A novel therapeutic effect of mannitol-rich extract from the brown seaweed Sargassum ilicifolium using in vitro and in vivo models.

BACKGROUND: Wound healing is an active, complex, integrated series of cellular, physiological, and biochemical changes initiated by the stimulus of injury in a tissue. The present study was performed to investigate the potential wound healing abilities of Sargassum ilicifolium crude extracts (CE) that were characterized by 1H NMR and FTIR Spectrometric measurements. MATERIALS AND METHODS: Seaweed samples were collected from southern coastal sites of Sri Lanka. To determine the cytotoxicity and proliferation of S. ilicifolium CE were used for the MTT and alamarBlue assays respectively. The scratch and exclusion wound models were used to HaCaT and HDF cells to assess the cell proliferation and migration. RAW 264.7 cells (macrophages) were used to evaluate Nitric Oxide (NO) production and phagocytosis activities. Moreover, Fifteen, 8-week-old, female, New Zealand rabbits were selected and divided into five groups: excision skin wounds (10.40 ± 0.60 mm) were induced in groups I, II, and III. Rabbits in groups I and IV were given S. ilicifolium CE (orally, 100 mg/kg day, two weeks), whereas groups II and V were given equal amounts of distilled water. Wound healing properties were measured and wound tissue samples were collated, formalin-fixed, wax-embedded, stained (Hematoxylin and Eosin; Van Gieson) and examined for the healing process. RESULTS: Anti-inflammatory and wound healing activities were observed in RAW 264.7, HDF and HaCaT cells treated with S. ilicifolium aqueous extracts when compared to the control groups. S. ilicifolium extracts concentration 8 - 4 µg/µL, (P<0.05) had remarkable the highest proliferative and migratory effects on RAW 264.7, HDF and HaCaT cells when compared with the control. RAW 264.7 cell proliferation and/or migration were higher in S. ilicifolium extracts (4 µg/µL, 232.8 ± 10.07%) compared with the control (100 %). Scratch wound healing were remarkably enhanced in 24 h, 48 h (P<0.05) when treated with S. ilicifolium on HaCaT cells. Rabbits treated with the CE of S. ilicifolium showed a significantly increased wound healing activities (P<0.05) within three days with a close wound area of 57.21 ± 0.77 % compared with control group (26.63 ± 1.09 %). Histopathology, aspartate aminotransferase and alanine aminotransferase levels evidenced no toxic effects on seaweed treated groups. Histopathological results also revealed that the healing process was significantly faster in the rabbit groups which were as treated with CE of S. ilicifolium orally with the evidence of enhanced early granulation tissue (connective tissue and angiogenesis) and significant epithelization compared to the control. CONCLUSIONS: Cell proliferation and migration are significantly faster when treated with S. ilicifolium aqueous extracts. Moreover, there are no toxic effect of S. ilicifolium aqueous extracts on RAW 264.7, HDF and HaCaT cell lines. In this study, it is revealed that S. ilicifolium has potential remedial agent; D-Mannitol for skin wound healing properties that by promote keratinocyte and fibroblast proliferation and migration. These findings show that S. ilicifolium have promising wound healing properties.

Comparative analysis of proximate compositions, mineral and functional chemical groups of 15 different seaweed species.

Seaweed is a popular edible source and is associated with many foods and pharmaceutical industries around the world. The current research aims to provide information on the chemical composition of 15 seaweed species, consisted of Chlorophyta, Ochrophyta/Phaeophyceae, and Rhodophyta macroalgae, collected from coastal areas of Sri Lanka. Seaweed samples were subjected to the analysis of lipids, proteins, ash and macro, micro, trace and ultra-trace elements. The highest protein content was recorded in the brown algae. Maximum dietary fiber and ash contents were recorded from green algae. The highest predominant fatty acids were observed from green seaweeds (Caulerpa racemosa); however, linoleic acid (C18:2n6) is the dominant fatty acid of all macroalgae. Mineral contents were highest in the red macroalga; however, copper, zinc and magnesium were also comparatively higher in green alga Ulva lactuca. In conclusion, 15 seaweed species belonging to the three different classes of seaweeds are investigated in details to obtain their biochemical, mineral and fatty acid compositions for the synthesis of novel therapeutic agents. In order to explore biorefinery processes for these seaweeds, as well as how they can potentially be cultivated, more extensive studies are required. Studying and determining the nutritional values of seaweeds will be beneficial with the potentials for future industrial uses and research.

