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Ethnopharmacological relevance Citrus fruit peels are traditionally used in folk medicine for the treatment of skin disorders but it lacks proper pharmacological intervention. Citrus limetta Risso (Rutaceae) is an important commercial fruit crops used by juice processing industries in all continents. Ethnopharmacological validation of an essential oil isolated from its peels may play a key role in converting the fruit waste materials into therapeutic value added products. Aim of the study To evaluate the chemical and pharmacological ( in-vitro and in-vivo ) profile of essential oil isolated from Citrus limetta peels ( Clp-EO) against skin inflammation for its ethnopharmacological validation. Materials and methods Hydro-distilled essential oil extracted from Citrus limetta peels ( Clp-EO) was subjected to gas chromatography (GC) analysis for identification of essential oil constituents and its anti-inflammatory evaluation through in vitro and in vivo models. Results Chemical fingerprint of Clp-EO revealed the presence of monoterpene hydrocarbon and limonene is the major component. Pre-treatment of Clp-EO to the macrophages was able to inhibit the production of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) in LPS-induced inflammation as well as the production of reactive oxygen species (ROS) in H 2 O 2 -induced oxidative stress. In in-vivo study, topical application of Clp-EO was also able to reduce the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear thickness, ear weight, lipid peroxidation, pro-inflammatory cytokines production and ameliorate the histological damage in the ear tissue. In-vitro and in-vivo toxicity study indicate that it is safe for topical application on skin. Conclusion These findings suggested the preventive potential of Clp-EO for the treatment of inflammation linked skin diseases. [ABSTRACT FROM AUTHOR]

In an effort to evaluate novel pharmacological activity of 1-chloro-2-formyl indene and tetralene analogues possessing potential antitubercular and antistaphylococcal agents, we explored its anti-inflammatory potential against lipopolysaccharide(LPS)-induced inflammation using in-vitro and in-vivo bioassay. Synthesized analogues significantly inhibited the production and expression of pro-inflammatory cytokines against LPS-induced inflammation in macrophages isolated from mice. Among all the analogues, TAF-5 (1-Chloro-2-formyl-1-tetralene) exhibited most potent anti-inflammatory activity without any cytotoxic effect. We have further evaluated the therapeutic efficacy and safety of TAF-5 in in-vivo system using LPS-induced sepsis, a systemic inflammation model and acute oral toxicity respectively in mice. Oral administration of TAF-5 inhibited the pro-inflammatory cytokines in serum, attenuated the organs injuries and improved host survival in dose dependent manner. Acute oral toxicity study showed TAF-5 is non–toxic at higher dose in mice. These results suggest the suitability of indene and tetralene analogues as new chemical entities for further investigation towards the management of inflammation related diseases. [ABSTRACT FROM AUTHOR]

Abstract: Ethnopharmacological relevance: Many of the effective therapeutic strategies have been derived from ethnopharmacologically used natural products. Pluchea lanceolata is an herb employed in Indian folk medicine for malaria like fever but it lacks proper pharmacological intervention. Aim of the study: To evaluate antimalarial and safety profile of Pluchea lanceolata: an in-vitro, in-vivo for its ethnopharmacological validation. Materials and methods: Methanol, butanol, ethyl acetate, chloroform, hexane extracts and its isolate, taraxasterol acetate (TxAc) were obtained from air dried aerial part of Pluchea lanceolata. These were tested in-vitro against chloroquine-sensitive strain of Plasmodium falciparum NF54 by measuring the parasite specific lactate dehydrogenase activity. The most potent hexane extract and TxAc were further validated for in-vivo antimalarial and safety evaluation. TxAc, a pentacyclic-triterpene isolated from the most active fraction was further evaluated with special emphasis on inflammatory mediators involved in malaria pathogenesis. Murine malaria was induced by intra-peritoneal injection of Plasmodium berghei infected red blood cells to the male Swiss inbred mice. Mice were orally treated following Peters 4-Day suppression test. In-vivo antimalarial efficacy was examined by evaluating the parasitaemia, percent survival, mean survival time, blood glucose, haemoglobin and pro-inflammatory mediators involved in malaria pathogenesis. Results: Hexane extract and TxAc showed promising antimalarial activity in-vitro and in-vivo condition. TxAc attributed in inhibition of the pro-inflammatory cytokines as well as afford to significant increase in the blood glucose and haemoglobin level when compared with vehicle treated infected mice. We have not observed the synergistic action of combinations of chloroquine and TxAc from our experimental results. In-vitro and in-vivo safety evaluation study revealed that hexane extract is non toxic at higher concentration. Conclusion: Present study further validates the ancient Indian traditional knowledge and use of Pluchea lanceolata as an antimalarial agent. Study confirms the suitability of Pluchea lanceolata as a candidate for further studies to obtain a prototype for antimalarial medicine. [Copyright &y& Elsevier]

Streptokinase is a well-established cost-effective therapeutic molecule for thrombo-embolic complications. In the current study, a tag-free variant of streptokinase with a native N-terminus (N-rSK) was developed using the Pichia expression system. A three-copy clone was screened that secreted 1062 mg/L of N-rSK in the complex medium at shake flask level. The biologically active (67,552.61 IU/mg) N-rSK recovered by anion exchange chromatography was predicted to contain 15.43% α-helices, 26.43% β-sheets. The fermentation run in a complex medium yielded a poor quality product due to excessive N-rSK degradation. Therefore, modified basal salt medium was also employed during fermentation operations to reduce the proteolytic processing of the recombinant product. The concomitant feeding of 1 g/L/h soya flour hydrolysate with methanol during the protein synthesis phase reduced the proteolysis and yielded 2.29 g/L of N-rSK. The fermentation medium was also supplemented with urea during growth and induction phases. The combined feeding approach of nitrogen-rich soya flour hydrolysate and urea during bioreactor operations showed significant improvement in protein stability and resulted in a 4-fold increase in N-rSK concentration to a level of 4.03 g/L over shake flask. Under optimized conditions, the volumetric productivity and specific product yield were 52.33 mg/L/h and 33.24 mg/g DCW, respectively. [ABSTRACT FROM AUTHOR]

