Nisachon Jangpromma

In Vivo Wound Healing Activity of Crocodile (Crocodylus siamensis) Hemoglobin and Evaluation of Antibacterial and Antioxidant Properties of Hemoglobin and Hemoglobin Hydrolysate.

The hydrolysis of proteins constitutes an invaluable tool, granting access to a variety of peptide fragments with potentially interesting biological properties. Therefore, a hemoglobin (Hb) hydrolysate of Crocodylus siamensis was generated by digestion under acidic conditions. The antibacterial and antioxidant activities of the Hb hydrolysate were assessed in comparison with intact Hb. A disc diffusion assay revealed that the Hb hydrolysate exhibited antibacterial activity against eight strains of gram-positive bacteria and showed a higher efficacy than intact Hb. Moreover, the antioxidant activity of intact Hb and its hydrolysate was evaluated using ABTS and DPPH radical scavenging assays. The Hb hydrolysate exhibited free radical scavenging rates of 6-32%, whereas intact Hb showed a slightly higher activity. In addition, non-toxicity to human erythrocytes was observed after treatment with quantities of Hb hydrolysate up to 10 μg. Moreover, active fragmented Hb (P3) was obtained after purifying the Hb hydrolysate by reversed-phase HPLC. Scanning electron microscopy demonstrated the induction of bacterial cell membrane abnormalities after exposure to P3. Antibacterial and antioxidant activities play crucial roles for supporting the wound healing activity. Consequently, an in vivo mice excisional skin wound healing assay was carried out to investigate the effects of intact Hb treatment on wound healing in more detail. The results clearly demonstrate that intact Hb is capable of promoting 75% wound closure within 6 days. These findings imply that intact Hb of C. siamensis and its acid hydrolysate may serve as valuable precursors for food supplementary products benefitting human health.

Comparative proteomic analysis of leaves, leaf sheaths, and roots of drought-contrasting sugarcane cultivars in response to drought stress

A better understanding of drought response proteins may improve our understanding of the mechanisms underlying drought tolerance in sugarcane. In this research, drought-tolerant (K86-161) and drought-sensitive (B34-164) sugarcane cultivars were grown and exposed to drought stress. The changes in protein expression in leafs, leaf sheaths and roots were analyzed using proteomics techniques. Proteins that responded to drought in both cultivars could be classified into four major categories, including energy and metabolism, photosynthesis, antioxidant, and defense protein. Interestingly, an increased abundance of fructose-bisphosphate aldolase under drought was observed in all three organs of K86-161. Elevated expression of oxygen-evolving enhancer protein was also found in leaves and leaf sheaths of K86-161, when compared with their controls. Additionally, SOD was abundant in the leaves and roots of K86-161. Importantly, the expression level of these proteins decreased in B34-164 under drought stress. These contrasting results suggest that these proteins were inhibited by drought stress in the drought-sensitive cultivar. This proteomic research is the first to combine analyses of leaves, leaf sheaths and roots in sugarcane, which may enhance our understanding of drought responses at the molecular level and lead to selective breeding for enhanced drought tolerance.

Protective Effect of Crocodile Hemoglobin and Whole Blood Against Hydrogen Peroxide-Induced Oxidative Damage in Human Lung Fibroblasts (MRC-5) and Inflammation in Mice

A putative protective effect of cHb and cWb against H2O2-induced oxidative damage was evaluated in detail using MRC-5 cells. In addition, the carrageenan (Carr)-induced mouse paw edema model and the cotton pellet-induced granuloma model were employed to examine the in vivo anti-inflammatory activity of cHb and cWb in mice. It was demonstrated that both cHb and cWb treatments significantly increased cell viability and inhibited morphology alterations in MRC-5 cells exposed to H2O2. Orally administered cHb and cWb significantly reduced Carr-induced paw edema volume and cotton pellet-induced granuloma formation. Moreover, cHb and cWb decreased the expression levels of important pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α), while only cWb was found to increase the expression of the anti-inflammatory cytokine IL-10 significantly. Finally, the activity of antioxidant enzymes (SOD, CAT, and GPx) in the liver improved after cHb and cWb treatment under acute and chronic inflammation. Taken collectively, the results of this study suggest that both cHb and cWb protect against hydrogen peroxide-induced damage in fibroblast cells. Moreover, cHb and cWb were found to exhibit anti-inflammatory activity in both the acute and chronic stages of inflammation and appear to enhance antioxidant enzyme activity and decrease lipid peroxidation in the livers of mice. Therefore, this study indicates that cHb and cWb have great potential to be used in the development of dietary supplements for the prevention of oxidative stress related to inflammatory disorders.

