Antimicrobial Activity of Extracts from In Vivo and In Vitro Propagated Lamium Album L. Plants

The antimicrobial activity of 18 different extracts from in vivo and in vitro grown L. album L. plants was evaluated against clinical bacteria and yeasts using the well diffusion method. All the used extracts demonstrated antibacterial activity, whereas only the water extracts from leaves (in vivo) possessed antifungal activity against Candida albicans NBIMCC 72 and Candida glabrata NBIMCC 8673 (14 and 20 mm diameter of inhibition zones and MIC 10 mg/ml, respectively). The methanol and ethanol extracts obtained from the in vitro propagated plants had a broader spectrum of antibacterial activity than those from in vivo plants, while the opposite tendency was observed for the chloroform extracts. All tested flower extracts possessed antimicrobial activity. The chloroform extract from in vivo flowers demonstrated the highest activity against E. faecalis NBIMCC 3915, S. aureus NBIMCC 3703, P. hauseri NBIMCC 1339 and P. aeruginosa NBIMCC 3700 (22 mm, 13 mm, 11 mm, 23 mm zone diameter of inhibition and MIC 0.313 mg/ml, respectively). The water extracts from leaves (both in vivo and in vitro) possessed higher antibacterial activity than extract from flowers. The obtained results showed that both in vivo and in vitro propagated L. album L. could be used as a source of antibacterial substances.     

Antioxidant Activity In Vitro and Hepatoprotective Effect of Phlomis Maximowiczii In Vivo

Background

A number of medicinal plants and there compounds played a major role in the treatment of hepatic disorders. They were widely used for the treatment of these disorders, and oxidant stress injury was one of the liver injury mechanisms. The present study evaluated the antioxidant activity and the hepatoprotective effect of each extracts of Phlomis maximowiczii.

Materials and Methods

The antioxidant activity was assayed by the methods of ABTS, FRAP and DPPH in vitro. Hepatoprotective effect of P. maximowiczii extracts was examined using carbon tetrachloride-induced acute liver injury in mice.

Results

P. maximowiczii n-butanol (PMBU) extract, ABTS (IC₅₀=18.96 µg/mL), DPPH (IC₅₀=25.15 µg/mL), and FRAP (RACT₅₀=2775.6±144.18 µmol/g), showed higher scavenging capacity than that of P. maximowiczii ethyl acetate (PMEA). The n-butanol extract could significantly reduce the level of GPT, GOT and MDA (P<0.05, P<0.001 and P<0.001, respectively) and increase the level of SOD (P<0.001), respectively.

Conclusion

The antioxidant activity of n-butanol extract in vitro was related with the level of MDA and SOD in vivo, and hepatoprotective effect of n-butanol extract also had relationship with its antioxidant activity in vivo.     

Characterization of the progressive sublines derived from a weakly malignant cloned cell line, ER-1, co-inoculated subcutaneously with a foreign body

We previously established an experimental model of tumor progression using a weakly malignant rat mammary carcinoma cell line, ER-1. Using this model, we demonstrated that ER-1 cells converted into highly tumorigenic and metastatic cells, ERpP, by s.c. co-inoculation with plastic plates. We here compared in vitro biological properties associated with malignancy of ER-1 cells with those of ERpP cells which were highly malignant when inoculated into syngeneic rats. In vitro growth rate of ERpP cells was higher than that of ER-1 cells under a low nutrient condition. Invasion capacity of ERpP cells to rat lung endothelial cell monolayer or reconstituted basement membrane, Matrigel, was higher than that of ER-1 cells. Migration of ERpP cells toward fibronectin or laminin was also significantly higher than that of ER-1 cells.There was no difference in gelatinolytic or plasminogen activator activity detected in conditioned media between ER-1 and ERpP cells. Furthermore, we found that ER-1 cells communicated better among themselves and with normal fibroblasts through gap junctions compared to ERpP cells. These results suggest that growth advan-tage in a poor nutrient condition, enhancement of cell motility, and loss or decrease of junctional communication may be associated with tumor progression of ER-1 cells. © Rapid Science 1998     

