Nematicidal Activity of Cyclopiazonic Acid Derived From Penicillium commune Against Root-Knot Nematodes and Optimization of the Culture Fermentation Process

Among 200 fungal strains isolated from the soil, only one culture filtrate of Aspergillus flavus JCK-4087 showed strong nematicidal activity against Meloidogyne incognita. The nematicidal metabolite isolated from the culture filtrate of JCK-4087 was identified as cyclopiazonic acid (CPA). Because JCK-4087 also produced aflatoxins, six strains of Penicillium commune, which have been reported to be CPA producers, were obtained from the bank and then tested for their CPA productivity. CPA was isolated from the culture filtrate of P. commune KACC 45973. CPA killed the second-stage juveniles of M. incognita, M. hapla, and M. arearia with EC50–3 days 4.50, 18.82, and 60.51 μg mL–1, respectively. CPA also significantly inhibited egg hatch of M. incognita and M. hapla after a total of 28 days of treatment with the concentrations > 25 μg mL–1. The enhancement of CPA production by P. commune KACC 45973 was explored using an optimized medium based on Plackett–Burman design (PBD) and central composite design (CCD). The highest CPA production (381.48 μg mL–1) was obtained from the optimized medium, exhibiting an increase of 7.88 times when compared with that from potato dextrose broth culture. Application of the wettable power-type formulation of the ethyl acetate extract of the culture filtrate of KACC 45973 reduced gall formation and nematode populations in tomato roots and soils under greenhouse conditions. These results suggest that CPA produced by P. commune KACC 45973 can be used as either a biochemical nematicide or a lead molecule for developing chemical nematicides to control root-knot nematodes.

Optimization of the Extraction of Total Flavonoids and Antioxidant Activity from Lippia multiflora Moldenke (Verbenaceae) Leaves using Experimental Design

The leaves of Lippia multiflora contain secondary metabolites including flavonoids which have an important antioxidant activity. This study aims to optimize the extraction conditions of total flavonoids and the antioxidant activity of these leaves. To achieve this, the Plackett-Burman design was used for the screening of the factors influencing the extraction, then the central composite design was implemented for the optimization itself. The effects of five factors, such as the plant-to-solvent ratio, the nature of the extraction solvent, the extraction time, the extraction method and the size of the L. multiflora leaves, on the extraction of total flavonoids and the antioxidant activity were studied. Results of Plackett-Burman design indicated that factors influencing both flavonoids extraction and antioxidant activity were the solid-liquid ratio and extraction time. The predicted optimal conditions for the highest flavonoids content from L. multiflora leaf with better antioxidant activity were found with aqueous decoction for 30 min with 3.5 g of cut leaves in 100 mL of distilled water. Using the predicted conditions, experimental responses were 87.18 ± 1.03 mg/g QE and 372.34 ± 4.04 µmol/g TE for total flavonoids and antioxidant capacity, respectively. Under the above-mentioned conditions, the experimental results are very close to predicted one. Thus, L. multiflora leaf can be considered as a natural source of flavonoids content with good antioxidant activity.

Application of Plackett-Burman design for screening of factors affecting pitavastatin nanoparticle formulation development

Introduction: Nanoparticle formulation of pitavastatin calcium is a potential alternative to solve the solubility related problem. However, the formulation of nanoparticle involves various parameters that affect product quality. Plackett-Burman design could facilitate an economical experimental plan that focuses on determining the relative significance of many. Aim: The objective of this study was to screen the variables which could significantly affect the pitavastatin nanoparticle formulation. Materials and methods: The pitavastatin nanoparticles were formulated by preparing nanosuspension using the emulsion solvent evaporation technique followed by freeze-drying. A Plackett-Burman screening design methodology was employed in which seven factors at two levels were tested at 12 runs to study the effect of formulation and process variables on particle size and polydispersity index of nanoparticles. The surface morphology and crystalline nature of nanoparticle were also evaluated. Results: The particle size and polydispersity index of nanosuspension was found in the range of 113.1 to 768.5 nm and 0.068 to 0.508, respectively. Statistical analysis of various variables revealed that stabilizer concentration, injection flow rate, and stirring rate were the most influential factors affecting the particle size and polydispersity index of the formulation. X-ray diffraction (XRD) and scanning electron microscopy (SEM) study suggested the amorphous nature of nanoparticles. Conclusions: This study concluded that the Plackett-Burman design was an efficient tool for screening the process and formulation variables affecting the properties of pitavastatin nanoparticles and also for the identification of the most prominent factor.

