Cliv-92-Loaded Glycyrrhetinic Acid-Modified Chitosan Nanoparticles for Enhanced Hepatoprotection-Preparation, Characterization, and In Vivo Evaluation.

Cliv-92 is a mixture of three structurally similar coumarinolignoids and a proven hepatoprotective agent. Low aqueous solubility and poor bioavailability are notable hindrances for its further use. Therefore, glycyrrhetinic acid-linked chitosan nanoparticles loaded with Cliv-92 were prepared for active targeting to the liver. The nanoparticles were prepared by the ionic gelation method to avoid the use of toxic solvents/rigorous agitation. The method of preparation was optimized using a central composite design with independent variables, namely polymer: drug ratio (3:1, w/w), crosslinker concentration (0.5%), and stirring speed (750 rpm). The optimized nanoparticles had a mean particle size of 185.17 nm, a polydispersity index of 0.41, a zeta potential of 30.93 mV, and a drug loading of 16.30%. The prepared formulation showed sustained release of approximately 63% of loaded Cliv-92 over 72 h. The nanoparticles were freeze-dried for long-term storage and further characterized. The formulation was found to be biocompatible for parenteral delivery. In vivo imaging study showed that optimized nanoparticles were preferentially accumulated in the liver and successfully targeting the liver. The present study successfully demonstrated the improved pharmacokinetic properties (≈12% relative bioavailability) and efficacy profile (evidenced by in vivo and histopathological studies) of fabricated Cliv-92 nanoparticles.

Phytoremediation of sewage sludge by Cymbopogon martinii (Roxb.) Wats. var. motia Burk. grown under soil amended with varying levels of sewage sludge.

Sewage sludge used as agriculture fertilizers contains a conspicuous amount of potentially toxic metals. In order to prevent the contamination in the food chain, there is an urgent need for the development of sewage sludge clean up technology. The use of non-food, multi-harvest aromatic crops for phytoremediation of sewage sludge has many benefits. Besides the eco-friendly approach, plant biomass generated can be used to extract economically important essential oil free of heavy metals. Cymbopogon martinii was grown in soil (s) amended with different ratios of sewage sludge (ss), that is, 100s:0ss (control), 80s:20ss, 60s:40ss, 40s:60ss, 20s:80ss, and 0s:100ss. The experiment was conducted in a plastic sack under an open environment for 1 year and harvesting was done thrice. Plant growth and essential oil yield were significantly increased with the increasing dose of sewage sludge. Accumulation of toxic metal (Cd, Cr, Pb, Ni) and micronutrient (Fe, Zn, Cu, Mn) increased significantly in the shoot tissues confirmed by estimation of bioaccumulation and bioconcentration, and scanning electron microscopy and X-ray microanalyses. Soil enzyme activities were significantly improved with the plant growth period and increased doses of sludge. Results showed C. martinii acts as hyper-accumulator and thus could be used for phytoremediation of sewage sludge.

Microbial interference mitigates Meloidogyne incognita mediated oxidative stress and augments bacoside content in Bacopa monnieri L.

Microbial interference plays an imperative role in plant development and response to various stresses. However, its involvement in mitigation of oxidative stress generated by plant parasitic nematode in plants remains elusive. In the present investigation, the efficacy of microbe's viz., Chitiniphilus sp. MTN22 and Streptomyces sp. MTN14 single and in combinations was examined to mitigate oxidative stress generated by M. incognita in medicinal plant, Bacopa monnieri. Microbial combination with and without pathogen also enhanced the growth parameters along with secondary metabolites (bacoside) of B. monnieri than the pathogen inoculated control. The study showed that initially the production of hydrogen peroxide (H2O2) was higher in dual microbes infected with pathogen which further declined over M. incognita inoculated control plants. Superoxide dismutase and free radical scavenging activity were also highest in the same treatment which was linearly related with least lipid peroxidation and root gall formation in B. monnieri under the biotic stress. Microscopic visualization of total reactive oxygen species (ROS), H2O2, superoxide radical and programmed cell death in host plant further extended our knowledge and corroborated well with the above findings. Furthermore, scanning electron microscopy confirmed good microbial colonization on the host root surface around nematode penetration sites in plants treated with dual microbes under pathogenic stress. The findings offer novel insight into the mechanism adopted by the synergistic microbial strains in mitigating oxidative stress and simultaneously stimulating bacoside production under pathogenic stress.