In Vitro and in Vivo Wound Healing Properties of Plasma and Serum from Crocodylus siamensis Blood

The plasma and serum of Crocodylus siamensis have previously been reported to exhibit potent antimicrobial, antioxidant, and anti-inflammatory activities. During wound healing, these biological properties play a crucial role for supporting the formation of new tissue around the injured skin in the recovery process. Thus, this study aimed to evaluate the wound healing properties of C. siamensis plasma and serum. The collected data demonstrate that crocodile plasma and serum were able to activate in vitro proliferation and migration of HaCaT, a human keratinocyte cell line, which represents an essential phase in the wound healing process. With respect to investigating cell migration, a scratch wound experiment was performed which revealed the ability of plasma and serum to decrease the gap of wounds in a dose-dependent manner. Consistent with the in vitro results, remarkably enhanced wound repair was also observed in a mouse excisional skin wound model after treatment with plasma or serum. The effects of C. siamensis plasma and serum on wound healing were further elucidated by treating wound infections by Staphylococcus aureus ATCC 25923 on mice skin coupled with a histological method. The results indicate that crocodile plasma and serum promote the prevention of wound infection and boost the re-epithelialization necessary for the formation of new skin. Therefore, this work represents the first study to demonstrate the efficiency of C. siamensis plasma and serum with respect to their wound healing properties and strongly supports the utilization of C. siamensis plasma and serum as therapeutic products for injured skin treatment.

Novel antioxidant and anti-inflammatory peptides from the Siamese crocodile (Crocodylus siamensis) hemoglobin hydrolysate

Novel antioxidant and anti‐inflammatory peptides were isolated from hydrolysates of Siamese crocodile (Crocodylus siamensis) hemoglobin. C. siamensis hemoglobin hydrolysates (CHHs) were obtained by pepsin digestion at different incubation times (2, 4, 6, and 8 H) at 37 °C and subjected to antioxidant and anti‐inflammatory activity assessment. CHH obtained by 2‐H hydrolysis (2H‐CHH) showed the highest anti‐inflammatory activity with respect to decreasing nitric oxide (NO) production, whereas the strongest antioxidant activity was found for 6‐H hydrolysis (6H‐CHH) against nitric oxide radicals. To evaluate the anti‐inflammatory and antioxidant activity of individual peptide components, 2H‐CHH and 6H‐CHH were purified by semipreparative HPLC. Peptide fraction P57 isolated from 6H‐CHH was found to exhibit the highest nitric oxide radical inhibition activity (32.0%). Moreover, purification of 2H‐CHH yielded peptide fraction P16, which displayed a high efficacy in decreasing NO production of macrophage RAW 264.7 cells (83.2%) and significantly reduced proinflammatory cytokines and inflammatory mediators interleukin‐6 (IL‐6), interleukin‐1 beta (IL‐1β), and prostaglandin‐E2 (PGE2) production to about 2.0, 0.3, and 1.9 ng/mL, respectively. Using LTQ orbitrap XL mass spectrometry, active peptide sequences were identified as antioxidant KIYFPHF (KF7), anti‐inflammatory SAFNPHEKQ (SQ9), and IIHNEKVQAHGKKVL (IL15). Additionally, CHHs simulated gastric and intestinal in vitro digestion positively contributed to antioxidant and anti‐inflammatory activity. Taken collectively, the results of this work demonstrate that CHHs contain several peptides with anti‐inflammatory and antioxidant properties, which may prove valuable as treatment or supplement against diseases associated with inflammation and oxidative stress.

Purification, characterization, cloning and structural analysis of Crocodylus siamensis ovotransferrin for insight into functions of iron binding and autocleavage

Ovotransferrin (OTf), the major protein constituent of egg white, is of great interest due to its pivotal role in biological iron transport and storage processes and its spontaneous autocleavage into peptidic fragments with alternative biological properties, such as antibacterial and antioxidant activities. However, despite being well-investigated in avian, a detailed elucidation of the structure-function relationship of ovotransferrins in the closely related order of Crocodilia has not been reported to date. In this study, electron paramagnetic resonance (EPR) confirmed the presence of two spectroscopically distinct ferric iron binding sites in Crocodylus siamensis OTf (cOTf), but implied a five-fold lower quantity of bound iron than in hen OTf (hOTf). In addition, quantitative estimation of free sulfhydryl groups revealed slight differences to hOTf. To gain a better structural understanding of cOTf, we found a cOTf gene consisting of an open reading frame of 2040bp and encoding a protein of 679 amino acids. In silico prediction of the three-dimensional structure of cOTf and comparison with hOTf revealed four evolutionarily conserved iron-binding sites in both N- and C-lobes, as well as the presence of only 13 of the 15 disulfide bonds in hOTf. This evolutionary loss of disulfide linkages in conjunction with the lack of hydrogen bonding from a dilysine trigger in the C-lobe are presumed to affect the iron binding and autocleavage character of cOTf. As a result, cOTf may be capable of exerting a more diverse array of functions compared to its avian counterparts; for instance, ion buffering, antioxidant and antimicrobial activities.

