Polysaccharides isolated from Morinda officinalis How roots inhibits cyclophosphamide-induced leukopenia in mice

Purpose: To investigate the optimum parameters for extracting polysaccharides from Morinda officinalis How (MOP), and explore their inhibitory effects on leukopenia in mice.Methods: Orthogonal design was performed to investigate the optimum parameters for extracting MOP. A leukopenia mouse model was established by injection of cyclophosphamide (CTX) for three days. Thereafter, MOP (100, 200 and 400 mg/kg) was administered orally for 10 days. Furthermore, blood cells (leukocytes, neutrophil, lymphocyte and mononuclear cell) were analyzed, while serum IL-3 and IL- 6 were determined by ELISA. The thymus and spleen of the mice were separated and weighed to determine viscera indices.Results: Orthogonal design showed that the influence order of the four factors was extraction times (C) > ratio of water to raw material (RWM, D) > extraction time (B) > extraction temperature (A). The optimum extraction parameters for MOP were: extraction temperature (80 °C), extraction duration (2 h), no. of extractions (3), and ratio of water to raw material (30 mL/g). Furthermore, the results indicate that MOP (100, 200 and 400 mg/kg) elevated the levels of leukocyte (p < 0.01), neutrophil (p < 0.01), lymphocyte (p < 0.01) and mononuclear cell (p < 0.01) in leukopenia mice. Besides, MOP (100, 200 and 400 mg/kg) also increased thymus (p < 0.01) and spleen (p < 0.05) indices and serum levels of IL-3 (p < 0.05) and IL-6 (p < 0.01).Conclusion: Orthogonal design is a good strategy for optimizing extraction parameters of MOP. Furthermore, MOP stimulated synthesis of leukocytes in CTX-induced leukopenia in mice. Thus, MOP is a potential adjunct for the treatment of tumors/cancers.Keywords: Morinda officinalis, Polyscacharide, Orthogonal design, Leukopenia, Thymus index, Spleen inde

An in silico approach for modelling T-helper polarizing iNKT cell agonists

Many analogues of the glycolipid alpha-galactosylceramide (α-GalCer) are known to activate iNKT cells through their interaction with CD1d-expressing antigen-presenting cells, inducing the release of Th1 and Th2 cytokines. Because of iNKT cell involvement and associated Th1/Th2 cytokine changes in a broad spectrum of human diseases, the design of iNKT cell ligands with selective Th1 and Th2 properties has been the subject of extensive research. This search for novel iNKT cell ligands requires refined structural insights. Here we will visualize the chemical space of 333 currently known iNKT cell activators, including several newly tested analogues, by more than 3000 chemical descriptors which were calculated for each individual analogue. To evaluate the immunological responses we analyzed five different cytokines in five different test-systems. We linked the chemical space to the immunological space using a system biology computational approach resulting in highly sensitive and specific predictive models. Moreover, these models correspond with the current insights of iNKT cell activation by α-GalCer analogues, explaining the Th1 and Th2 biased responses, downstream of iNKT cell activation. We anticipate that such models will be of great value for the future design of iNKT cell agonists

Extraction-condition Optimization of Baicalein and Schisandrin from Hu-gan-kang-yuan Formula Using Orthogonal Array Design

Purpose: To optimize the extraction conditions for Hu-gan-kang-yuan Formula based on extraction rates of baicalein and schisandrin using an orthogonal array design.Methods: Ethanol concentration (50 - 70 %), ratio of solvent to raw material (8 - 12 mL/g), and extraction time (1 - 3 h) were examined with a three-factor and three-level L9(3)3 orthogonal array design. In addition, analysis of variance (ANOVA) was used to evaluate the statistical significance of the effects of individual factors on extraction rates of baicalein and schisandrin determined by high performance liquid chromatography (HPLC). The number of extractions was further investigated to confirm the extraction rate of target compounds based on the optimized conditions.Results: The optimized conditions were an ethanol concentration of 70 %; ratio of solvent to raw material, 12:1 mL/g; and extraction time of 60 min. Ethanol concentration and ratio of solvent to raw material showed significant effects on the extraction of the two compounds (p < 0.05). The number of extraction steps, two, was reasonable. The final optimum extraction conditions resulted in 79.48 ± 1.40 and 88.55 ± 1.85 % of extraction for baicalein and schisandrin, respectively.Conclusion: The optimized extraction conditions for baicalein and schisandrin are practicable and repeatable, and can be upgraded for pilot-scale production of Hu-gan-kang-yuan preparations.Keywords: Hu-gan-kang-yuan Formula, Extract-condition optimization, Orthogonal array design, Baicalein, Schisandri

Effects of flavonoids extracted from the whole plant of Patrinia Villosa (Thunb) Juss in a rat model of chronic pelvic inflammation

