendáveis (23,6 ± 4,3 %) para saúde, a potência aeróbia estimada foi considerada boa (34,8 ± 1,1 ml/kg/min), a RML de membros superiores (21 ± 8 repetições) e foi obtida por realização dos testes de abdominal e flexão de braço, respectivamente (28 ± 8 repetições) foram classificadas como média e uma parcela importante dos oficiais (23 %) mostraram-se com níveis pressóricos elevados. Conclusão: Os policiais militares rodoviários mostraram-se com níveis inadequados de condição física, apresentando excesso de peso e adiposidade corporais, e, uma parcela importante, exibiu níveis pressóricos elevados, sugerindo elevado risco cardiovascular
Objetivo. Evaluar los niveles de aptitud, composición corporal y de la presión arterial de la policía de carreteras en el estado de Paraná, Brasil. Método. Se analizó a 52 oficiales del sexo masculino (38,3 ± 6,3 años, y 89,6 ± 18,4 kg). Fueron realizadas diferentes mediciones para la obtención del índice de masa corporal (IMC); la circunferencia de cintura (CC); el índice cintura/cadera (ICC). La composición corporal se midió por medio del espesor del pliegue cutáneo; la potencia aeróbica se estimó indirectamente mediante una prueba de esfuerzo en una cinta ergométrica; la fuerza muscular de los miembros superiores se midió mediante el número de flexiones y la del abdomen por el número de abdominales y la presión arterial fue medida por auscultación. Resultados. Los policías presentaban un IMC clasificado como obesidad leve (28,6 ± 4,8 kg/m2); la CC de alto riesgo (95,4 ± 10,8 cm); el ICC alto (0,92 ± 0,05); el porcentaje de grasa corporal por encima de los valores recomendados (23,6 ± 4,3 %); la potencia aeróbica buena (34,8 ± 1,1 ml/kg/min); la RM de los miembros superiores (21 ± 8 repeticiones) y del abdomen (28 ± 8 repeticiones) medias, y una proporción significativa (23 %) de los policías presentaron niveles de presión arterial alterada. Conclusión. En base a estos resultados, fue posible verificar que aunque la policía ha mantenido buenos niveles de potencia aeróbica y fuerza muscular, presentaron exceso de peso e índices que indican alto riesgo cardiovascular (AU)
Objective. The aim of this study was to assess the physical fitness, body composition and blood pressure of highway police officers in the state of Paraná, Brazil. Method. The sample consisted of 52 male (38.3 ± 6.3 years old, 89.6 ± 18.4 kg) where the following determinations were performed: body mass index (BMI); waist circumference (WC); waist/hip ratio (WHR); body composition (skinfold thickness); aerobic power (indirectly estimated in a treadmill test); muscle strength of the upper limbs was measured by the number of push-ups and abdominal strength by the number of crunches (ES) and blood pressure (measured by auscultatory method). Results. The highway police officers had a BMI classified as mild obesity (28.6 ± 4.8 kg/m2), and a higher cardiovascular risk as determined by WC (95.4 ± 10.8 cm) and WHR (0.92 ± 0.05). The percentage of body fat was above the recommended values (23.6 ± 4.3 %) but the aerobic power was considered good (34.8 ± 1.1 ml/kg/min). Mean ES upper body (21 ± 8 repetitions) and abdomen (28 ± 8 repetitions) were qualified as fair but mean blood pressure was considered high in 23 % of the police officers. Conclusion. Based on our results it was possible to conclude that although the police officers presented good levels of aerobic power and muscle strength, they are overweight and showed a higher cardiovascular risk (AU)
Subject(s)
Humans , Male , Physical Conditioning, Human/physiology , Cardiovascular Diseases/epidemiology , Overweight/epidemiology , Hypertension/epidemiology , Police , Risk Factors , Adiposity , Body Composition , Body Weights and Measures/statistics & numerical data , Body Mass IndexABSTRACT