In silico and in vitro Studies on Begomovirus Induced Andrographolide Biosynthesis Pathway in Andrographis Paniculata for Combating Inflammation and Cancer.

Andrographolide and neoandrographolide are major bioactive molecules of Andrographis paniculata, a well-known medicinal plant. These molecules exhibited varying degrees of anti-inflammatory and anticancer activities in-vitro and in-vivo. Role of begomovirus protein C2/TrAP in biosynthesis of andrographolide was identified through molecular modeling, docking and predicted results were substantiated by in vitro studies. Homology molecular modeling and molecular docking were performed to study the binding conformations and different bonding behaviors, in order to reveal the possible mechanism of action behind higher accumulation of andrographolide. It was concluded that C2/TrAP inhibit the activation of SNF1-Related Protein Kinase-1 (SnRK1) in terpenoid pathway and removes the negative regulation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) by SnRK1, leading to higher accumulation of andrographolide and neoandrographolide in begomovirus infected plants. The binding site residues of SnRK1 docked with C2/TrAP were found to be associated with ATP binding site, substrate binding site and activation loop. Predicted results were also validated by HPTLC. This study provides important insights into understanding the role of viral protein in altering the regulation of biosynthesis of andrographolide and could be used in future research to develop biomimetic methods for increasing the production of such phytometabolites having anti-cancerous and anti-inflammatory properties.

Diffuse reflectance near infrared-chemometric methods development and validation of amoxicillin capsule formulations.

OBJECTIVE: The aim of present study was to establish near infrared-chemometric methods that could be effectively used for quality profiling through identification and quantification of amoxicillin (AMOX) in formulated capsule which were similar to commercial products. In order to evaluate a large number of market products easily and quickly, these methods were modeled. MATERIALS AND METHODS: Thermo Scientific Antaris II near infrared analyzer with TQ Analyst Chemometric Software were used for the development and validation of the identification and quantification models. Several AMOX formulations were composed with four excipients microcrystalline cellulose, magnesium stearate, croscarmellose sodium and colloidal silicon dioxide. Development includes quadratic mixture formulation design, near infrared spectrum acquisition, spectral pretreatment and outlier detection. According to prescribed guidelines by International Conference on Harmonization (ICH) and European Medicine Agency (EMA) developed methods were validated in terms of specificity, accuracy, precision, linearity, and robustness. RESULTS: On diffuse reflectance mode, an identification model based on discriminant analysis was successfully processed with 76 formulations; and same samples were also used for quantitative analysis using partial least square algorithm with four latent variables and 0.9937 correlation of coefficient followed by 2.17% root mean square error of calibration (RMSEC), 2.38% root mean square error of prediction (RMSEP), 2.43% root mean square error of cross-validation (RMSECV). CONCLUSION: Proposed model established a good relationship between the spectral information and AMOX identity as well as content. Resulted values show the performance of the proposed models which offers alternate choice for AMOX capsule evaluation, relative to that of well-established high-performance liquid chromatography method. Ultimately three commercial products were successfully evaluated using developed methods.

Solvent-based extraction optimisation for efficient ultrasonication-assisted ginsenoside recovery from Panax quinquefolius and P. sikkimensis cell suspension lines.

The present study aims at developing an extraction protocol for efficient ginsenoside recovery from cell suspensions of Panax quinquefolius and P. sikkimensis. Methanol (100%, 70% and 30%), water (40°C, 90°C), water-saturated butanol and butanol-saturated water were compared for their ultrasonication-assisted ginsenoside retrieval efficacy. HPLC and HP-TLC analysis revealed 100% methanol as the best solvent for maximum retrieval of Rb (diol) and Rg (triol) ginsenosides (P. quinquefolius: Rb: 0.189, Rg: 3.163 mg/g DW; P. sikkimensis: Rb: 0.245, Rg: 4.073 mg/g DW), followed by water (90°C). Methanolic solutions, especially 70%, proved to be significant retrievers of Rg1 (1.812 and 1.327 mg/g DW in P. quinquefolius and P. sikkimensis), with poor Re recovery (0.328 and 0.342 mg/g DW). Water-saturated butanol also led to significant ginsenoside extraction (72.4% of content extracted by methanol), selectively in P. quinquefolius, with a less than 50% of total content extracted by methanol, in P. sikkimensis.