Hepatitis B Virus Vector Carries a Foreign Gene into Liver Cells In Vitro

Hepatitis B viruses (HBV) specifically target the liver, where they efficiently infect quiescent hepatocytes. Thus, HBV virus has potential to be used as vectors for liver-directed gene transfer. We constructed a new HBV-based vector system. It is composed of transfer vector for transferring a foreign gene, green fluorescence protein (GFP) gene, and a helper vector. When the transfer vector and the helper vector were cotransfected into HepG2 cells, the recombinant HBV (rHBV) particles were generated by trans-complementation between two vectors. The rHBV particles carrying the foreign gene were identified by the Southern blot assay. To test gene delivery and the transduction of the rHBV, we infected primary human hepatocytes and immortalized, HepG2 cells with rHBV in vitro. The results using fluorescence microscopy confirmed that the inserted GFP gene was successfully transferred and expressed both in primary human hepatocytes and HepG2 cells.     

In Vivo oligonucleotide-protein interactions in the blood

The interaction of oligonucleotides with serum proteins was studiedin vivo by the method of affinity modification. Alkylating oligonucleotide derivatives administered to animals induced the formation of oligonucleotide-protein complexes circulating in the blood for a long-time. Oligonucleotides in these complexes were partially protected from nucleases. The major oligonucleotide-binding proteins identified with specific antibodies were albumins and IgG. In the erythrocyte fraction no specific oligonucleotide-binding proteins were detected, while membrane-cytosolic leukocyte fraction contained an oligonucleotide-binding protein with a molecular weight of 72 kD. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 6, pp. 654–657, June, 1999     

Modeling of Microfabricated Microneedles for Minimally Invasive Drug Delivery, Sampling and Analysis

We compare simulation to analysis and experiments for flows in three microneedle geometries—straight, bent and filtered. The bent microneedle was found to have the highest fluid carrying capacity of 0.082 ml/sec at 138 kPa with a Reynolds number of 738. A microneedle with a built in microfilter had a flow rate of 0.07 ml/sec. Although the throughput of these microneedles is low they compare favorably with other microneedle designs. Laminar flow models were found to accurately predict the flow behavior through the microneedles. All computational modeling was performed with the CFDRC CFD-ACE + suite of software tools.     

In Vivo Accumulation of nitric oxide in blood vessels

Against the background of NO-synthase blockade, diethyldithiocarbamate had no effect on the tone of isolated rat aorta, but induced relaxation of aorta preparations isolated afterin vivo NO accumulation and isolated aorta incubated with dinitrosyl iron complex. Guanylate cyclase inhibitor methylene blue prevented the relaxation induced by diethyldithiocarbamate. These data suggest that accumulation of NO in the organism can result in its accumulation in the vessel wall. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 6, pp. 629–632, June, 1999     

Antioxidant Activities In Vitro and Hepatoprotective Effects of Nelumbo Nucifera Leaves In Vivo

Background

Herbal medicines played a major role in the treatment of hepatic disorders, and a number of medicinal plants and their compounds were widely used for the treatment of these disorders, and oxidant stress injury was one of the mechanism of liver injury.

Materials and Methods

Antioxidant activity of Nelumbo nucifera leaves (NU) extracts was assayed by the methods of scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzo-thiazoline-6-sulfonicacid) (ABTS) radical and ferric reducing antioxidant power (FRAP) in vitro. By intraperitoneal injection carbon tetrachloride (CCl₄) to establish acute liver injury model in mice, the levels of Glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), superoxide dismutase (SOD) and the content of and maleicdialdehyde (MDA) were detected to evaluate hepatoprotective effect of NU using corresponding test kit.