In vitro and in vivo evaluation of the wound healing properties and safety assessment of two seaweeds (Sargassum ilicifolium and Ulva lactuca).

Seaweeds have been regarded as a reservoir of biologically active molecules that are important in the pharmaceutical industry. The aim of the present study was to explore the wound healing properties and to assess the safety of the seaweed Sargassum ilicifolium and Ulva lactuca. Enhanced cell proliferation and cell migration activities were observed in L929 cells treated with S. ilicifolium extract compared to U. lactuca extract treated cells and the control group. In-vivo experiments were conducted using five groups (10 in each) of Albino mice (BALB/c). Mice in group I and group II were treated (Orally, 100 mg/kg BW/day) with aqueous extracts of S. ilicifolium and U. lactuca, respectively for 14 days. Treatment group III received a topical application of the aqueous extract of S. ilicifolium (25% w/w) and ointment base (75% w/w) (2 g/kg BW/day, for 14 days). Group IV (Control) received an equal amount of distilled water, orally and mice in group V kept without wounds. The extract from S. ilicifolium showed stronger wound healing properties than the one from Ulva lactuca. Histopathological findings also revealed that the healing process was significantly enhanced in the mice group treated orally with S. ilicifolium aqueous extract. These findings show that S. ilicifolium species possess promising wound healing properties in-vitro and in-vivo.

Preliminary screening of the aqueous extracts of twenty-three different seaweed species in Sri Lanka with in-vitro and in-vivo assays.

BACKGROUND: Seaweeds are an important source of bioactive compounds which are applied in various aspects of medicinal investigations. The present study was conducted to investigate cytoxicity (in-vitro and in-vivo) and wound healing activity of different seaweed species in Sri Lanka. METHODS: Twenty-three seaweed samples, belonging to Phaeophyta (Brown), Chlorophyta (Green) and Rhodophyta (Red) were used for the experiments. Samples were collected from the inter-tidal and the sub-tidal habitats around Sri Lankan coast (Southern, Northern and North-western). Aqueous seaweed extracts were tested for cytotoxic and wound healing activity; in-vitro and in-vivo. To determine toxicity of aqueous seaweed extracts, brine shrimp lethality assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay on mouse fibroblasts (L929) cell line were performed. Cell migration induction of seaweed extracts was assessed by scratch wound healing assay using L929 cell line. Based on the our previous experiments S.ilicifolium (SW23) was selected for the in vivo study to confirm our hypothesis. Albino mice (BALB/c) were divided into three groups (12 in each) and a circular area (44.07 ± 02.51 mm2) of full skin was excised to create a wound in mice group II and III. Group III received aqueous extract of Sargasum illicifolium (400 mg/kg BW/day for 12 days, orally), Group II received distilled water for 12 days whereas Group I was used as the control group and it was tested without forming wounds and without providing any treatment. Further, the expression level of Tumor Necrosis Factor (TNF-α) and Transforming Growth Factor-ß (TGF-ß) via RT-PCR were measured every three days until the end of the experiment. RESULTS: Phytochemical tests showed positive results to flavonoids in all the selected green seaweeds and alkaloids were observed in red seaweeds. In the toxicity assay, red seaweed, Acanthophora spicifera (SW17) was found to be highly effective on nauplii of brine shrimp (LC50 = 0.072 µg/µl). LC50 value of green seaweed species, Caulerpa racemosa (SW02 and SW08) and Caulerpa sertularioides (SW10) was not found within the tested concentration series. The highest cytotoxic effect on L929 cell line was exhibited by aqueous extracts of red seaweed; Jania adhaereus with 50.70 ± 7.304% cell viability compared with control group. The highest cell migration activity was observed in L929 cell line group treated with extracts of green seaweed namely; Halimeda opuntin (SW07) and extracts of brown seaweed namely; Stoechospermum polypodioides (SW11). Extracts of S. illicifolium (SW23) exhibited a significantly enhanced wound healing activity in mice group III within three days (P < 0.05) with an open wound area of 17.35 ± 1.94 mm2 compared with control group (26.29 ± 2.42 mm2). TGF-ß gene expression peaked on 6th day of post-wound and subsequently decreased on 9th day of post-wound in mice group III. TNF-α expression was suppressed in mice group III whereas it was elevated in group II. TGF-ß expression is enhanced in the treatment group compared to the control group. CONCLUSIONS: Aqueous extracts of selected seaweeds are a significant source of potential compounds with wound healing properties, which might be helpful in the healing of various wounds. This also infers that many species of brown and red seaweeds have the potential of wound healing, specifically, Sargasum illicifolium and Jania adhaereus could be a potential candidate for in-vivo studies related to wound healing and cancer therapy in the near future.