Creatine is widely used by athletes as an ergogenic resource. The aim of this study was to evaluate the influence of creatine supplementation on the duodenum of rats submitted to physical training. The number and myenteric neuronal cell bodies as well mucosal and muscular tunic morphometry were evaluated. Control animals received a standard chow for 8 weeks, and the treated ones received the standard chow for 4 weeks and were later fed with the same chow but added with 2% creatine. Animals were divided in groups: sedentary, sedentary supplemented with creatine, trained and trained supplemented with creatine. The training consisted in treadmill running for 8 weeks. Duodenal samples were either processed for whole mount preparations or for paraffin embedding and hematoxylin-eosin staining for histological and morpho metric studies of the mucosa, the muscular tunic and myenteric neurons. It was observed that neither creatine nor physical training alone promoted alterations in muscular tunic thickness, villus height or crypts depth, however, a reduction in these parameters was observed when both were associated. The number of myenteric neurons was unchanged, but the neuronal cell body area was reduced in trained animals but not when training and creatine was associated, suggesting a neuroprotector role of this substance.(AU)
Subject(s)
Male , Animals , Rats , Myenteric Plexus/drug effects , Neurons/drug effects , Physical Conditioning, Animal/physiology , Myenteric Plexus/cytology , Rats, WistarABSTRACT
Creatine is widely used by athletes as an ergogenic resource. The aim of this study was to evaluate the influence of creatine supplementation on the duodenum of rats submitted to physical training. The number and myenteric neuronal cell bodies as well mucosal and muscular tunic morphometry were evaluated. Control animals received a standard chow for 8 weeks, and the treated ones received the standard chow for 4 weeks and were later fed with the same chow but added with 2% creatine. Animals were divided in groups: sedentary, sedentary supplemented with creatine, trained and trained supplemented with creatine. The training consisted in treadmill running for 8 weeks. Duodenal samples were either processed for whole mount preparations or for paraffin embedding and hematoxylin-eosin staining for histological and morpho metric studies of the mucosa, the muscular tunic and myenteric neurons. It was observed that neither creatine nor physical training alone promoted alterations in muscular tunic thickness, villus height or crypts depth, however, a reduction in these parameters was observed when both were associated. The number of myenteric neurons was unchanged, but the neuronal cell body area was reduced in trained animals but not when training and creatine was associated, suggesting a neuroprotector role of this substance.
Subject(s)
Male , Animals , Rats , Physical Conditioning, Animal/physiology , Neurons , Myenteric Plexus , Myenteric Plexus/cytology , Rats, WistarABSTRACT
The present study examined whether the running speed at the lactate threshold estimated by the maximal deviation method (LT (Dmax)) is highly correlated and in agreement with 10-km road race performance (S (10 km)) in middle-aged female runners. Additionally, the LT (Dmax) was compared with the visual detection of the inflection point (LT (Visual)), the fixed lactate level of 4 mmol.L (-1) (LT (4)) and the peak speed (S (peak)) in relation to performance. Sixteen middle-aged, recreational female runners performed a discontinuous, incremental treadmill test. The initial speed was set at 7 km.h (-1), and this speed was increased every 3 min by 1 km.h (-1) with a 30-s rest between the stages used for earlobe capillary blood sample collection. All of the participants took part in the same local 10-km road race, and S (10 km) mean speed was calculated. The speeds (mean ± SD) were 10.5 ± 1.0 (S (10 km)), 10.5 ± 1.0 (LT (Visual)), 10.9 ± 0.9 (LT (Dmax)), 11.4 ± 1.3 (LT (4)) and 13.5 ± 1.1 km.h (-1) (S (peak)). The LT (Dmax) had the narrowest limits of agreement (0.3 ± 0.4 km.h (-1)) and was the most highly correlated with the S (10 km) ( R=0.98), followed by the S (peak) ( R=0.95), LT (4) ( R=0.85) and LT (Visual) ( R=0.81). In conclusion, the LT (Dmax) should be more widely used to estimate long-distance performance and to verify improvements in training.