Purpose: To investigate the effects of total flavonoids (PLV) extracted from the  whole plant of Patrinia Villosa (Thunb.) Juss (PTJ) in a rat model of chronic pelvic inflammation.Methods: An orthogonal test design was employed to optimize the extraction  conditions of PLV via reflux extraction by ethanol. Rats were randomly divided into control group, model group and PLV groups. An absorbable gelatin sponge with  pathogens was inserted into the cervix of the rat to establish a pelvic inflammatory model. The PLV groups were orally administered PLV at doses of 25, 50 and 100 mg/kg for eight days. Enzyme-linked immunosorbent assay (ELISA) was used for the determination of inflammatory cytokines in rat serum and the culture  supernatant of RAW264.7 cells. Real-time reverse transcription polymerase chain reaction (real-time PCR) was employed to determine mRNA levels.Results: The optimum extraction conditions for PLV by orthogonal test were  obtained: extraction time (120 min), ratio of liquid to raw material (20 mL/g) and ethanol concentration (50 %). By treating with PLV, the levels of TNF-α, IL-6 and IL-1β significantly decreased (p < 0.01), while IL-10 level significantly increased (p < 0.01) in the serum of chronic pelvic inflammatory rats and LPS-stimulated  macrophages. In addition, a similar trend was observed in the mRNA levels of LPS-stimulated macrophages treated with PLV.Conclusion: PLV showes significant anti-inflammatory effects on chronic pelvic inflammation. The potential mechanism is related to regulating the expression of inflammatory factorsKeywords: Patrinia Villosa (Thunb.) Juss, Total flavonoids, Chronic pelvic  inflammation, Inflammatory cytokine

Optimizing glycerosome formulations via an orthogonal experimental design to enhance transdermal triptolide delivery

Triptolide exerts strong anti-inflammatory and immunomodulatory effects; however, its oral administration might be associated with side effects. Transdermal administration can improve the safety of triptolide. In this study, glycerosomes were prepared as the transdermal vehicle to enhance the transdermal delivery of triptolide. With entrapment efficiency and drug loading as dependent variables, the glycerosome formulation was optimized using an orthogonal experimental design. Phospholipid-to-cholesterol and phospholipid-to-triptolide mass ratios of 30:1 and 5:1, respectively and a glycerol concentration of 20 % (v/v) were used in the optimization. The glycerosomes prepared with the optimized formulation showed good stability, with an average particle size of 153.10 ± 2.69 nm, a zeta potential of –45.73 ± 0.60 mV and an entrapment greater than 75 %. Glycerosomes significantly increased the transdermal delivery of triptolide compared to conventional liposomes. As efficient carriers for the transdermal delivery of drugs, glycerosomes can potentially be used as an alternative to oral triptolide administration

What, who and when? Incorporating a discrete choice experiment into an economic evaluation

Acknowledgements The Medman study was funded by the Department of Health for England and Wales and managed by a collaboration of the National Pharmaceutical Association, the Royal Pharmaceutical Society of Great Britain, the Company Chemist Association and the Co-operative Pharmacy Technical Panel, led by the Pharmaceutical Services Negotiating Committee. The research in this paper was undertaken while the lead author MT was undertaking a doctoral research fellowship jointly funded by the Economic and Social Research Council (ESRC) and the Medical Research Council (MRC). The Health Economics Research Unit (HERU), University of Aberdeen is funded by the Chief Scientific Office of the Scottish Government Health and Social Care Directorate.Peer reviewedPublisher PD

Optimization of Clostridium tyrobutyricum encapsulation by extrusion method and characterization of the formulation

Purpose: To optimize the process parameters for the encapsulation of Clostridium tyrobutyricum (Ct) and to determine its in vitro characteristics.Methods: The process parameters, including the concentration of the wall and hardening material, Ct to gelatin ratio and hardening time, were studied by single factor analysis, while optimization was performed by orthogonal experimental design for the encapsulation rate of Ct.Results: Optimal conditions exhibited by orthogonal experimental design at a 92.17 % encapsulation rate with a viable count of 9.61 ± 0.06 lgCFU/g were: 6 % modified starch, 3 % sodium alginate, and 2 % CaCl2 at a Ct to gelatin ratio of 1:1 with a hardening time of 30 min. The survival rates of encapsulated Ct were higher than free Ct in simulated gastric (6.22 %) and intestinal juices (15.55 %). Reduction in viable counts of Ct at 90 °C were higher for free cells (44.76 %) than encapsulated cells (28.09 %) after 30 min of heat treatment. Correspondingly, encapsulation boosted the capacity of Ct to withstand the strong acidic conditions of the stomach and improved the storage properties of Ct.Conclusion: The results suggested that extrusion is a good technique for the encapsulation of Ct, as it enhances the viability of Ct during their transit through the gastrointestinal tract. Furthermore, encapsulation is favorable for Ct if planned for use in formulations where high temperature treatment is required

Immunoprotective evaluation of Escherichia coli outer membrane protein A against the main pathogens of animal mastitis

