Garima Pandey

Quercetin Sensitizes Fluconazole-Resistant Candida albicans To Induce Apoptotic Cell Death by Modulating Quorum Sensing

ABSTRACT Quorum sensing (QS) regulates group behaviors of Candida albicans such as biofilm, hyphal growth, and virulence factors. The sesquiterpene alcohol farnesol, a QS molecule produced by C. albicans, is known to regulate the expression of virulence weapons of this fungus. Fluconazole (FCZ) is a broad-spectrum antifungal drug that is used for the treatment of C. albicans infections. While FCZ can be cytotoxic at high concentrations, our results show that at much lower concentrations, quercetin (QC), a dietary flavonoid isolated from an edible lichen (Usnea longissima), can be implemented as a sensitizing agent for FCZ-resistant C. albicans NBC099, enhancing the efficacy of FCZ. QC enhanced FCZ-mediated cell killing of NBC099 and also induced cell death. These experiments indicated that the combined application of both drugs was FCZ dose dependent rather than QC dose dependent. In addition, we found that QC strongly suppressed the production of virulence weapons—biofilm formation, hyphal development, phospholipase, proteinase, esterase, and hemolytic activity. Treatment with QC also increased FCZ-mediated cell death in NBC099 biofilms. Interestingly, we also found that QC enhances the anticandidal activity of FCZ by inducing apoptotic cell death. We have also established that this sensitization is reliant on the farnesol response generated by QC. Molecular docking studies also support this conclusion and suggest that QC can form hydrogen bonds with Gln969, Thr1105, Ser1108, Arg1109, Asn1110, and Gly1061 in the ATP binding pocket of adenylate cyclase. Thus, this QS-mediated combined sensitizer (QC)-anticandidal agent (FCZ) strategy may be a novel way to enhance the efficacy of FCZ-based therapy of C. albicans infections.

Development and characterization of a novel Swarna-based herbo-metallic colloidal nano-formulation – inhibitor of Streptococcus mutans quorum sensing

Herbo-metallic preparations such as bhasmas (ash) are used traditionally in Indian and Chinese medicinal systems. In Ayurveda, Swarna (gold) nanoparticles are used as Swarna bhasma to treat several clinical manifestations. While Usnea longissima, a medicinal lichen, ethnobotanical is known for the treatment of tooth cleaning and infectious diseases. The study aims to develop a herbo-metallic colloidal nano-formulation containing Swarna nanoparticles and polyphenols rich U. longissima extract (ULE) and evaluate its anti-quorum sensing (QS) property against Streptococcus mutans, an idea that has never been explored before, with a view towards combating the emergence of antibiotic resistance often linked with QS-regulated virulence factors and biofilms. The synthesized Uh-Au@Nano-CF was confirmed by a peak at 550 nm in the UV-visible spectrum. The obtained XRD data confirmed the crystalline nature of nanoparticles of 28 nm size. TEM image revealed that all the particles were spherical with a narrow size range of 5–23 nm. The FTIR result clearly showed that the ULE containing secondary OH as functional groups induces encapsulation of nanoparticles. HPTLC and HPLC fingerprinting of ULE confirmed the presence of polyphenols, including orcinol, arabitol, apigenin, and usnic acid. The data from the inhibition of violacein production in C. violaceum 12u2006472 revealed that the Uh-Au@Nano-CF at sub-lethal concentrations (5, 10 and 15%) show potent anti-QS activity. The treatment of Uh-Au@Nano-CF was found to inhibit the secretion of S. mutans virulence factors, including acid production, ATPase, enolase, lactate dehydrogenase, protease, total exopolysaccharide content, and glucosidase. The Uh-Au@Nano-CF in a concentration dependent manner showed anti-biofilm activity, inhibiting biofilm formation. Eventually, it was also documented that the Uh-Au@Nano-CF at 15% dilution enhanced the susceptibility of S. mutans to its conventional antibiotics. This study introduces not only a novel antimicrobial herbo-metallic colloidal nano-formulation, but also explores its new biomedical application, which targets QS-regulated virulence factors and biofilm of S. mutans rather than its viability.

Antioxidant and antibacterial activities of leaf extract of Achyranthes aspera Linn. (prickly chaff flower).