• Native PIR1 gene signal used for streptokinase secretory expression in P. pastoris. • Streptokinase gene dosage to 4 copies led to a higher product yield of 1206.48 mg/L. • Cell retention strategy further improved the product concentration to 3049.53 mg/L. • Cell recycling prevented the proteolytic degradation of recombinant streptokinase. • Streptokinase was biologically active with a specific activity of 62,782.40 IU/mg. Streptokinase is a therapeutically important thrombolytic agent which is toxic to its heterologous expression hosts and also prone to proteolytic degradation. In the current study, its secretory expression was targeted in P. pastoris using the native protein with internal repeats 1 (PIR1) secretion signal where the optimization of streptokinase gene dosage resulted in an increase in the volumetric product yield from 339.69 to 1206.48 mg/L. The cell retention strategy was applied during the induction phase to maintain sustained productivity where up to a 2.4 fold enhancement in the overall product yield was achieved at shake flask to a level of 3049.53 mg/L. Moreover, the cell recycling approach prevented the streptokinase proteolytic degradation and also helped in achieving a high specific product yield (Y P/X) in a range of 167.65–176.44 mg/g DCW even after five cycles. Similarly, the cell recycling strategy was also employed using S. cerevisiae α-mating factor (α-MF) signal sequence which also resulted in improved product stability and volumetric product concentration confirming its wide applicability in obtaining enhanced product quality and yield. The recombinant streptokinase produced using PIR1 signal sequence was biologically active possessing a specific activity of 62,782.40 IU/mg and was predicted to contain 10.45% α-helix and 29.09% β-sheet. [ABSTRACT FROM AUTHOR]

Highlights • ScUlp1 (402–621 aa) catalytic domain was cloned and expressed under T7 promoter. • Soluble expression was achieved at 30 °C to a level of 170 mg/L at shake flask. • ScUlp1 was purified near 95% homogeneity with specific activity of 3.986 × 105 U/mg. • The catalytic efficiency was demonstrated upto 1:10,000 enzyme to substrate ratio. • Bioprocess optimization resulted in a ScUlp1 concentration of 3.25 g/L at fermentor. Abstract The exploitation of SUMO (small ubiquitin-related modifier) fusion technology at a large scale for the production of therapeutic proteins with an authentic N-terminus is majorly limited due to the higher cost of ScUlp1 protease. Therefore, the cost-effective production of Saccharomyces cerevisiae Ulp1 protease catalytic domain (402–621 aa) was targeted via its cloning under strong T7 promoter with and without histidine tag. The optimization of cultivation conditions at shake flask resulted in ScUlp1 expression of 195 mg/L in TB medium with a specific product yield of 98 mg/g DCW. The leaky expression of the ScUlp1 protease was controlled using the chemically defined minimal medium. The Ni-NTA affinity purification of ScUlp1 was done near homogeneity using different additives (0.1% Triton X-100, 0.01 mM DTT, 0.02 mM EDTA and 1% glycerol) where a product purity of ∼95% with a recovery yield of 80% was obtained. The specific activity of purified ScUlp1 was found to be 3.986 × 105 U/mg. The ScUlp1 protease successfully cleaved the SUMO tag even at 1:10,000 enzyme to substrate ratio with high efficacy and also showed a comparable catalytic efficiency as of commercial control. Moreover, the in vivo cleavage of SUMO tag via co-expression strategy also resulted in more than 80% cleavage of SUMO fusion protein. The optimization of high cell density cultivation strategies and maintenance of higher plasmid stability at bioreactor level resulted in the ScUlp1 production of 3.25 g/L with a specific product yield of 45.41 mg/g DCW when cells were induced at an OD 600 of 132 (63.66 g/L DCW). [ABSTRACT FROM AUTHOR]

Diarylheptanoids, a group of plant secondary metabolites are increasingly recognized as potential therapeutic agents. The aim of study was to ascertain the anti-inflammatory profile of diarylheptanoids from Alnus nepalensis against lipopolysaccharide (LPS)-induced inflammation in macrophages and endotoxic shock in mice. Extracts prepared from dried leaves of A. nepalensis using standard solvents were tested against LPS-induced inflammation in macrophages. Among all, butanol extract ( ANB ) has shown most significant inhibition of pro-inflammatory cytokines without any cytotoxicity. HPLC analysis of ANB showed the presence of diarylheptanoids. The diarylheptanoids were further isolated and tested in-vitro for anti-inflammatory activity. Treatment of isolated diarylheptanoids ( HOG , ORE and PLS ) was able to reduce the production and mRNA level of pro-inflammatory cytokines (TNF-α and IL-6). Furthermore, we demonstrated that it inhibited the expression of NF-kB protein in LPS-induced inflammation in macrophages. In-vivo efficacy and safety profile of ANB revealed that oral treatment of ANB was able to improve the survival rate, and inhibited the production of pro-inflammatory cytokines in serum, attenuated vital organ injury in a dose dependent manner without any toxic effect at higher dose in mice. The results suggest that diarylheptanoids from A. nepalensis can be considered as potential therapeutic candidates for the management of inflammation related diseases. [ABSTRACT FROM AUTHOR]