Results

EtOAC (NUEA) and n-BuOH extracts (NUBU) of N. nucifera leaves had good scavenging DPPH and ABTS radical activity and ferric reducing antioxidant power in vitro. DPPH radical scavenging activity and ferric reducing antioxidant power of NUEA (IC₅₀= 6.68±0.29 µg/mL, RACT₅₀=1749.82±67.03 µmol/g) and NUBU (IC₅₀= 4.61±0.01 µg/mL, RACT₅₀=1995.27±135.71 µmol/g ) were higher than that of BHT (IC₅₀=8.76±0.20 µg/mL, RACT₅₀=1581.68±97.41 µmol/g) and Dangfeiliganning (IC₅₀=28.06±0.17 µg/mL, RACT₅₀=1028.55±3.28 µmol/g). ABTS radical scavenging activity of NUEA (IC₅₀= 5.32±0.12 µg/mL) and NUBU (IC₅₀= 8.16±0.27 µg/mL) were higher than that of Dangfeiliganning (IC₅₀= 9.76±0.16 µg/mL). Thus, hepatoprotective effect of NUEA and NUBU was evaluated on CCl₄-induced acute liver injury mice. The results showed that the levels of GOT and GPT in each treatment group significantly decreased (p<0.001 and p<0.01, p<0.05, respectively) except for the group of NUEA (130.8 mg/kg) (p>0.05). The contents of malondialdehyde (MDA) in liver in groups of NUEA (523 mg/kg), NUBU (840.5 and 420.5 mg/kg, repectively) had significant decrease (p<0.001 and p<0.05, respectively), and the level of SOD in liver for each treatment group could significantly decrease (p<0.001, p<0.05, respectively).

Conclusion

NUEA and NUBU had significantly hepatoprotective effect for Calcium tetrachloride CCl₄-induced liver injury, which might be attributable to its antioxidant activity.     

Microneedle-based drug delivery: studies on delivery parameters and biocompatibility

There is a significant interest in the application of microneedles in intradermal drug delivery systems. Previous studies have demonstrated that skin permeation of drugs can be increased by orders of magnitude with microneedle insertion. In this study, emphasis is placed on the development of low cost, painless intradermal microneedle systems that can enhance the percutaneous drug permeation. Microneedles of octagonal pyramidal shape with the length of 150 mum were employed, and the capabilities of skin permeation enhancement under different delivery conditions were examined. The delivery parameters taken into account included the insertion time and the area of insertion. It was found that when solid microneedle arrays of 150 mum in length were pierced into human dermatomed skin for 5 to 60 s, microconduits with the depth of 50 to 80 mum were created to facilitate the percutaneous permeation of drugs. In percutaneous tests, it was demonstrated that the permeability coefficient of calcein (MW = 622.55) was significantly increased by 10(4) to 10(5) times compared to that on intact skin. In terms of biocompatibility, biological evaluation indicated a broad spectrum of safety for the microneedle system. These results suggest that the octagonal pyramidal microneedles can be an effective tool in developing novel intradermal drug delivery system.     

Particle Motion Within In Vitro Models of Stenosed Internal Carotid and Left Anterior Descending Coronary Arteries

Asymmetric 75% and 95% area reduction, transparent Sylgard stenotic models were operated under internal carotid artery (ICA) (Womersley parameter, =5.36, Re_mean=213 and 180, respectively, and Re_peak=734 and 410, respectively) and left anterior descending coronary artery (LAD) flow wave forms (=2.65,Re_mean=59 and 57, respectively, and Re_peak=137 and 94, respectively) to evaluate the effect of these conditions on particle residence times downstream of the stenoses. Amberlite particles (1.05 g/cm³, 400 m) were added to the fluid to simulate platelets and their motion through the stenotic region and were traced using a laser light sheet flow visualization method with pseudo-color display. Two-dimensional (2D) particle motions were recorded and particle washout in the stenotic throat and downstream section were computed for all cases. All four model cases demonstrated jetting through the stenosis which followed an arching pattern around a large separation zone downstream. Considerable mixing was observed within these vortex regions during high flow phases. Particle washout profiles showed no clear trend between the degrees of stenosis although particles downstream of the stenoses tended to remain longer for LAD conditions. The critical washout cycle (1% of particles remaining downstream of the stenosis), however, was longer for the 95% stenoses cases under each flow condition due to the larger protected region immediately downstream and maximal for the LAD 95% case. Results of this study suggest that particle residence times downstream of 75% and 95% stenoses (~ 3–6 s for ICA and ~ 8–10 s for LAD) exceed the minimum time for platelet adhesion (~ 1 s) for at least 1% of cells and, thus, may be sufficient to initiate thrombus formation under resting conditions. © 1998 Biomedical Engineering Society. PAC98: 8745Hw, 8722-q, 4727Wg, 4732Cc     

