Titpawan Nakpheng

The effect of sericin from various extraction methods on cell viability and collagen production.

Silk sericin (SS) can accelerate cell proliferation and attachment; however, SS can be extracted by various methods, which result in SS exhibiting different physical and biological properties. We found that SS produced from various extraction methods has different molecular weights, zeta potential, particle size and amino acid content. The MTT assay indicated that SS from all extraction methods had no toxicity to mouse fibroblast cells at concentrations up to 40 μg/mL after 24 h incubation, but SS obtained from some extraction methods can be toxic at higher concentrations. Heat-degraded SS was the least toxic to cells and activated the highest collagen production, while urea-extracted SS showed the lowest cell viability and collagen production. SS from urea extraction was severely harmful to cells at concentrations higher than 100 μg/mL. SS from all extraction methods could still promote collagen production in a concentration-dependent manner, even at high concentrations that are toxic to cells.

Evaluation of the wound healing potential of Wedelia trilobata (L.) leaves

ETHNOPHARMACOLOGICAL RELEVANCE Wedelia trilobata (L.) Hitchc (Asteraceae) leaves are used in the treatment of wounds by traditional healers. Despite the use of this plant in wound healing, there is a scarcity of scientific data to support its therapeutic application. AIM OF THE STUDY To investigate the wound healing potential of Wedelia trilobata (L.) leaves commonly employed by traditional healers and to clarify its traditional use in a scientific investigation. MATERIALS AND METHODS An ethanolic extract of Wedelia trilobata leaves was subjected to column chromatography. Hexane, ethyl acetate (WEA) and chloroform:methanol (50:50) (WCM) fractions were obtained. The fractions were tested using relevant in vitro wound healing assays. Antioxidant activity was measured by the DPPH assay. The fibroblast proliferation, oxidative stress using hydrogen peroxide, an in vitro scratch assay, and increasing collagen content was determined using fibroblast L929. Minimum inhibitory concentrations (MICs) were determined against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa. RESULTS WEA (3 μg/mL) promoted fibroblast L929 survivability up to more than 90% before and more than 85% after hydrogen peroxide induced oxidative stress. WEA (3 μg/mL) induced a 70% migration rate in the in vitro scratch assay and the collagen content was increased to 261 μg/mL compared to the control (57.5 μg/mL). WCM exhibited a scavenging activity for DPPH with an IC(50) value of 179.5 μg/mL comparable to BHT (139.3 μg/mL). WEA was active against gram positive bacteria Staphylococcus aureus, Staphylococcus epidermidis with MIC values of 62.5 and 31.25 μg/mL, respectively. CONCLUSION These scientific findings of wound healing activity supports the traditional claims for Wedelia trilobata (L.) leaves. The WEA displayed antibacterial and fibroblast stimulatory activities while WCM exhibited antioxidant to indicate its potential wound healing properties. However further studies to isolate the antibacterial, antioxidant and fibroblast stimulatory compounds that contribute to the wound healing properties of this plant are needed.

Inhaled pyrazinamide proliposome for targeting alveolar macrophages

Objective: In this study, pyrazinamide (PZA)-proliposome in a dry powder aerosol form was developed for delivering drugs to alveolar macrophages (AMs) infected with mycobacteria. Materials and methods: PZA-proliposomes consisting of pyrazinamide, soybean phosphatidylcholine, cholesterol and porous mannitol were prepared by a spray drying method. The PZA-proliposome physicochemical properties were determined using a cascade impactor, X-ray diffraction, differential scanning calorimetry and infrared spectroscopy. The toxicity of proliposomes to respiratory-associated cell lines (Calu-3, A549 and NR8383) and its potential to provoke immunological responses from AMs were determined. In vivo repeated dose toxicity in rats was evaluated. Results and discussion: PZA-proliposomes were successfully prepared. For the aerosolization properties of PZA-proliposomes at 60 l/min, the powders showed mass median aerodynamic diameters of 4.26–4.39 µm, with fine particle fractions (aerosolized particles less than 4.4 µm) of 20–30%. Encapsulation of PZA was 26–45%. PZA-proliposomes were less toxic to respiratory-associated cells, and did not activate AMs to produce inflammatory mediators, including interleukin-1β, tumor necrosis factor-α, and nitric oxide, at a toxic level. Renal and liver toxicity in rats were not observed. Conclusions: We suggest that PZA-proliposomes are potential candidates for pulmonary tuberculosis treatment.