In vitro antimalarial studies of novel artemisinin biotransformed products and its derivatives.

Biotransformation of antimalarial drug artemisinin by fungi Rhizopus stolonifer afforded three sesquiterpenoid derivatives. The transformed products were 1α-hydroxyartemisinin (3), 3.0%, a new compound, 10ß-hydroxyartemisinin, 54.5% (4) and deoxyartemisinin (2) in 9% yield. The fungus expressed high-metabolism activity (66.5%). The chemical structures of the compounds were elucidated by 1D, 2D NMR spectrometry and mass spectral data. The major compound 10ß-hydroxyartemisinin (4) was chemically converted to five new derivatives 5-9. All the compounds 3-9 were subjected for in vitro anti-malarial activity. 10ß-Hydroxy-12ß-arteether (8), IC50 at 18.29nM was found to be 10 times better active than its precursor 4 (184.56nM) and equipotent antimalarial with natural drug artemisinin whereas the α-derivative 9 is 3 times better than 4 under in vitro conditions. Therefore, the major biotransformation product 4 can be exploited for further modification into new clinically potent molecules. The results show the versatility of microbial-catalyzed biotransformations leading to the introduction of a hydroxyl group at tertiary position in artemisinin in derivative (3).

Box-Behnken design for optimum extraction of biogenetic chemicals from P. lanceolata with an energy audit (thermal × microwave × acoustic): a case study of HPTLC determination with additional specificity using on-line/off-line coupling with DAD/NIR/ESI-MS.

INTRODUCTION: The genus Pluchea comprises about 80 species distributed worldwide, out of them, only Pluchea lanceolata (DC.) Oliv. & Hiern, is used extensively in the traditional system of India. No chromatographic method is available for its quality. OBJECTIVES: To perform the energy audit for the extraction of biogenetic pentacyclic triterpene, its acetate and sterol from P. lanceolata utilising organic and four alternative solvents. Additionally to resolve the uncertainty of TLC determination, on-line/off-line coupling with a diode-array detector (DAD), and near-infrared (NIR) and electrospray ionisation (ESI) MS was introduced. METHODS: The extraction of taraxasterol (Tx), taraxasterol acetate (TxAc) and stigmasterol (St) from P. lanceolata was performed using three energy modes. The effects of different operating parameters were studied for optimum extraction yield using the design of experiments, that is, the central composite design and Box-Behnken design. In addition to the retention factor (Rf ) and visible spectral matching, two additional optical spectroscopic techniques, that is, NIR and ESI-MS, were applied for extended specificity. RESULTS: The method was developed for Tx, TxAc and St determination using HPTLC at 645 nm. The optimum extraction yield of targeted compounds was found to be higher with organic solvents than eco-friendly surfactants. The pulse ultrasonic assisted extraction (PUAE) has resulted in optimum extraction of compounds comparable to hot extraction. Both NIR and ESI-MS provided extended specificity in determination. CONCLUSION: The 5/1-PUAE was determined to be effective, reproducible, simple and energy efficient for the determination of Tx, TxAc and St in P. lanceolata. The offline coupling of NIR and ESI-MS with HPTLC led to considerable improvement in specificity.

First Report of Chilli leaf curl India virus Infecting Mentha spicata (Neera) in India.