In Vitro secretion of tumor necrosis factor-α and interleukin-1 by placental macrophages in various outcomes of pregnancy

Production of tumor necrosis factor-α and interleukin-1β by placental cells obtained after term and premature labors with or without labor activity increases with an increase in the gestational period. The secretory activity of macrophages significantly increases during spontaneous premature labor in the second trimester of pregnancy (abortion) and decreases during normal term labor. The use of phorbol myristate acetate and lipopolysaccharide for stimulating functional activity of macrophages revealed the differences in cell responses in case of the presence or absence of spontaneous labor activity. Translated fromByulleten', Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 7, pp. 97–100, July, 1999     

Qualitative Phytochemical Screening and In Vitro Antimicrobial Effects of Methanol Stem Bark Extract of Ficus Thonningii (Moraceae)

The methanolic stem bark extract of Ficus thonningii (Moraceae) was subjected to preliminary phytochemical screening and in vitro antimicrobial tests. The phytochemical tests was carried out using standard methods of analysis and these investigations revealed the presence of alkaloids, anthraquinones, carbohydrates, flavonoids, saponins and tannins. The antimicrobial activity of the plant extract was assayed using the agar plate disc diffusion and nutrient broth dilution techniques. Test micro organisms were: Escherichia coli, Klebsiella spp, Pseudomonas aeruginosa, Salmonella typhi (Gram-negative), Staphylococcus aureus and Streptococcus spp. (Gram-positive). The extracts inhibited the growth of all the test organisms at different concentrations especially against Pseudomonas aeruginosa and Streptococcus spp. which had mean inhibition zone of 33.33±7.33 mm and 32.33±2.51 mm respectively. The results showed the MIC of 10 mg ml⁻¹ against pseudomonas and 1.25 against remaining organisms tested. The MBC against Staphylococcus aureus was 2.5 mg ml⁻¹ and that of Streptococcus spp. was found to be 0.625mg ml⁻¹. The extracts showed varied inhibitory activity against the organisms studied.     

In Vitro Cell Shearing Device to Investigate the Dynamic Response of Cells in a Controlled Hydrodynamic Environment

Mechanical stresses and strains play important roles in the normal growth and development of biological tissues, yet the cellular mechanisms of mechanotransduction have not been identified. A variety of in vitro systems for applying mechanical loads to cell populations have been developed to gain insight into these mechanisms. However, limitations in the ability to control precisely relevant aspects of the mechanical stimuli have obscured the physical relationships between mechanical loading and the biochemical signals that mediate the cellular response. We present a novel in vitro cell shearing device based on the principles of a cone and plate viscometer that utilizes microstepper motor technology to control independently the dynamic and steady components of a hydrodynamic shear-stress environment. Physical measurements of the cone velocity demonstrated faithful reproduction of user-defined input wave forms. Computational modeling of the fluid environment for the unsteady startup confirmed small inertial contributions and negligible secondary flows. Finally, we present experimental results demonstrating the onset rate dependence of functional and structural responses of endothelial cell cultures to dynamically applied shear stress. The controlled cell shearing device is a novel tool for elucidating mechanisms by which mechanical forces give rise to the biological signals that modulate cellular morphology and metabolism. © 2000 Biomedical Engineering Society. PAC00: 8780Rb, 8717-d     

In Vitro Antioxidant and In Vivo Hepatoprotective Activity of Leave Extract of Raphanus Sativus in Rats Using CCL4 Model

Background

Raphanus sativus is reported to have a variety of biological activities. This work screened the hepato-protective and antioxidant activity of ethanol (ERS), and aqueous (ARS), extracts of leaves of Raphanus sativus in Carbon tetrachloride (CCl₄), model in rats.