Levofloxacin-Proliposomes: Opportunities for Use in Lung Tuberculosis

Levofloxacin (LEV) is a relatively new-generation fluoroquinolone antibiotic that has good activity against Mycobacterium tuberculosis. The aims of this study were to develop and evaluate LEV-proliposomes in a dry powder aerosol form for pulmonary delivery. LEV-proliposomes containing LEV, soybean phosphatidylcholine, cholesterol and porous mannitol were prepared by a spray drying technique. The physicochemical properties of LEV-proliposomes were determined using a cascade impactor, X-ray diffraction (XRD), differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FT-IR). The toxicity of proliposomes to respiratory-associated cell lines and its potential to provoke immunological responses from alveolar macrophages (AMs) were evaluated. Antimycobacterial activity using flow cytometry and an in vivo repeated dose toxicity test in rats were carried out. LEV-proliposomes were successfully prepared with mass median aerodynamic diameters of 4.15–4.44 μm and with fine particle fractions (aerosolized particles of less than 4.4 µm) of 13%–38% at 60 L/min. LEV-proliposomes were less toxic to respiratory-associated cells than LEV, and did not activate AMs to produce inflammatory mediators that included interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and nitric oxide. The minimum inhibitory concentration (MIC) against M. bovis of LEV and LEV-proliposomes containing LEV 10% were 1 and 0.5 µg/mL, respectively. The efficacy of LEV-proliposomes against M. bovis was significantly higher than that of free LEV (p < 0.05). The efficacy of the LEV-proliposomes against M. tuberculosis was equal to that of the free LEV (MIC = 0.195 µg/mL). In a repeated dose toxicity study in rats, renal and liver toxicity was not observed. LEV-proliposomes should now be tested as an alternative formulation for delivering LEV to the lower airways.

Wound healing activity of ent-kaura-9(11),16-dien-19-oic acid isolated from Wedelia trilobata (L.) leaves.

Wedelia trilobata (L.) Hitchc (Asteraceae) has been used in traditional medicine in the Caribbean and Central America for stubborn wounds, sores, swelling, arthritic painful joints. The present study was carried out to derive bioactive compounds from ethanolic extracts of W. trilobata (L.) leaves that could influence wound healing. W. trilobata leaves extract were subjected to bioassay-guided fractionation. The five fractions (WEA1-A, B, C, D, and E) obtained were tested for antimicrobial activity. Out of the five fractions only the fraction (WEA1-B) containing ent-kaura-9(11),16-dien-19-oic acid showed promising antibacterial activity with MIC value of 15.62μg/ml against S. aureus and 7.81μg/ml against S. epidermidis. It was then further assessed for its possible activity on fibroblasts by measuring their percentage cell viability and on oxidative stress induced by hydrogen peroxide. WEA1-B (2.5-0.08μg/ml) produced an increase in the percentage viability of mouse fibroblast L929 cells from 97 to 117% and protection of the fibroblast L929 cells against oxidative stress induced by hydrogen peroxide (94-80%). The present study provides some scientific evidence for the traditional use of W. trilobata in the management of wound healing due to a combination of antimicrobial, stimulation of fibroblast growth and protection of the cells from hydrogen peroxide-induced injury, all of which could play some role in its effect on tissue repair.