Medium optimization and subsequent fermentative regulation for the scaled-up production of anti-tuberculosis drug leads ilamycin-E1/E2

Tuberculosis (TB) and its emerged drug resistance exert huge threats on the global health, therefore development of novel anti-TB antibiotics is very essential. Ilamycin-E1/E2 is a pair of cycloheptapeptide enantiomers obtained from a marine-derived Streptomyces atratus SCSIO ZH16-ΔilaR mutant, and become promising anti-TB lead compounds due to their significant anti-TB activities, but their low titer hampered the further clinical development. In this work, the statistical Plackett-Burman design (PBD) model was applied to screen out bacterial peptone as the only significant but negative factor affecting the ilamycin-E1/E2 production. Subsequent single factor optimization revealed that replacement of bacterial peptone with malt extract eliminated the accumulation of porphyrin-type competitive byproduct, and the titer of ilamycin-E1/E2 in shaking flasks was improved from original 13.6±0.8 to 142.7±5.7 mg/L for about 10.5 folds. Furthermore, a pH coordinated feeding strategy was first adopted in scaled-up production of ilamycin-E1/E2. The obtained titer of ilamycin-E1/E2 in 30L was 169.8±2.5 mg/L, while in 300L fermentor was only 131.5±7.5 mg/L due to the unsynchronization of feeding response and pH change. Therefore, the continuous pulse feeding strategy was further applied in 300L fermentor and finally achieved 415.7±29.2 mg/L ilamycin-E1/E2, which represented about 30.5 folds improvement at last. Our work provided the solid basis to achieve sufficient ilamycin-E1/E2 lead compounds and support their potential anti-TB drug development.

Optimization of Extraction or Purification Process of Multiple Components from Natural Products: Entropy Weight Method Combined with Plackett–Burman Design and Central Composite Design

In this paper, the optimization of the extraction/purification process of multiple components was performed by the entropy weight method (EWM) combined with Plackett–Burman design (PBD) and central composite design (CCD). We took the macroporous resin purification of Astragalus saponins as an example to discuss the practicability of this method. Firstly, the weight of each component was given by EWM and the sum of the product between the componential content and its weight was defined as the comprehensive score, which was taken as the evaluation index. Then, the single factor method was adopted for determining the value range of each factor. PBD was applied for screening the significant factors. Important variables were further optimized by CCD to determine the optimal process parameters. After the combination of EWM, PBD and CCD, the resulting optimal purification conditions were as follows: pH value of 6.0, the extraction solvent concentration of 0.15 g/mL, and the ethanol volume fraction of 75%. Under the optimal conditions, the practical comprehensive score of recoveries of saponins was close to the predicted value (n = 3). Therefore, the present study provided a convenient and efficient method for extraction and purification optimization technology of multiple components from natural products.

Identification of Effective Factors for Immobilized Lipase Catalysed Biodiesel Production from Pongamia pinnata Seed Oil Using Plackett-Burman Design

Biodiesel a fatty acid alkyl esteris one of the promising biofuel and a clean energy source as an alternative to petroleum-based diesel fuels. The Enzymatic transesterification reaction is influenced by many factors such as amount of biocatalyst, molar ratio of oil to alcohol, temperature, pH, rpm, time etc. Effective variables for transesterification may vary based on the type of feedstock and catalyst used, therefore it is essential to optimise the process suitable for each type of feedstock to achieve higher yield of biodiesel. The statistically-based Plackett-Burman experimental design was adopted in this study to identify effective factors for transesterification reaction of Pongamiapinnata seed oil using immobilized lipase. The factors used in the present study for Plackett-Burman design are molar ratio, amount of immobilized lipase, temperature, time, pH, agitation and water content. The result showed that among seven variables, pH (p-value0.003), agitation speed(p-value 0.024) and amount of immobilized lipase(p-value 0.041) having p<0.05 are statistically significant, positively affecting the transesterification process of Pongamia seed oil. Further the variables which had significant effect on transesterification process will be selected for Response Surface Methodology studies to enhance the yield of biodiesel will be the future scope of work.