Material and Methods

The extracts were subjected to antioxidant tests (Total reducing power and Total phenolic content), and preliminary phytochemical screening. A pilot study was done on 100 and 300 mg/kg extracts, form which 300 mg was chosen for further experiments. The albino rats (200–250 grams), were divided into 5 groups of 6 animals each (n=6). There were three control groups comprising of normal control (normal saline −1ml/kg), negative control group (CCl₄ 1ml/kg in olive oil in a ratio of 1:1 v/v), and positive control group (Silymarin 50mg/kg). The Test drugs were given in a dose of 300 mg/kg for both ERS and ARS extract for 7 days. Biochemical parameters (AST, ALT, Alkaline phosphatase, Total Bilirubin), histo-pathological examination of liver and in vivo antioxidant tests [CAT, GSH and MDA] were done.

Results

The phytochemical study showed the presence of flavanoids, terpenoids, alkaloids, saponins and sterols. A dose dependent increase in the oxidative potential was observed in both the extracts with total phenolic content 70.1 and 44.4 GAE/g extract for ERS and ARS respectively. ERS 300mg/kg showed a significant (p<0.001) increase in levels of AST, ALT and alkaline phosphatase as compared to negative control (percentage hepatoprotection =45.3%) while ARS 300 mg/kg (p<.01) group showed 30% hepatoprotection. The GSH (p<0.001) and CAT (p<0.05) in ERS and ARS were significantly increased while MDA levels were decreased (P< 0.01), as compared negative control. The findings were confirmed histo-pathological examination.

Conclusion

The ethanol and aqueous extract of Raphanus sativus have partial hepatoprotection against CCl₄ toxicity.     

Phytochemical and In Vitro Antimicrobial Assay of the Leaf Extract of Newbouldia Laevis

The methanolic leaf extract of Newbouldia laevis was subjected to preliminary phytochemical screening and in-vitro antimicrobial tests. The extract revealed the presence of flavonoids, tannins, terpenes, steroidal and cardiac glycosides. The antimicrobial activity of the plant extract was assayed by the agar plate disc diffusion and nutrient broth dilution techniques. Test microorganisms were Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Salmonella typhi, Klebsiella spp. and Candida albicans; all the organisms were laboratory isolates. The extract inhibited the growth of all the test organisms especially against Klebsiella spp. and S. aureus which had mean inhibition zone of 42.3±1.5 and 32.3±1.5 mm respectively. The results showed minimum inhibitory concentration (MIC) of 1.563 mg/ml against Escherichia coli and Klebsiella spp. and 3.125 mg/ml against Pseudomonas aeruginosa, Staphylococcus aureus and Salmonella typhi. The minimal bactericidal concentration (MBC) against Escherichia coli and Staphylococcus aureus was 0.39 mg/ml. This study has justified the traditional use of this plant for the treatment of stomach discomfort, diarrhea, dysentery and as a remedy for wound healing whose causative agents are some of the organisms used in this study.     

Anthelmintic Efficacy of Nauclea Latifolia Extract Against Gastrointestinal Nematodes of Sheep: In Vitro and In Vivo Studies

Direct effects of Nauclea latifolia extracts on different gastrointestinal nematodes of sheep is described. In vivo and in vitro studies were conducted to determine possible anthelmintic effect of leaf extracts of Nauclea latifolia toward different ovine gastro intestinal nematodes. A larval development assay was used to investigate in vitro, the effect of aqueous and ethanolic extracts of N. latifolia towards strongyles larvae. The development and survival of infective larvae (L₃) was assessed and best-fit LC₅₀ values were computed by global model of non-linear regression analysis curve-fitting (95% CI). Twenty sheep harbouring naturally acquired gastrointestinal nematodes were treated with oral administration of ethanolic extracts at a dose rate of 125 mg/kg, 250 mg/kg and 500mg/kg to evaluate therapeutic efficacy, in vivo.The presence of the extracts in the cultures decreased the survival of larvae. The LC₅₀ of aqueous and ethanolic extract were 0.704 and 0.650 mg/ml respectively and differ significantly (P<0.05, paired t test). Faecal egg counts (FEC) on day 12 after treatment showed that the extract is effective, relative to control (1-way ANOVA, Dunnett''s multiple comparison test), at 500mg/kg against Haemonchus spp, Trichostrongylus spp (p<0.05), Strongyloides spp (P < 0.01); at 250mg/kg against Trichuris spp (P < 0.01) and ineffective against Oesophagostomum spp (p>0.05). The effect of doses is extremely significant; the day after treatment is sometimes significant while interaction between dose and day after treatment is insignificant (2-way ANOVA).N. latifolia extract could therefore find application in the control of helminth in livestock, by the ethnoveterinary medicine approach.     