Development and Characterization of an Anti-Acne Gel Containing Siamese Crocodile (Crocodylus siamensis) Leukocyte Extract

Leukocytes are reportedly the first line of the innate immune defense and essential for the control of common bacterial infections. Therefore, in this work, the antibacterial activity of crocodile leukocyte extract against Propionibacterium acnes was evaluated, and we also characterized the related activity of skin infection. The leukocyte extract showed the minimum inhibitory concentration to be 100 μg/ml to P. acnes. SEM imaging demonstrated that the leukocyte extract adversely affected P. acnes cell permeability in a concentration-dependent manner. Furthermore, the crocodile leukocyte extract could significantly reduce proinflammatory markers and decrease inflammatory signs in infected mouse ears. The crude leukocyte extract was further purified using FPLC and RP-HPLC. The resulting fraction F5 was indicated as the anti-acne peptide-containing fraction. The molecular mass of the peptide contained in F5 was calculated to be 4,790.5 Da. N-Terminal sequencing revealed the amino acid sequence as GPEPVPAIYQ, which displays similarities to immunoglobulin A and leucine-rich repeat neuronal protein. This is the first reported amino acid sequence of a crocodile leukocyte extract that possesses anti-acne activity. To attempt to use it in a prototype cosmetic, an anti-acne gel containing crude crocodile leukocyte extract was formulated, resulting in seven gel formulations (G1, G2, G3, G4, G5, G6, and G7). The formulations G5, G6, and G7 exhibited 2-fold higher anti-acne activity than G1-G4. Investigation of accelerating stability studies of anti-acne gel formulations G5, G6, and G7 demonstrated that a low storage temperature (4°C) is suitable for maintaining the physical properties and biological activity of the anti-acne gel products.

KT2 and RT2 modified antimicrobial peptides derived from Crocodylus siamensis Leucrocin I show activity against human colon cancer HCT-116 cells

Conventional colon cancer treatments have been associated with side effects. Consequently, the discovery of novel effective and safe therapies is urgently needed. Hence, cationic antimicrobial peptides KT2 and RT2 were evaluated towards human colon cancer HCT-116 cells. The MTT assay indicated that both KT2 and RT2 exhibited anticancer activity with good therapeutic indices, and were found to be non-toxic to non-cancerous Vero cells. The IC50 values of KT2 were determined as 111.96 and 90.25 μg/mL while RT2 showed IC50 as 104.07 and 87.84 μg/mL after 12 and 24 h treatments, respectively. Moreover, KT2 and RT2 treatment caused a significant reduction in PI3K, AKT1 and mTOR mRNA expression levels, which resulted in suppression either of HCT-116 proliferation or migration. The mechanism involved in apoptosis induction were due to decreased Bcl-2 and XIAP and increased p53, cytochrome c, caspase-2, caspase-3, caspase-8, and caspase-9 mRNA expression levels. These effects increased the level of cell cycle associated gene p21 and decreased cyclin B1 and cyclin D1 expression.

Purification and characterization of macrolactins and amicoumacins from Bacillus licheniformis BFP011: a new source of food antimicrobial substances

ABSTRACT In this study, Bacillus licheniformis BFP011 has been cultivated for producing antimicrobial compounds, which were concentrated from the crude supernatant and purified using thin-layer chromatography. The antimicrobial activity was investigated in the presence of several organic solvents and detergents, and inhibiting effects to various Gram-negative and Gram-positive human pathogenic and food spoilage bacteria were observed. Three bands (F4, F5 and F6) showing antimicrobial activity against Salmonella typhi ATCC 5784 were subjected to reversed-phase high-performance liquid chromatography purification. Two peaks of fraction F5, F5-P3 and F5-P4, showed 100% inhibition against S. typhi ATCC 5784. The contained mixture of antimicrobial compounds consists of macrolactins and amicoumacins, which induced the collapse and breaking of S. typhi ATCC 5784 cell membranes in a time-dependent manner. Taken collectively, the results indicate that these compounds may represent promising candidates for the development of food preservative agents of natural origin, as well as novel antimicrobial drug candidates against multiresistant bacterial strains.

A novel antibacterial peptide derived from Crocodylus siamensis haemoglobin hydrolysate induces membrane permeabilization causing iron dysregulation, oxidative stress and bacterial death

A novel antibacterial peptide from Crocodylus siamensis haemoglobin hydrolysate (CHH) was characterized for antimicrobial activity.