Hematological parameters and morphological characteristics of blood cells in turtle and tortoise species within captivity in Sri Lanka.

OBJECTIVE: This study was conducted to determine hematological reference ranges and morphological characteristics blood cells in healthy captive Sri Lankan fresh water turtle and tortoise species. MATERIALS AND METHODS: Following turtle species, namely, Melanochelys trijuga parkeri (n = 06), M. trijuga thermalis (n = 06), and Lissemys punctata punctata (n = 06) and a tortoise species Geochelone elegans (n = 06) were evaluated. Blood smears were stained using Leishman-Gram staining protocol. The differential white blood cell counts and morphology were assessed using the standard protocols. RESULTS: Both red blood cells and their nuclei were irregular in M. trijuga parkeri, whereas M. trijuga thermalis had oval-shaped and the nuclei were irregular in shape. L. punctata punctata and G. elegans had oval-shaped red blood cells and their nuclei were round in shape. In terms of differential counts, heterophils were the most abundant leukocyte type in all the species. Melanochelys trijuga parkeri had the highest heterophils value of 55% and the lowest value of 48.5% was recorded with G. elegans. Lymphocytes count was significantly high in G. elegans. Characteristic morphological features were observed in different leukocytes. In terms of thrombocytes, M. trijuga thermalis and L. punctata punctata had single platelets, whereas M. trijuga parkeri and G. elegans had platelets as clumps. Thrombocytes are ellipsoidal with centrally located dark-stained nuclei and their cytoplasm is clear. CONCLUSION: The findings in this study can be used as the reference values in the assessment of health of the above species and also will be useful in future hematological studies related to these species.

How to safeguard an appropriate "all trans retinoic acid" concentration to keep cell division on track: Exploring therapeutic hotspots from metabolomics.

In this letter to editor, I hypothesize a potential affinity of retinol saturase (RetSat) enzyme towards a conjugated trienoic fatty acid; alpha-eleostearic acid (α-ESA) and subsequent hindrance of the action on its usual substrate; all trans retinol. Hence, RetSat is speculated to be involved in a rapid unusual conversion of α-ESA to conjugated linoleic acid (CLA), giving a less priority to its usual substrate all trans retinol, which would subsequently be converted into "all trans retinoic acid" (atRA). Otherwise, all trans retinol is converted by RetSat into all-trans-13,14-dihydroretinol and eventually forms all-trans-13,14-dihydroretinoic acid, but not the atRA. The atRA controls differentiation, proliferation and apoptosis of cells and it's deficiencies end up as neoplasms. Thus, here it is emphasized that safeguarding atRA would help controlling cell division and growth in a favourable manner. Hence, inhibition of RetSat could be a hot target to control unwarranted cell growths within the body. This hypothesis could be easily tested in a RetSat ablated (RetSat -/-) animal model or using antagonists on RetSat activity or α-ESA.

Fish oil induced hyperlipidemia and oxidative stress in BioF1B hamsters is attenuated by elderberry extract.

We have previously reported fish oil induced hyperlipidemia in BioF1B hamsters compared with Golden Syrian (GS) hamsters. Elderberry (Sambucus nigra L.) extract is abundant in anthocyanins and is believed to exert cardioprotective effects primarily by virtue of its hypolipidemic and antioxidant potential. In the current study, high-fat fish oil feeding increased oxidative stress in BioF1B hamsters compared with GS hamsters; this increase was associated with increased levels of omega-3 polyunsaturated fatty acids in plasma and liver. We then investigated whether cosupplementation with anthocyanin-rich elderberry extract would reverse fish oil induced hyperlipidemia and reduce lipid peroxidation in BioF1B hamsters. Plasma and hepatic lipids decreased significantly when hamsters were fed diets containing elderberry extract along with fish oil. Both plasma and liver thiobarbituric acid reactive substances showed significant reductions upon cosupplementation with elderberry extract in fish oil fed BioF1B hamsters. Our findings demonstrate that cosupplementation with elderberry extract reverses hyperlipidemia and lipid peroxidation observed with dietary fish oil alone in BioF1B hamsters.