In - Vitro Propagation and Antimycotic Potential of Extracts and Essential Oil of Roots of Aristolochia Bracteolata Linn. (Aristolochiaceae)

In spite of the therapeutic importance of Aristolochia bracteolata Linn. in Nigerian ethnomedicine, it is largely collected from the wild. Owing to the acclaimed potency of the plant and the difficulty in treating candidiasis, the anticandidal activity and in vitro propagation of the plant were investigated. Phytochemical screening and preparation of extracts of the roots were done using standard procedures. Clinical isolates of Candida albicans were screened against extracts and essential oil of Aristolochia bracteolata root using agar-well diffusion method. Minimum Inhibitory Concentration (MIC) of the ethanol extract was determined using broth dilution method. The nodal cuttings of A. bracteolata were cultured on Murashige and Skoog (MS) basal media. A. bracteolata contained alkaloids, saponins and cardenolides. The water extract was inactive on all isolates. The ethanol extract (500 mg/ml) and essential oil (undiluted) exhibited anticandidal activity on 9 out of 10 isolates at 10¹ − 10⁶ cfu/ml inoculums concentration. Green growth and callus formation were observed in explants cultured on MS basal media after 30 days. A. bracteolata could be a source of anticandidal phytomedicine and the in vitro propagation confirmed its sustainability as anticandidal agent.     

Characterization of Superparamagnetic Nanoparticle Interactions with Extracellular Matrix in an in Vitro System

Controlled dispersion of therapeutic agents within liquid- and gel-filled cavities represents a barrier to treatment of some cancers and other pathological states. Interstitial delivery is compromised by the poor mobility of macromolecules and larger nanoscale structures. We developed an in vitro system to quantify the suitability of superparamagnetic nanoparticles (SPM NPs) as a site-specific therapeutic vehicle for delivery through fluid- and gel-based systems. SPM NP motion was induced by an external magnetic field. NP migration was modulated by NP concentration and surface coating. 135 nanometer radius PEGylated NPs moved through the extracellular matrix with an average velocity of 1.5 mm h⁻¹, suitable for some clinical applications. Increasing the SPM NP radius to 400 nm while maintaining the same per NP magnetic susceptibility resulted in a greater than 1000-fold reduction in magnetic mobility, to less than 0.01 mm h⁻¹. The critical influence of NP size on gel permeation was also observed in silica-coated 135 nm SPM NPs that aggregated under the experimental conditions. Aggregation played a critical role in determining the behavior of the nanoparticles. SPM NPs allow significant free-solution mobility to specific sites within a cavity and generate sufficient force to penetrate common in vivo gels.     

In vitro validation of a right ventricular thermodilution ejection fraction system

Right ventricular ejection fraction (RVEF) is used clinically as an index of right ventricular (RV) pump function. Clinical measurements of RVEF are complicated by the need for complex imaging equipment to compute RV volumes. Recently, the use of thermodilution (TD) methods have been suggested as a simplified means to measure RVEF (RVEF_TD) in patients using rapid response thermistors. Validation, however, by comparison of RVEF_TD and other methodsin vivo, is difficult. Accordingly, thermodilution derived EF measurements (EF_TD) were compared to known values using anin vitro system, with known ejection fractions (EF) set from 17–78% and stroke rates varying independently from 50–100 strokes/min. EF_TD was computed by fitting the downslope of the TD curve to a monoexponential function and computing the time constant of thermal decay. A significant correlation existed between EF_TD and actual EF over the entire study (r=0.96, p<0.001). Bias analysis showed that the points were within a 95% confidence interval of ±12%. Multivariate analysis showed that stroke rate did not significantly affect TD measurements (r=0.03, p>0.7). This study demonstrates that TD accurately predicts EF using anin vitro system and appears to be independent of stroke rate. Thus, TD methods may provide an accurate, simple and reliable means to serially measure RVEF in the clinical setting. This work was supported in part by a Grant-in-Aid from the American Heart Association, and the Bioengineering Alliance of South Carolina.