Cloning and expression of recombinant arazyme with anti-inflammatory and anti-breast cancer potential.

Arazyme is an extracellular metalloprotease which is secreted by a Gram-negative symbiotic bacterium called Serratia proteomaculans. There are limited studies on various biological activities of arazyme. This preliminary study was designed to investigate the anti-cancer and anti-inflammatory capacities of recombinant arazyme (rAra) in vitro and in vivo. Arazyme gene, araA was cloned and expressed in E. coli BL21 (DE3) using pET-28a as a vector. Nickel column purification was used to obtain pure rAra. SDS-PAGE and protein assay were used to identify the product and to measure protein content, respectively. Skimmed milk test and casein assay were carried out to assess protease activity. MCF7 cells as a breast cancer cell model were exposed to different concentrations of rAra to study anti-breast cancer potentials using MTT assay. The anti-inflammatory property of rAra was investigated using a murine air-pouch model. PCR and SDS-PAGE data showed that cloning and expression of rAra was successful and the enzyme of interest was observed at 52 KDa. Protein assay indicated that 1 mg/ml of rAra was obtained through purification. A clear zone around the enzyme on skimmed milk agar confirmed the proteolytic activity of rAra and the enzymatic activity was 320 U/mg protein in the casein assay. Cytotoxic effects of rAra reported as IC50 were 16.2 µg/ml and 13.2 mg/ml after 24 h and 48 h, respectively. In the air-pouch model, both the neutrophil count and myeloperoxidase activity, which are measures of inflammation, were significantly reduced. The results showed that rAra can be used in future mechanistic studies and R&D activities in the pharmaceutical industry to investigate the safety and efficacy of the recombinant arazyme.

Wound healing and anti-inflammatory effects of bacterial cellulose coated with Pistacia atlantica fruit oil.

BACKGROUND: Biological activities of Pistacia atlantica have been investigated for few decades. The fruit oil of the plant has been used for treatment of wounds, inflammation, and other ailments in Traditional Persian Medicine (TPM). OBJECTIVES: The main objectives of this study were to analyze the chemical composition of Pistacia atlantica fruit oil and to study wound healing and anti-inflammatory effects of oil-absorbed bacterial cellulose in an in vivo burn wound model. METHOD: Bacterial cellulose membrane was prepared from Kombucha culture and Fourier-transform infrared was used to characterize the bacterial cellulose. Cold press technique was used to obtain Pistacia atlantica fruit oil and the chemical composition was analyzed by gas chromatography. Bacterial cellulose membrane was impregnated with the Pistacia atlantica fruit oil. Pistacia atlantica hydrogel was prepared using specific Carbopol. Burn wound model was used to evaluate in vivo wound healing and anti-inflammatory effects of the wound dressings containing either silver sulfadiazine as positive control, Pistacia atlantica hydrogel or bacterial cellulose membrane coated with the Pistacia atlantica fruit oil. Blank dressing was used as negative control. RESULTS: FT-IR analysis showed that the structure of the bacterial cellulose corresponded with the standard FT-IR spectrum. The major components of Pistacia atlantica fruit oil constituted linoleic acid (38.1%), oleic acid (36.9%) and stearic acid (3.8%). Histological analysis showed that bacterial cellulose coated with fruit oil significantly decreased the number of neutrophils as a measure of inflammation compared to either negative control or positive control (p < 0.05). Wound closure occurred faster in the treated group with fruit oil-coated bacterial cellulose compared to the other treatments (p < 0.05). CONCLUSION: The results showed that bacterial cellulose coated with Pistacia atlantica fruit oil can be a potential bio-safe dressing for wound management.