Mint (Mentha spp.; family Lamiaceae) is an important essential oil-bearing crop cultivated on the Indian subcontinent as a cash crop for the international market and industrial purposes. Since May 2010, typical symptoms such as yellow vein, leaf yellowing, mosaic, crinkling, and cupping were observed, which led to significant yield loss in spearmint (M. spicata var. Neera) at CIMAP experimental fields and farmers' fields of Badaun, Rampur, and Moradabad regions of Uttar Pradesh province, India. Disease incidence was recorded in the range of 40 to 50%. Mentha spp. has been reported to be affected by many viral diseases (3). Due to the absence of fungal/bacterial infection, lack of mechanical transmission of the pathogen, and presence of whiteflies in the fields, the causal pathogen was suspected to be a begomovirus. Total genomic DNA was extracted from the leaves of naturally infected and healthy samples of Mentha by the CTAB protocol. Eighteen symptomatic samples were collected from different location of fields and screened for the presence of begomovirus. DNA from these samples was used as PCR template to amplify a 771-bp fragment using begomovirus coat protein (CP) gene specific primers. Eleven of 18 (61.1%) samples were found positive. PCR products were cloned into the pGEM-T Easy (Promega) and sequenced using the universal M13F/M13R primers showed sequence similarity with Chilli leaf curl India virus. To amplify the full-length DNA-A/B and a possible ß-satellite, a second detection method was used: rolling circle amplification (RCA) using the TempliPhi 100 Amplification System (GE Healthcare). RCA products were digested independently with various restriction enzymes: BamHI, EcoRI, EcoRV, HincII, HindIII, SacI, and KpnI. Digested products were resolved on 1% agarose gel and the bands corresponding to ~2.7 and ~1.3 kb were purified using Nucleospin Gel and PCR Clean-up Kit and cloned into the respective sites of pGreen0029 vector. The sequence of full-length DNA-A (2,749 bp) and ß-satellite component (1,347-bp) were obtained and deposited in NCBI GenBank with accession nos. KF312364 and KF364485, respectively. The sequence analysis showed maximum nucleotide identity (99%) with Chilli leaf curl India virus (FM877858) and distant affinities (≤88%) with other begomoviruses. The sequence analysis of isolated ß-satellite showed 93% identity with Ageratum yellow vein virus satellite (AJ252072.1). No presence of DNA-B was detected using the universal primer PBL1v2040/PCRc1 (2), thus confirming it to be a monopartite begomovirus (1). Viruliferous whiteflies (Bemisia tabaci) proved Koch's postulation by inducing similar symptoms on healthy plants while aphids (Myzus persicae) failed to transmit the virus. To our knowledge, this is the first report of Chilli leaf curl India virus infecting M. spicata var. Neera in India. Mint is widely grown together with other reported hosts of begomoviruses, and thus could pose a serious threat as future expansion of begomovirus to new crops. Hence, the development of resistant varieties coupled with the implementation of adapted integrated pest management strategies would be essential for successful production of mint crops. References: (1) Y. Kumar et al. Plant Pathol. 60:1040, 2011. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993. (3) I. E. Tzanetakis et al. Plant Dis. 94:4, 2010.

Atropa belladonna hairy roots: orchestration of concurrent oxidation and reduction reactions for biotransformation of carbonyl compounds.

The biotransformation potential of a selected Atropa belladonna hairy root clone (AB-09) had been evaluated with regard to three different aromatic carbonyl compounds, i.e., 3,4,5-trimethoxybenzaldehyde (1), 3,4,5-trimethoxyacetophenone (2), and 3,4,5-trimethoxy benzoic acid (3). The results demonstrated for the first time the untapped potentials of the selected hairy root clone to perform simultaneous oxidation (34.49%) and reduction (32.68%) of 3,4,5-trimethoxy benzaldehyde (1) into 3,4,5-trimethoxy benzoic acid (3), and 3,4,5-trimethoxy benzyl alcohol (4), respectively, without any intermediate separation or addition of reagents. The same hairy root clone also demonstrated reduction (<5%) of a 3,4,5-trimethoxyacetophenone (2) into a secondary alcohol, i.e., 1-(3,4,5-trimethoxyphenyl) ethanol (5), while in the case of aromatic carboxylic acid substrate (3), no biotransformation could be obtained under the similar conditions. The current observations revealed oxidation and reduction of the formyl group of the aromatic ring, and only reduction of the carbonyl group of acetophenone through the specific hairy root clone. The concurrent oxidation and reduction reactions by the selected hairy root clone highlight the importance of this study, which, as per our observations, is the first of its kind relating the hairy root culture of A. belladonna.

FT-NIR spectroscopy for rapid and simple determination of nimesulide in rabbit plasma for pharmacokinetic analysis.

High-throughput analysis of a large number of samples for pharmacokinetic study is necessary in drug development and pharmacovigilance. Usually, drug quantification for pharmacokinetics and bio-availability is achieved through matrix extraction and HPLC analysis, which is time, labour and cost intensive method. A prompt and solvent free method is the quest for such analysis in the present times. Pharmacokinetic analysis of nimesulide from plasma samples of rabbits through Fourier transform near infrared (FT-NIR) spectroscopy analysis combined with partial least squares (PLS) regression model was undertaken with validation through HPLC analysis. Pharmacokinetic parameters obtained through FT-NIR and HPLC were found to be statistically similar with errors below the acceptable limits. The study demonstrates the use of FT-NIR for pharmacokinetics and bio-availability studies. This high throughput method analyses more than 50 samples in an hour without solvents usage and provide ample scope for automation and commercial utilization.