Diets Enriched in Fish-Oil or Seal-Oil have Distinct Effects on Lipid Levels and Peroxidation in BioF1B Hamsters.

AIM: Fish-oil omega-3 polyunsaturated fatty acids (n-3 PUFAs) are mostly esterified to the sn-2 position of triglycerides, while in seal-oil triglycerides, these are mostly esterified to the sn-1 and -3 positions. We investigated whether fish-oil and seal-oil feeding has a different effect on the regulation of lipid metabolism and oxidative stress in BioF1B hamsters. METHODS: BioF1B hamsters were fed high fat diets rich in fish-oil or seal-oil for 4 weeks, and fasted for 14 hours prior to blood and tissue collection. RESULTS: Plasma and hepatic lipids and lipid peroxidation levels were significantly lower in seal-oil-fed hamsters as compared to those fed fish-oil. There was a selective hindrance of clearance of lipids in fish-oil-fed hamsters as reflected by higher levels of plasma apoB48. CONCLUSION: Differences in the fatty acid composition and positional distribution of n-3 PUFAs in triglycerides of fish-oil and seal-oil are suggested to trigger metabolic differences.

Mice lacking angiotensin-converting enzyme have increased energy expenditure, with reduced fat mass and improved glucose clearance.

In addition to its role in the storage of fat, adipose tissue acts as an endocrine organ, and it contains a functional renin-angiotensin system (RAS). Angiotensin-converting enzyme (ACE) plays a key role in the RAS by converting angiotensin I to the bioactive peptide angiotensin II (Ang II). In the present study, the effect of targeting the RAS in body energy homeostasis and glucose tolerance was determined in homozygous mice in which the gene for ACE had been deleted (ACE(-/-)) and compared with wild-type littermates. Compared with wild-type littermates, ACE(-/-) mice had lower body weight and a lower proportion of body fat, especially in the abdomen. ACE(-/-) mice had greater fed-state total energy expenditure (TEE) and resting energy expenditure (REE) than wild-type littermates. There were pronounced increases in gene expression of enzymes related to lipolysis and fatty acid oxidation (lipoprotein lipase, carnitine palmitoyl transferase, long-chain acetyl CoA dehydrogenase) in the liver of ACE(-/-) mice and also lower plasma leptin. In contrast, no differences were detected in daily food intake, activity, fed-state plasma lipids, or proportion of fat excreted in fecal matter. In conclusion, the reduction in ACE activity is associated with a decreased accumulation of body fat, especially in abdominal fat depots. The decreased body fat in ACE(-/-) mice is independent of food intake and appears to be due to a high energy expenditure related to increased metabolism of fatty acids in the liver, with the additional effect of increased glucose tolerance.

Angiotensin converting enzyme inhibition from birth reduces body weight and body fat in Sprague-Dawley rats.

In vitro studies have demonstrated that angiotensin II (ANG II) induces adipocyte hyperplasia and hypertrophy. The aim of the present study was to determine the effect of angiotensin-converting enzyme inhibition on body weight, adiposity and blood pressure in Sprague-Dawley rats. From birth half of the animals (n=15) were given water to drink, while the remainder were administered perindopril in their drinking water (2 mg/kg/day). Food intake, water intake and body weight were measured weekly. Blood pressure was measured by tail cuff plethysmography at 11-weeks. Body fat content and distribution were assessed using dual energy X-ray absorptiometry and Magnetic Resonance Imaging at 12 weeks. Animals administered with perindopril had a body fat proportion that was half that of controls. This was consistent with, but disproportionately greater than the observed differences in food intake and body weight. Perindopril treatment completely removed hypertension. We conclude that the chronic inhibition of ANG II synthesis from birth specifically reduces the development of adiposity in the rat.

Perinatal omega-3 polyunsaturated fatty acid supply modifies brain zinc homeostasis during adulthood.