Purpose: To evaluate prokaryotic expression of the Escherichia coli (E. coli) outer membrane protein A (OmpA) and its immunoprotective function against the main pathogens of animal mastitis.Methods: A molecular cloning method was used to develop a prokaryotic strain expressing OmpA protein, which was purified by Ni-affinity  chromatography. Polyclonal antiserum was generated in mice immunized with OmpA protein. Enzyme-linked immunosorbent assay (ELISA) and western blotting were used to determine the titer and verify anti-OmpA serum specificity, respectively. Interaction between OmpA antiserum and main pathogens of animal mastitis was verified by ELISA and a pull-down method. The immune protective function of OmpA protein was evaluated in mice challenged with pathogens of animal mastitis. Optimal fermentation conditions to produce OmpA protein were determined by the L9(34) orthogonal test.Results: A prokaryotic strain expressing OmpA protein was developed, and purified OmpA was used to develop a mouse polyclonal antibody. The anti-OmpA serum exhibited high specificity and a titer of 1:1600. Anti-OmpA serum directly interacted with E. coli and Staphylococcus aureus (S. aureus). OmpA demonstrated a significant immune protective function of 58.33 % against E. coli and 46.15 % against S. aureus. The optimal conditions for expressing fermentation OmpA were a strain absorbance of 0.5 at a wavelength of 600 nm, IPTG final concentration of 0.3 mmol/L, induction time of 12 h, and induction temperature of 28 °C.Conclusion: OmpA possesses selective immunogenicity and a significant immune protective effect against the main pathogens of animal mastitis. The results suggest that OmpA may potentially be used as a vaccine for animal mastitis. Keywords: E. coli, OmpA protein, Immunoprotection, Animal mastitis, Protein fermentatio

Construction and evaluation of a novel triple cell epitopebased polypeptide vaccine against cow mastitis induced by Staphylococcus aureus, Escherichia coli and Streptococcus

Purpose: To construct a novel triple cell epitope-based polypeptide vaccine against cow mastitis induced by Staphylococcus aureus, Escherichia coli and  Streptococcus and to reduce the use of antibiotics.Methods: Based on bioinformatics approach, a novel triple epitope-based polypeptide (CM-TEP) was designed and subjected to Ni-NTA flow resin purification. Purified CM-TEP was immunized into mice to prepare a polyclonal antibody. Pull-down assays and enzyme-linked immunosorbent assay (ELISA) were used to detect the interaction between CM-TEP antibodies and S. aureus, E. coli and Streptococcus. Active immunity mice and challenge of bacterial pathogens were used to detect immune protection of CM-TEP. Additionally, the optimal expressing conditions of CM-TEP strain were analyzed using orthogonal test design.Results: A novel cow mastitis triple cell epitope-based polypeptide (CM-TEP) with a MW of 36 kDa was designed, purified and used to immunize mice to prepare a  polyclonal antibody. Pull-down assays and ELISA data showed that CM-TEP  antibodies directly interacted with S. aureus, E. coli and Streptococcus. CM-TEP displayed a significant immune protective effect against infection by S. aureus (50 %, p < 0.05) and E. coli (54.54 %, p < 0.05) and provided some immune protective effect (30.78 %, p > 0.05) against Streptococcus. The optimum expressing conditions of CM-TEP were as follows: IPTG concentration of 0.3 mmol/L, strain OD600 value of 1, inducing temperature of 37 oC, and inducing time of 8 h.Conclusion: The findings suggest that epitope-based vaccine of CM-TEP may be a useful strategy for treating cow mastitis induced by S. aureus, E. coli and Streptococcus.Keywords: Cow mastitis, Epitope vaccine, Immunogenicity, Immune protectiv

Utjecaj dodatka ekstrakta maslačka, saharoze i starter kulture na viskoznost, sposobnost zadržavanja vode i pH jogurta

Dandelion extract is a traditional Chinese medicine and contains significant nutritional value. The aim of this study was to research the optimum fermentation conditions for dandelion addition to plain yogurt using a single factor experiments and orthogonal experiment. The results of the present study demonstrated that the addition of dandelion extract affected the viscosity, water-holding capacity and pH of yogurt. Optimized conditions for dandelion addition to plain yogurt based on viscosity, incubation time, pH and sensory score were 10 % sucrose, 0.3 % of the starter cultures, incubation time of 6.5 hours and 3 % dandelion extract. A new kind of dandelion yogurt with high viscosity, good water-holding capacity and good taste was prepared in this study.Ekstrakt maslačka je tradicionalni kineski lijek, a također ima i značajnu nutritivnu vrijednost. Cilj ovog istraživanja bio je ispitati optimalne uvjete fermentacije za dodavanje maslačka u jogurt korištenjem pojedinih faktorskih eksperimenata i ortogonalnog eksperimenta. Rezultati ove studije pokazali su da dodavanje ekstrakta maslačaka utječe na viskoznost, sposobnost zadržavanja vode i pH jogurta. Optimalni uvjeti za dodavanje maslačka u jogurt na temelju viskoznosti, vremena inkubacije, pH i senzorskog rezultata bili su 10 % saharoze, 0,3 % starter kulture, vrijeme inkubacije od 6,5 sati i dodatak 3 % ekstrakta maslačka. U ovoj studiji pripremljena je nova vrsta jogurta s dodatkom maslačka karakteriziranog visokom viskoznošću, dobrom sposobnošću zadržavanja vode i dobrim okusom