In vitro evaluation of caffeic acid derivatives as efflux pump inhibitor in Pseudomonas aeruginosa and Burkholderia pseudomallei

Aims: Bacterial pathogens such as Pseudomonas aeruginosa and Burkholderia pseudomallei are intrinsically resistant to many classes of antibiotics. This is not only due to the poor permeability of their outer membrane but also because of expression of multiple efflux pumps. A promising strategy to minimize the efflux of drugs by these pumps is the use of efflux pump inhibitors (EPIs). In this study, the potential of caffeic acid derivatives as EPIs in P. aeruginosa and B. pseudomallei were evaluated. Methodology and results: The potential of caffeic acid and its derivatives, i.e. chlorogenic acid, caffeic acid phenethyl ester (CAPE) and caffeic acid phenethyl amide (CAPA) to act as EPIs in P. aeruginosa and B. pseudomallei were assessed using the ethidium bromide (EtBr) accumulation and minimum inhibitory concentration (MIC) validation assays. Among the four test compounds, CAPE was found to significantly increased intracellular accumulation of EtBr in both P. aeruginosa and B. pseudomallei. An increase of 21.4% and 16.8% in cell fluorescence, over a 5-min time frame was observed in P. aeruginosa and B. pseudomallei respectively. Combination of CAPE with kanamycin significantly reduced MICs of this aminoglycoside by a factor of 8-fold in P. aeruginosa and 2-fold in B. pseudomallei. Combination of CAPE with gentamicin also led to a reduction of 4-fold MIC value of this antibiotic in B. pseudomallei. Conclusion, significance and impact of study: The in-vitro results suggest that CAPE has the potential to act as an EPI in P. aeruginosa and B. pseudomallei, thus improving the efficacy of aminoglycosides as antimicrobial agents.

Hypoglycemic and anti-hyperglycemic study of Gynura procumbens leaf extracts

<p><b>OBJECTIVE</b>To study the antidiabetic activity of Gynura procumbens (G. procumbens) used in the traditional management of diabetes in Southern Asia.</p><p><b>METHODS</b>G. procumbens leaves were extracted sequentially with graded percentage of ethanol in water (95%, 75%, 50%, 25% and 0%), and the extracts were tested for antidiabetic activity using acute (7 h), subcutaneous glucose tolerance test and sub-chronic (14 d) test in non-diabetic and streptozotocin-induced diabetic rats. The extracts were further subjected to phytochemical studies.</p><p><b>RESULTS</b>In acute dose (1 g/kg), the extracts significantly lowered fasting blood glucose (FBG) in streptozotocin-induced diabetic rats (P<0.05). However, the FBG-lowering effect of the 25% extract compared to the other extracts, was rapid (47% after 2 h) and the highest: 53%, 53% and 60% in the 3rd, 5th, and 7th h, respectively (P<0.05), comparable only to the effect of metformin. Furthermore, the extracts suppressed peak FBG in subcutaneous glucose tolerance test, but only the 0% and 25% extracts, and metformin sustained the decrease until the 90th min (P<0.05). Moreover, in the 14 days study, the 25% extract exerted the highest FBG-lowering effect, namely 49.38% and 65.43% on days 7 and 14, respectively (P<0.05), similar to the effect of metformin (46.26% and 65.42%). Total flavanoid and phenolic contents in the extracts were found to decrease with increase in polarity of extraction solvents. The composition of reference compounds (chlorogenic acid, rutin, astragalin and kaempferol-3-O-rutinoside) followed a similar trend.</p><p><b>CONCLUSIONS</b>G. procumbens contains antidiabetic principles, most extracted in 25% ethanol. Interaction among active components appears to determine the antidiabetic efficacy, achieved likely by a metformin-like mechanism.</p>

Rapid separation and determination of betulinic acid from a complex matrix using combination of TLC and RP-HPLC

Hitherto, only a few studies are reported about using the combination of TLC and RP-HPLC for the separation and determination of analyte[s] from a complex matrix. The present study is aimed to develop a simple and rapid method for the separation and determination of betulinic acid from a complex matrix, extracts of Orthosiphon stamineus, using a combination of the two techniques. The samples having higher contents of the analyte and fewer interfering species were prepared using TLC. The samples were then eluted through C[18] column using isocratic solvent system comprising acetonitrile, methanol and acetic acid acidified water of pH 2.8 in a ratio of 70: 20: 10 [v/v/v], respectively, and detection was carried out at 210 nm. The method was validated and applied successfully to quantify betulinic acid in various types of extracts of the plant. The limit of detection [LOD] and limit of quantification [LOQ] were found to be 0.0005 and 0.0050 micro g/ml, respectively. The method exhibited linearity in a concentration range of 0.005-100.00 micro g/ml [R[2]= 0.9999]. The recovery was found to be 97.10 - 97.60% [RSD < 5%], whereas, intra-day and inter-days accuracy values were 97.13 - 98.67% [RSD < 5%] and 96.45 - 98.00% [RSD < 5%], respectively. The results of the present study indicate that the developed method is simple, rapid, sensitive and accurate, and may be of a value to natural product industry and researchers for the standardization of extracts containing betulinic acid in a lesser time and consuming fewer solvents