Subject(s)
Exercise Test/methods , Lactic Acid/metabolism , Physical Endurance/physiology , Running/physiology , Adult , Female , Humans , Middle AgedABSTRACT
Over the past century, the Brazilian Atlantic forest has been reduced to small, isolated fragments of forest. Reproductive isolation theories predict a loss of genetic diversity and increases in inbreeding and spatial genetic structure (SGS) in such populations. We analysed eight microsatellite loci to investigate the pollen and seed dispersal patterns, genetic diversity, inbreeding and SGS of the tropical tree Copaifera langsdorffii in a small (4.8 ha), isolated population. All 112 adult trees and 128 seedlings found in the stand were sampled, mapped and genotyped. Seedlings had significantly lower levels of genetic diversity (A=16.5±0.45, mean±95% s.e.; H(e)=0.838±0.006) than did adult trees (A=23.2±0.81; H(e)=0.893±0.030). Parentage analysis did not indicate any seed immigration (m(seeds)=0) and the pollen immigration rate was very low (m(pollen)=0.047). The average distance of realized pollen dispersal within the stand was 94 m, with 81% of the pollen travelling <150 m. A significant negative correlation was found between the frequency and distance of pollen dispersal (r=-0.79, P<0.01), indicating that short-distance pollinations were more frequent. A significant SGS for both adults (â¼50 m) and seedlings (â¼20 m) was also found, indicating that most of the seeds were dispersed over short distances. The results suggested that the spatial isolation of populations by habitat fragmentation can restrict seed and pollen gene flow, increase SGS and affect the genetic diversity of future generations.
Subject(s)
Fabaceae/genetics , Gene Flow , Genetic Variation , Pollen/genetics , Seeds/genetics , Trees/genetics , Brazil , Population , Seedlings/genetics , Seedlings/physiologyABSTRACT
Acidic soft drinks are potentially erosive for dental hard tissues. This in vitro study evaluated the effect of calcium, fluoride, iron and phosphate, supplemented alone or in combination to a commercial citric acid-based carbonated beverage on dental erosion. Ninety enamel samples (4 x 4 x 3 mm) were randomly allocated to nine groups (n = 10): G1-pure beverage (control); G2-with 1 mM Ca; G3-with 0.047 mM F; G4-with 1 mM Fe; G5-with 1 mM P; G6 - with 1 mM Ca and 0.047 mM F; G7-with 1 mM Ca and 1 mM P; G8-with 1 mM Fe and 0.047 mM F; and G9-with 1 mM Ca, 1 mM P, 0.047 mM F and 1.0 mM Fe. The samples were subjected to six pH cycles over a 24-h period. In each cycle, the samples were immersed in pure or modified beverage (1 min) and in artificial saliva (59 min). During the remaining period (18 h), the samples were maintained in artificial saliva. Enamel loss was assessed by profilometry (microm). Data were tested using ANOVA and Tukey's tests (p < 0.05). Highest enamel losses were observed in the control group (G1) and in the groups containing Fe (G4 and G8). The groups containing Ca (G2 and G6) showed significantly less wear compared to control. In conclusion, the modification of an erosive soft drink with low concentrations of Ca with or without F may reduced its erosive potential.
Subject(s)
Calcium, Dietary/adverse effects , Carbonated Beverages/adverse effects , Dental Enamel/drug effects , Fluorides/administration & dosage , Iron, Dietary/adverse effects , Phosphates/administration & dosage , Analysis of Variance , Animals , Cattle , Dental Enamel Solubility/drug effects , Hardness , Hydrogen-Ion Concentration , Random Allocation , Saliva, Artificial , Tooth Erosion/prevention & controlABSTRACT
UNLABELLED: The chronic pancreatitis (CP) may evolve with low insulin levels and develop clinical picture of diabetes mellitus. Low seric levels of insulin and C peptide after stimulus has also been described in asymptomatic alcoholics even with normal glycemic curves. It is known that the chronic alcoholism is the main etiological factor of CP and hepatic diseases, and that the insulin produced by the pancreas is metabolized mainly by the liver. High levels of periferic insulin are described in hepatic cirrhosis due to decrease of hepatic metabolization alone or associated to increase of periferic resistance. AIM: In alcoholics with minimal hepatic lesions to evaluate the seric insulin and glucose levels after stimulus with intravenous glucose. METHODS: In 8 alcoholic patients with minimal hepatic lesions characterized by hepatic biopsy, and 26 non-alcoholics, healthy controls, it was studied the serum glucose and insulin levels in basal time, 1, 3, 5, and 10 minutes after stimulus with intravenous glucose (0.5 g/kg). RESULTS: The insulin means in time 1, 3 minutes and total integrated response after stimulus were lower (p < 0.05) in alcoholic group than in control, even with normal glucose curves. CONCLUSION: Alcoholics with minimal hepatic lesions showed low seric insulin levels after glucose stimulus, similar to former observations in asymptomatic alcoholics, indicating hypofunction of pancreatic B cells.