Evaluation of the wound healing property of Boesenbergia longiflora rhizomes

ETHNOPHARMACOLOGICAL RELEVANCE The rhizomes of Boesenbergia longiflora (Wall.) Kuntze (Zingiberaceae) have been traditionally used for treatment of inflammatory bowel disease, ulcerative colitis, aphthous ulcer and abscess by decoction with alcohol. AIM OF THE STUDY The rhizomes of Boesenbergia longiflora were carried out to investigate for anti-inflammatory and wound healing activities in order to support the traditional use. MATERIAL AND METHODS The ethanolic extract of Boesenbergia longiflora and its fractions were tested using relevant in vitro anti-inflammatory and wound healing assays. For the in vitro studies, murine macrophage RAW264.7 cells and mouse fibroblast L929 cells were assessed for anti-inflammatory and fibroblast stimulatory activities, respectively. In vivo anti-inflammatory activity was determined by carrageenan-induced rat paw edema model as well as acute toxicity estimated by the up-and-down method in mice. RESULTS The present study has demonstrated that the ethanolic extract of Boesenbergia longiflora rhizomes possesses a potent anti-inflammatory and wound healing activities. Among the isolated fractions, the CHCl3 fraction showed potent anti-inflammatory effect through nitric oxide inhibitory activity (IC50=5.5 μg/ml) and reduction of carrageenan-induced rat paw edema (ED50=222.7 mg/kg), whereas this fraction exhibited wound healing property via fibroblast migration on both day 1 (77.3%) and day 2 (100%) as well as enhanced collagen production (187.5 μg/ml) at concentration of 3 μg/ml, compared to that of the controls, 39.4% for fibroblast and 60.8 μg/ml for collagen, respectively. The anti-inflammatory mechanism of the CHCl3 fraction is found to suppress the iNOS and COX-2 mRNA expression. CONCLUSION The scientific investigation of wound healing activity of Boesenbergia longiflora rhizomes support the Thai traditional uses for treatment of inflammatory bowel disease, ulcerative colitis, aphthous ulcer and abscess. The EtOH extract and CHCl3 fraction exert potential wound healing property through NO inhibition, anti-oxidant effect and stimulation of fibroblast migration and collagen production. The phytochemical screening revealed that the CHCl3 fraction of Boesenbergia longiflora rhizomes contains diarylheptanoids, flavonoids and terpenes. The isolation of the compounds responsible for the wound healing effect is now in progress.

The safety of ethambutol dihydrochloride dry powder formulations containing chitosan for the possibility of treating lung tuberculosis

Abstract Objective: The aim of this study was to conduct in vitro studies of a dry powder formulation of ethambutol dihydrochloride (EDH) to determine if it was an acceptable candidate for further in vivo studies to target alveolar macrophages for the treatment of lung tuberculosis. Materials and methods: Nanosized drug particles were prepared by optimizing the spray drying conditions. The cell toxicities were determined by interacting the formulations with respiratory cell lines (A549, calu-3 and NR8383 cell lines), and phagocytosis of the formulations was tested on a macrophage cell line. Permeations of the EDH formulations across a lipid bilayer were studied using the Ussing chamber and HPLC. Bioactivity tests of the formulations were carried out by using the resazurin method on M. bovis cells. Result and discussion: Spray rate and inlet temperature were the two most important factors that affected the size and % yield of the product. The % cell viability of A549 cells with all EDH formulations, pure EDH and chitosan carrier was higher than 80%, the calu-3 cell line had % viabilities of between 85 and 99%, and the % viability of NR8383 cells was between 81 and 100%. The pure EDH had a minimum inhibitory concentration (MIC) of 2 µg/mL while the EDH formulations had MIC values of less than 1 µg/mL when tested against M. bovis. The formulation was completely phagocytized by the macrophage cells after 30 min. The permeability of pure EDH across lipid bilayer was 48.7% after 2 h while in the EDH formulations it was enhanced to 71%. Conclusion: The EDH formulations showed a lower toxicity, higher potency and better permeation than the pure EDH. Thus, EDH DPI formulations could help to minimize the duration of treatment and the risk of developing multidrug resistance tuberculosis compared to the non-formulated EDH.