Dietary omega-3 polyunsaturated fatty acid (PUFA) influences the expression of a number of genes in the brain. Zinc transporter (ZnT) 3 has been identified as a putative transporter of zinc into synaptic vesicles of neurons and is found in brain areas such as hippocampus and cortex. Neuronal zinc is involved in the formation of amyloid plaques, a major characteristic of Alzheimer's disease. The present study evaluated the influence of dietary omega-3 PUFA on the expression of the ZnT3 gene in the brains of adult male Sprague-Dawley rats. The rats were raised and/or maintained on a control (CON) diet that contained omega-3 PUFA or a diet deficient (DEF) in omega-3 PUFA. ZnT3 gene expression was analyzed by using real-time PCR, free zinc in brain tissue was determined by zinquin staining, and total zinc concentrations in plasma and cerebrospinal fluid were determined by atomic absorption spectrophotometry. Compared with CON-raised animals, DEF-raised animals had increased expression of ZnT3 in the brain that was associated with an increased level of free zinc in the hippocampus. In addition, compared with CON-raised animals, DEF-raised animals had decreased plasma zinc level. No difference in cerebrospinal fluid zinc level was observed. The results suggest that overexpression of ZnT3 due to a perinatal omega-3 PUFA deficiency caused abnormal zinc metabolism in the brain. Conceivably, the influence of dietary omega-3 PUFA on brain zinc metabolism could explain the observation made in population studies that the consumption of fish is associated with a reduced risk of dementia and Alzheimer's disease.

Does perinatal omega-3 polyunsaturated fatty acid deficiency increase appetite signaling?

OBJECTIVE: To investigate the effect of maternal dietary omega-3 polyunsaturated fatty acid (PUFA) deficiency and repletion on food appetite signaling. RESEARCH METHODS AND PROCEDURES: Sprague-Dawley rat dams were maintained on diets either supplemented with (CON) or deficient in (DEF) omega-3 PUFA. All offspring were raised on the maternal diet until weaning. After weaning, two groups remained on the respective maternal diet (CON and DEF groups), whereas a third group, born of dams fed the DEF diet, were switched to the CON diet (REC). Experiments on food intake began when the male rats reached 16 weeks of age. Food intake was stimulated either by a period of food restriction, by blocking glucose utilization (by 2-deoxyglucose injection), or by blocking beta-oxidation of fatty acids (by beta-mercaptoacetate injection). RESULTS: DEF animals consumed more than CON animals in response to all stimuli, with the greatest difference (1.9-fold) demonstrated following administration of 2-deoxyglucose. REC animals also consumed more than CON animals in response to food restriction and 2-deoxyglucose but not to beta-mercaptoacetate. DISCUSSION: These findings indicate that supply of omega-3 PUFA, particularly during the perinatal period, plays a role in the normal development of mechanisms controlling food intake, especially glucoprivic (i.e. reduced glucose availability) appetite signaling. Dietary repletion of omega-3 PUFA from 3 weeks of age restored intake responses to fatty acid metabolite signaling but did not reverse those in response to food restriction or glucoprivic stimuli.

Effects of dietary omega-3 polyunsaturated fatty acids on brain gene expression.

Polyunsaturated fatty acids (PUFA) are essential structural components of the central nervous system. Their role in controlling learning and memory has been well documented. A nutrigenomic approach with high-density microarrays was used to reveal brain gene-expression changes in response to different PUFA-enriched diets in rats. In aged rats fed throughout life with PUFA-enriched diets, genes with altered expressions included transthyretin, alpha-synuclein, and calmodulins, which play important roles in synaptic plasticity and learning. The effect of perinatal omega-3 PUFA supply on gene expression later in life also was studied. Several genes showed similar changes in expression in rats fed omega-3-deficient diets in the perinatal period, regardless of whether they or their mothers were fed omega-3 PUFA-sufficient diets after giving birth. In this experiment, among the down-regulated genes were a kainate glutamate receptor and a DEAD-box polypeptide. Among the up-regulated genes were a chemokine-like factor, a tumor necrosis factor receptor, and cytochrome c. The possible involvement of the genes with altered expression attributable to different diets in different brain regions in young and aged rats and the possible mode of regulatory action of PUFA also are discussed. We conclude that PUFA-enriched diets lead to significant changes in expression of several genes in the central nervous tissue, and these effects appear to be mainly independent of their effects on membrane composition. The direct effects of PUFA on transcriptional modulators, the downstream developmentally and tissue-specifically activated elements might be one of the clues to understanding the beneficial effects of the omega-3 PUFA on the nervous system.