Subject(s)
Alcoholism/metabolism , Insulin/deficiency , Liver Diseases/metabolism , Adult , Blood Glucose/analysis , Female , Glucose/administration & dosage , Humans , Insulin/blood , Male , Middle AgedABSTRACT
OBJETIVO: Em alcoolistas portadores de lesões hepáticas mínimas avaliar os níveis de glicose e insulina séricas após estímulo com glicose intravenosa. MÉTODOS: Em oito etilistas, portadores de alterações hepáticas mínimas, caracteriza por biópsia hepática, e em 26 controles sadios não-alcoólicos, foram estudados os níveis glicêmicos e insulinêmicos (RIE) nos tempos 1, 3, 5, e 10 minutos após estímulo com glicose intravenosa (0.5g/Kg de peso). RESULTADOS: As médias da insulina sérica dos tempos 1, 3 minutos e resposta integrada total (RIT-10min) após estímulo foram menores no grupo alcoolista em relação ao controle (p < 0,05) apesar de curvas glicêmicas normais. CONCLUSÃO: Etilistas crônicos com lesões hepáticas mínimas, à semelhança do observado anteriormente em etilistas assintomáticos, apresentam níveis diminuídos de insulina sérica, mas com curvas glicêmicas normais, após estímulo com glicose intravenosa, indicando hipofunção de células beta do pâncreas.
Subject(s)
Humans , Male , Female , Adult , Middle Aged , Alcoholism/metabolism , Insulin/deficiency , Liver Diseases, Alcoholic/metabolism , Blood Glucose/analysis , Glucose/administration & dosage , Insulin/blood , Liver/pathologyABSTRACT
Although the pineal gland influences several physiological systems, only a few studies have investigated its role in the intermediary metabolism. In the present study, male Wistar rats, pinealectomized or sham-operated 6 wk before the experiment, were submitted to both intravenous glucose tolerance tests (IVGTT) and insulin binding as well as glucose transport assays in isolated adipocytes. The insulin receptor tyrosine kinase activity was assessed in liver and muscle. The insulin secretory response during the IVGTT was impaired, particularly in the afternoon, and the glucose transport responsiveness was 33% lower in pinealectomized rats. However, no difference was observed in the insulin receptor number of adipocytes between groups as well as in insulin-stimulated tyrosine kinase activity, indicating that the initial steps in the insulin signaling were well conserved. Conversely, a 40% reduction in adipose tissue GLUT-4 content was detected. In conclusion, pinealectomy is responsible for both impaired insulin secretion and action, emphasizing the influence of the pineal gland on glucose metabolism.