Aim: The present study was undertaken to establish the potential role of Achyranthes aspera Linn for cure of skin diseases. Study Design: The plant is traditionally used by various tribes for curing a wide range of diseases. A 50% ethanolic extract of the leaves was subjected to phytochemical studies and further investigated for in vitro antioxidant and antibacterial activities. Place and Duration of Study: CSIR-National Botanical Research Institute (NBRI), Lucknow, between December 2012 and November 2013. Methodology: In vitro antioxidant activity was determined by DPPH free radical scavenging assay, hydroxyl radical scavenging activity, β-Carotene-linoleic acid assay and reducing power assay. Antibacterial activity was studied by agar well diffusion method. Results: The total phenol and flavonoid content was estimated to be 3.363% and 6.36% respectively. The HPTLC analysis showed the presence of oleanolic acid, lupeol and βsitosterol. The free radical scavenging activity of the extract was concentration dependent Original Research Article European Journal of Medicinal Plants, 4(6): 695-708, 2014 696 and IC50 was observed at a concentration of 62.24μg/ml for DPPH free radical scavenging activity and 68.32μg/ml for hydroxyl radical scavenging activity. The extract showed significant total antioxidant activity and reducing power. Antibacterial activity was studied by well diffusion method and the MIC was recorded at 0.75 mg/ml for S. aureus, 0.8 mg/ml for M. luteus, 2.75 mg/ml for E. coli and 0.8 mg/ml for P. aeruginosa. Conclusion: The results obtained from current study demonstrate that the leaf extract of Achyranthes aspera L possess significant antioxidant and antibacterial properties. Presence of various classes of phytocompounds e.g. Phenols, flavonoids, saponins, alkaloids etc. contribute highly to its medicinal values, thus indicating its potential for cure of skin diseases.

Altitudinal variation of berberine, total phenolics and flavonoid content in Thalictrum foliolosum and their correlation with antimicrobial and antioxidant activities

Background The quality of herbal medicine is determined by its secondary metabolites, which may vary according to growth, season and altitude etc. Objective We studied the variation in phytochemistry and biological activities of Thalictrum foliolosum (TF) roots collected from four sites at different altitudes. Material and methods The berberine content in different extracts of T. foliolosum roots collected from various altitudes was estimated using HPTLC. Total phenolic and flavonoid contents were determined using Folin-Ciocalteau reagent and aluminum chloride method respectively. The sensitivity of microbes for the extracts was studied using disc diffusion and the MIC was estimated using broth dilution method. Antioxidant capacity of the plant was studied using β-carotene bleaching assay, lipid peroxidation assay using goat liver, reducing power assay and DPPH free radical scavenging activity. Results Berberine content varied inversely with altitude; while phenol and flavonoid content of TF increased at higher altitudes. All the TF extracts showed moderate to high activity against Candida albicans, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Extracts with high berberine content were most effective against C. albicans and S. aureus and also showed relatively significant anti-lipid peroxidation, β-carotene bleaching and reducing power. TF extracts with higher phenol and flavonoid content showed better scavenging of DPPH free radicals. Berberine was used as a standard in all the antioxidant and antimicrobial experiments performed. Conclusion Thalictrum from lower elevations can be explored as an alternate source of berberine and the plant has high antioxidant and antimicrobial properties owing to its berberine content.

Grilling enhances antidiarrheal activity of Terminalia bellerica Roxb. fruits.

ETHNOPHARMACOLOGICAL RELEVANCEnTerminalia bellerica Roxb. fruits are rich in a variety of biologically active ingredients. Tharu and Buksa tribes of Udham Singh Nagar, Uttarakhand, India use grilled fruits of Terminalia bellerica as an effective cure for diarrhea AIM OF THE STUDY: We validated the ethnobotanical claim by comparing the antidiarrheal effect of grilled fruits (GF) with dried fruits (DF).nnnMATERIALS AND METHODSnThe 50% ethanolic extracts of GF and DF were successively fractionated; the antioxidant and bacterial inhibition activity were studied using DPPH free radical scavenging, anti-lipid peroxidation and broth dilution method respectively. Difference in metabolites of ethyl acetate fractions of GF and DF was analyzed using GC-MS, gallic acid content was determined through HPTLC. Further the in-vivo antidiarrheal effect of ethyl acetate fractions of DF and GF was studied on castor oil induced diarrhea model.nnnRESULTSnThe ethyl acetate fractions showed potential DPPH free radical scavenging (IC50 11.13µg/ml in DF and 8.56µg/ml in GF), anti-lipid peroxidation and antibacterial activity. The non-targeted metabolic profiling showed higher content of tartaric acid, valeric acid, gallic acid, succinic acid, oxalic acid, malonic acid, malic acid, 1,2,3 trisbenzene, uridine and 11-eicosenoic acid in GF. The HPTLC results indicated that gallic acid content was 2.8 (±0.14) and 4.92 (±0.28) mg/g while ellagic acid content was 4.7 (±0.32) and 4.45 (±0.45) mg/g dry powder in DF and GF respectively. According to in vivo antidiarrheal activity DF and GF (100mg/kg oral) inhibited diarrhea by 41.87% and 71.72% respectively.nnnCONCLUSIONnGrilling significantly altered the levels of metabolites in T. bellerica fruits which could be responsible for its increased therapeutic potential.

Wound Repair and Anti-inflammatory Potential of Fumaria indica in Excision Wound-induced Rats.