Occurrence of a Clover Proliferation (16SrVI) Group Phytoplasma Associated with Little Leaf Disease of Portulaca grandiflora in India.

Portulaca grandiflora (family Portulacaceae), commonly known as moss rose purslane, is a popular ornamental plant widely grown in temperate climates because it blooms all summer. Portulaca is also used for medicinal purposes since it is rich in vitamins A, B1, and C and has antimicrobial and cytotoxic activity. Since March 2005, 30 to 50% of P. grandiflora plants in the ornamental gardens as well as in pots at the Central Institute of Medicinal and Aromatic Plants, Lucknow, India have displayed symptoms resembling phytoplasma infection. Disease symptoms start as a typical bud proliferation, downward curling, and diminishing size of leaves, followed by overall stunted growth and yellowing of the whole plant from April to June. Some plants also formed rosettes and a proliferation of axillary shoots resulting in a witches'-broom appearance. Typical pleomorphic bodies, mostly spherical to oval, ranging from 340 to 1,100 nm were observed only in sieve elements of infected plants by transmission electron microscopy (TEM). On the basis of symptoms, TEM observations, PCR, and response to antibiotic treatment, the causal organism was identified as phytoplasma (1). Total genomic DNA from healthy and infected plants was extracted with the CTAB buffer method (2). Of 27 suspected samples screened by PCR, 23 were phytoplasma positive. Presence of phytoplasmas in plants was demonstrated by a nested PCR assay employing primer pair P1/P6 followed by R16F2n/R16R2 that generated rDNA products of 1.5 and 1.2 kb, respectively, only from symptomatic plants. No differences among phytoplasmas in Portulaca plants were detected by restriction fragment length polymorphism (RFLP) analysis of nested rDNA (1.2 kb) products using endonucleases BamHI, RsaI, AluI, HpaII, and EcoRI. Comparative analysis of RFLP patterns with those derived from reference phytoplasmas tentatively identified the Portulaca little leaf (PLL) phytoplasma as a member of 16S rDNA RFLP group 16SrVI (3). A nested PCR product (1.25 kb) was cloned with a TOPO TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced. The sequence was deposited in the GenBank database (Accession No. EF651786). Sequence analysis revealed the PLL phytoplasma to be most similar (98%) to Indian brinjal little leaf (Accession No. EF186820) and 'Candidatus Phytoplasma trifolii' (Accession No. AY390261), two 16SrVI group phytoplasmas previously reported from India and Canada, respectively. The status of PLL (EF651786) was also verified by in silico RFLP analysis (4) of the F2n/R2 sequence of six closely related strains (Accession Nos. AF228052, AY390261, AY270156, AY409070, AY409069, and EF186820) of the 16SrVI group using 17 restriction enzymes (AluI, BamHI, BfaI, BsfUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, MseI, Sau3AI, RsaI, SspI, and TaqI). In silico restriction digestion and virtual gel plotting showed similar patterns for all enzymes. To our knowledge, this is the first report of a 16SrVI group phytoplasma infecting Portulaca plants in India. References: (1) P. V. Ajayakumar et al. Aust. Plant Dis. Notes 2:67, 2007. (2) S. P. S. Khanuja et al. Plant Mol. Biol. Rep. 17:74, 1999. (3) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (4) W. Wei et al. Int. J. Syst. Evol. Mic. 57:1855, 2007.

Characterization of an Indian isolate of cucumber mosaic virus infecting Egyptian henbane (Hyoscyamus muticus L.).

A cucumber mosaic virus isolate was found to be associated with mottle crinkle and severe mosaic disease of Egyptian henbane (Hyoscyamus muticus L.). The virus has been characterized as an Indian isolate of cucumber mosaic virus (CMV) based on non-persistent transmission by aphid, presence of 28-nm isometric particles, capsid protein of 26 K and single-stranded tripartite RNA genome with a subgenomic RNA (RNA 4). There was no evidence of satellite RNA genome. The isolate showed a strong serological relationship with S and A strains of CMV (CMV-S and CMV-A) in double diffusion test. A band of the 26 K capsid protein was also detected by Western blot analysis using antibodies specific to CMV-S.