Subject(s)
Glucose Intolerance/etiology , Hypoglycemic Agents/pharmacology , Insulin/pharmacology , Muscle Proteins , Pineal Gland/physiology , Adipose Tissue/cytology , Adipose Tissue/drug effects , Animals , Biological Transport/physiology , Deoxyglucose/pharmacokinetics , Glucose/metabolism , Glucose Tolerance Test , Glucose Transporter Type 4 , Insulin/metabolism , Liver/enzymology , Male , Monosaccharide Transport Proteins/metabolism , Muscle, Skeletal/enzymology , Rats , Rats, Wistar , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, Insulin/metabolismABSTRACT
A lectin was purified from seeds of Erythrina velutina forma aurantiaca by affinity chromatography on cross-linked guar gum. The lectin is a potent agglutinin for human (minimal concentration of protein able to cause visible agglutination of a 2% erythrocyte suspension varying from 1 to 4 micrograms/ml), rabbit (4 micrograms/ml) and chicken erythrocytes (8 micrograms/ml) but presented low activity against cow (250 micrograms/ml) or sheep (333 micrograms/ml) blood cells. Hemagglutination of human O+ erythrocytes was inhibited by D-lactose (0.2 mM) > D-galactose (0.8 mM) > D-raffinose (2.1 mM). At pH 7.5, chromatography on a Superose 12 HR 10/30 column showed that the lectin was primarily a dimer (56.0 kDa) composed of two identical subunits (31.6 kDa each). A small amount of a tetrameric form was also apparently present. The lectin is a glycoprotein (7.3% carbohydrate), has a pI of 4.5, contains high levels of acidic (Asp and Glu, 64.2 and 51.6 residues/mol, respectively) and hydroxy amino acids (Ser and Thr, 42.9 and 38.5 residues/mol, respectively) but relatively low amounts of sulfur amino acids (Cys and Met, 1.0 and 5.0 residues/mol, respectively) and has an N-terminal sequence of Val-Glu-Thr-Ile/Leu-Pro-Phe-Ser. Its hemagglutinating activity was abolished by heating at 70 degrees C for 10 min. The activation energy (delta G') required for denaturation measured by loss of hemagglutination activity was 24.87 kcal/mol. In rats, the purified lectin (100 micrograms) induced neutrophil migration into the peritoneal cavity (3.7 +/- 0.6 x 10(6) neutrophils/ml) or into the air pouch (2.75 +/- 0.25 x 10(6) neutrophils/ml), 8 and 10 times greater than the negative control, respectively.
Subject(s)
Erythrina/chemistry , Lectins/chemistry , Plants, Medicinal , Animals , Brazil , Humans , Lectins/isolation & purification , Plant Lectins , Rats , Rats, Wistar , Seeds/chemistryABSTRACT
A lectin was purified from seeds of Erythrina velutina forma aurantiaca by affinity chromatography on cross-linked guargum. The lectin is a potent agglutinin for human (minimal concentration of protein able to cause visible agglutination of a 2 per cent erythrocyte suspension varying from 1 to 4 mug/ml), rabbit(4 mug/ml) and chicken erythrocytes (8 mug/ml) but presented low activity against cow (250 mug/ml) or sheep (333 mug/ml) blood cells. Hemagglutination of human O+ erythrocytes was inhibited by D-lactose (0.2 mM) > D-galactose(0.8 mM) > D-raffinose (2.1 mM). At pH 7.5, chromatography on a Superose 12 HR 10/30 column showed that the lectin was primarily a dimer (56.0 kDa) composed of two identical subunits (31.6 kDa each). A small amount of a tetrameric form was also apparently present. The lectin is a glycoprotein (7.3 per cent carbohydrate), has a pI of 4.5, contains high levels of acidic (Asp and Glu, 64.2 and 51.6 residues/mol, respectively) and hydroxy amino acids (Ser and Thr, 42.9 and 38.5 residues/mol, respectively) but relatively low amounts of sulfur amino acids (Cys and Met, 1.0 and 5.0 residues/mol, respectively) and has an N-terminal sequence of Val-Glu-Thr-Ile/Leu-Pro-Phe-Ser. Its hemagglutinating activity was abolished by heating at 70 degrees Celsius for 10 min. The activation energy (delta G') required for denaturation measured by loss of hemagglutination activity was 24.87 kcal/mol. In rats, the purified lectin (100 mug) induced neutrophil migration into the peritoneal cavity (3.7 ñ 0.6 x 10(6) neutrophils/ml) or into the air pouch (2.75 ñ 0.25 x 10(6) neutrophils/ml), 8 and 10 times greater than the negative control, respectively.