Stabilization of luteinizing hormone-releasing hormone in a dry powder formulation and its bioactivity

Abstract Background: Luteinizing hormone-releasing hormone (LHRH) is a naturally occurring hormone that controls sex hormones in both men and women. In general, LHRH is poorly absorbed through the gastrointestinal tract due to its large molecular size, high polarity, and loss from enzymatic degradation. Objective: Prepare and develop LHRH in a dry power formulation with stability and biological activity. Methods: Mannitol (M) and glycine (G) were chosen as ingredients to stabilize and protect LHRH during the freeze drying processes and during storage. The physicochemical properties of LHRH dry powders were examined by capillary electrophoresis, fluorescence spectrophotometry, scanning electron microscopy, and photon correlation spectroscopy. The release of LHRH from the dry powder was carried out in dissolution apparatus. In addition, a rat model was employed to study the bioactivity of LHRH in the dry powder form. Results: The LHRH dry powder formulations using M and G in the ratios of 6:4 and 7:3 were more stable than other formulations. LHRH colloids containing M:G showed no aggregation after storage at 4°C for one month. The concentration of LHRH in the dry powder form was more stable than that of LHRH in solution form. All the LHRH dry powder formulations were instantly dissolved within 10 seconds in an aqueous medium. After the LHRH dry powder (13 mg) was reconstituted and administered intraperitoneally to male rats during a one-month period, the testosterone level in the plasma was significantly decreased compared with an untreated group (15.0±1.0 ng/mL, 15.0±1.0 ng/mL and 20.0±2.0 ng/mL for LHRH containing M:G; 6:4, 7:3, and 8:2, respectively, compared to the control of 35±2 ng/mL, p<0.05). Conclusion: The LHRH dry powder formulations had good physicochemical properties and bioactivity.

Evidences for salbutamol metabolism by respiratory and liver cell lines

This study aimed to investigate the enantiomeric biotransformation of salbutamol in the human respiratory and liver cells. The cells from the different cell growth cycles were treated with various concentrations of salbutamol sulfate. Salbutamol and its metabolites were analyzed using chiral liquid chromatography and mass spectrometry. There were no metabolites of salbutamol found in the extracellular medium, intracellular, and cell lysate of respiratory cell lines. The S/R ratios of salbutamol were found to be 0.99-1.10 in all cell lines, cell cycles, and salbutamol concentrations in this study. Salbutamol metabolites were found only in intracellular HepG2 cells. The S/R ratios of the salbutamol inside the liver cells were 10 times greater than the S/R ratios of the salbutamol in the liver extracellular medium (0.99-1.10). It is important to note that the S/R ratios of salbutamol in liver cell lysate enzyme were 0.99-1.10 whereas the S/R salbutamol metabolites inside the liver cell were around 1.91-2.14. Both salbutamol and sulfate conjugation metabolites were detected in MS chromatograms with an m/z of 239.2 and 317.6, respectively. Hence, the delivery of salbutamol directly to the respiratory system is a right target that can avoid first-pass metabolism.

Development of a topical mupirocin spray for antibacterial and wound healing applications

Abstract Objective: The aim of this study was to develop mupirocin topical spray using Eudragit E100 as a film-forming agent for the treatment of bacterial skin infections as well as to promote wound healing. Materials and methods: Twenty-seven of mupirocin formulations were formulated containing Eudragit E100 and other excipients. Mupirocin spray was prepared by aerosol crimping and filling machine using HFA-134a as a propellant. The formulations were evaluated for their stability and physicochemical properties. The factorial study was applied to evaluate the effects of glycerol and PEG400 on mupirocin-loaded Eudragit E100 films. The optimized formulation was assessed of drug release, antibacterial activities and in vitro cell line studies in comparison to the ointment formulation. Results and discussion: Mupirocin sprays were formulated and optimized to obtain the formulation with excellent physicochemical and mechanical properties of the dressing film. The formulation had an excellent stability up to a year with more than 80% of mupirocin content. Mupirocin was released from the film up to 90% within 2 h. The formulation had a potent antibacterial effect against S. aureus and S. epidermidis. The formulation was safe to use as a topical formulation that had no toxicity to keratinocytes, fibroblasts and monocytes. The formulation also had an antiendotoxin effect without stimulating the production of NO and inflammatory cytokines (IL-1β and TNF-α). Conclusions: Mupirocin topical spray was successful developed as a topical formulation and can be used instead of the ointment formulation. Animal experiments are warranted to further emphasize the safe use in the human skin.