Aims: Objective of the study was to investigate the wound contraction and antiinflammatory activity of the 50% ethanolic extract of Fumaria indica (Hausskn.) Pugsley (Fumariaceae) by excision wound model and estimation of pro-inflammatory and antiinflammatory cytokines. Study Design: Prospective. Place and Duration of Study: Department of Pharmacognosy and Ethnopharmacology, CSIR-National Botanical Research Institute, Lucknow, India. December 2012 to May 2013. Methodology: Dried powdered whole plant of Fumaria indica was extracted with 50% ethanolic extract. The extract was subjected to HPTLC fingerprinting, DPPH free radical scavenging and antibacterial activities. Further, 10% F. indica ointment was tested for its wound contraction, pro-inflammatory and anti-inflammatory potentials. Results: The 50% ethanolic extract showed presence of ellagic acid, ferulic acid andquercetin. The IC50 was 0.11mg/mL and significant antibacterial activity was observed against S. aureus and E. coli. The 10% F. indica ointment applied topically to the wound area reducedits size from 500 mm2 to 40 mm by the end of 9 day. These results were Original Research Article British Journal of Pharmaceutical Research, 4(2): 257-266, 2014 258 comparable to the effect of 0.2% nitrofurazone. The extract further showed a reduction in the release of pro-inflammatory cytokines (TNFα and IL-6) and an increase in antiinflammatory cytokine IL-10.

Wound Healing Activity of Premna latifolia Roxb.

Aims: The study aims to understand the wound healing potentials of a 50% aqueous ethanolic extract of Premna latifolia stem using excision wound model. Study Design: The wound healing potentials were simultaneously supported by observing the bacterial functional diversity of wound swabs using Biolog Eco plates. The antioxidant activity was performed using In vitro DPPH free radical scavenging assay. Place and Duration of Study: CSIR-National Botanical Research Institute (NBRI), Lucknow, between May 2013 and November 2013. Methodology: Wound healing activity of the plant was studied using excision wound model. Animals were divided into three groups of six male rats each as control group (GI) dressed with compound free simple ointment. Test group (GII) treated with 50% aqueous ethanolic extract of P. latifolia stem (10% w/w) in ointment vehicle and standard group (GIII) group treated Nitrofurazone ointment, Himedia (0.2%w/w). The wound healing potential was further supported by the DPPH free radical scavenging and antibacterial activity of the plant. The phytochemical estimations were done using standard methods. Results: Sugar and starch content in the plant was 3.55% and 5.54% respectively. Total tannins, phenol and flavonoid content were estimated to be 0.18%, 0.54% and 2.73%. Original Research Article ArticRe Article British Journal of Pharmaceutical Research, 4(8): 929-942, 2014 930 The 50% ethanolic extract of the plant showed moderate DPPH free radical scavenging activity with an IC50 of 188.02μg/ml. A 69.15% of wound closure was observed on 10 day post wounding of the rats treated with 200 mg/kg of extract. The results also indicated significant antibacterial activity of the extract. Conclusion: The 50% aqueous ethanolic extract of P. latifolia shows significant wound healing activity.

Effect of Andrographis serpyllifolia Leaves Extract on Experimentally Induced Typhoid Using Salmonella Typhi.

Aims: The aim of the study was to evaluate the effect of the standardized ethanolic extract of Andrographis serpyllifolia leaves on experimentally induced typhoid. Study Design: Single dose of 1 ml Salmonella Typhi (10 CFU/mL) was administered orally to rats to induce typhoid in rats. Blood culture test confirmed typhoid infectioned rats received orally the ethanolic extract of Andrographis serpyllifolia at dose levels of 200 and 400 mg/kg twice daily for 10 days, respectively and control animals received physiological saline. Place and Duration of Study: CSIR-National Botanical Research Institute (NBRI), Lucknow, between December 2011 and June 2013. Methodology: Leaves of Andrographis serpyllifolia was extracted with ethanol and concentrated on rotavapour. Single dose of 1 ml S. Typhi (10 CFU/mL) was administered orally to rats with the help of orogastric tube to induce typhoid in rats. After seven days, Original Research Article British Journal of Pharmaceutical Research, 4(2): 230-239, 2014 231 typhoid confirmed rats received the standardized extract subsequently subjected to in vitro antimicrobial susceptibility test. Results: The treatment with ethanolic extract of Andrographis serpyllifolia at dose level of 200 mg/kg showed 75.0% to 87.5% protection and 100% protection observed at higher dose of 400 mg/kg on widal, blood culture and typhidot test respectively. Biochemical test carried out on blood culture isolates confirmed the presence or absence of S. Typhi. A. serpyllifolia extract at a concentration of 1.50 mg/disc showed antimicrobial activity susceptibility against S. Typhi. Conclusion: Andrographis serpyllifolia leaves extract showed antimicrobial activity against S. Typhi and accomplished the extract of A. serpyllifolia is recommended for clinical applications in the treatment of typhoid.