Biological properties of mucus from land snails (Lissachatina fulica) and freshwater snails (Pomacea canaliculata) and histochemical study of mucous cells in their foot.

Background: Mucus derived from many land snails has been extensively utilised in medicine and cosmetics, but some biological activities of the mucus need to be well documented. Nevertheless, most mucus is obtained from land snails, while mucus from freshwater snails has yet to be attended. Methods: This study aims to determine and compare mucus's antioxidant and anti-inflammatory activities from the land snail Lissachatina fulica and the freshwater snail Pomacea canaliculata. ABTS, DPPH, reducing power and total antioxidant activity assays were used to evaluate the antioxidant capacity. Inhibition of nitric oxide production in lipopolysaccharide-activated RAW 264.7 cells was performed to determine the anti-inflammatory activity. Additionally, the histochemical analysis of mucous cells in each snail foot was conducted to compare the distribution of mucous cells and types of mucins using periodic acid-Schiff and Alcian blue staining. Results: Mucus from L. fulica and P. canaliculata exhibited antioxidant and anti-inflammatory activities in different parameters. L. fulica mucus has higher total antioxidant (44.71 ± 2.11 mg AAE/g) and nitric oxide inhibitory activities (IC50 = 9.67 ± 0.31 µg/ml), whereas P. canaliculata mucus has better-reducing power activity (43.63 ± 2.47 mg AAE/g) and protein denaturation inhibition (IC50 = 0.60 ± 0.03 mg/ml). Histochemically, both species' dorsal and ventral foot regions contained neutral and acid mucins in different quantities. In the dorsal region, the neutral mucins level in L. fulica (16.64 ± 3.46%) was significantly higher than that in P. canaliculata (11.19 ± 1.50%), while the acid mucins level showed no significant difference between species. Levels of both mucins in the ventral foot region of L. fulica (15.08 ± 3.97% and 10.76 ± 3.00%, respectively) were significantly higher than those of P. canaliculata (2.25 ± 0.48% and 2.71 ± 0.56%, respectively). This study revealed scientific evidence of the biological capacity of mucus from L. fulica and P. canaliculata as well as provided helpful information on the region of the foot which produces effective mucus.

Comparative histochemical study of the gastrointestinal tracts of the Nile tilapia (Oreochromis niloticus) and the hybrid catfish (Clarias batrachus x Clarias gariepinus).

Because of the wide variety of cells which are arrayed in the tissue of the gastrointestinal tracts of these two fish, basic histological examination is not appropriate to clarify the histological structure. But histochemical techniques can be applied to identify the chemical differences contained in the tissues. The present study aimed to compare the histochemical characteristics of the gastrointestinal tracts of adult fishes which consume different types of food: the Nile tilapia is herbivore and the hybrid catfish is carnivore. The esophagus, stomach and intestine of each fish was stained with 1) Masson's trichrome, for the muscular and connective tissues, 2) Periodic acid Schiff, for neutral mucin, 3) Alcian blue pH 2.5 and pH 1.0, for acid and sulfated acid mucin. The neutral and acid mucins were observed in the esophagus of the tilapia and catfish but sulfated acid mucin was found only in the catfish. Both neutral and acid mucins presented in the stomach of the tilapia, whereas the stomach of the catfish contained only the neutral mucin although it is contained more collagen in the submucosa. The tilapia intestine revealed that neutral and acid mucins were presented throughout the tissue, and small amounts of sulfated acid mucin were observed in the anterior intestine. However, sulfated acid mucin was very obviously located in posterior intestine of the catfish. Moreover, all parts of the catfish intestine showed both neutral and acid mucins with a thick collagen layer in the submucosa. This research will be useful for understanding the physiology of the relevant digestive systems and making a diagnosis of some gastrointestinal diseases in these two fish.

Lethal and sublethal effects of a methomyl-based insecticide in Hoplobatrachus rugulosus.

The aim of this study was to investigate the lethal and sublethal toxicity of a methomyl-based insecticide in Hoplobatrachus rugulosus, as methomyl-based insecticides are applied in massive amounts and agrochemicals have effects on the decline in amphibian populations. To evaluate the toxic effects of methomyl from agricultural application, a methomyl-based insecticide containing 40% methomyl was selected. The median lethal concentration of 96 hours of methomyl exposure was 8.69 ppm for H. rugulosus tadpoles. The lethal concentration also produced severe histological damage in the livers and kidneys of the exposed tadpoles. The sublethal concentration used for methomyl was 144 ppb during the metamorphosis period. It was found that the sublethal concentration of the methomyl compound could decrease growth, metamorphosis time, and size, disturb biochemical parameters, and produce histological damage. In livers, methomyl effects increased oxidative stress and dramatically decreased the glycogen level of the treated froglets. Mononuclear infiltration, blood congestion, amorphous substances, and hepatocytes vacuolization were observed throughout liver tissue. The methomyl-based insecticide also increased oxidative stress and decreased nitric oxide levels in the kidneys of the exposed froglets. Renal tissue damage including blood congestion, amorphous substances, and Bowman's capsule spaces reduction were found in the methomyl exposure group. The methomyl compound also produced vacuoles in various stages of oocytes, but no histological damage was found in testicular tissue. Our results indicated strong toxic effects of the methomyl-based insecticide on H. rugulosus, a broadly tolerant anuran.

Uvaria rufa Blume attenuates benign prostatic hyperplasia via inhibiting 5α-reductase and enhancing antioxidant status.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional medicine has used Uvaria rufa Blume as an ethnomedicinal plant for treating fever, skin allergies, intestinal ulcers and prostate disorders including BPH. However, no scientific evidence supports the traditional use. AIM OF THE STUDY: This study aimed to evaluate the therapeutic potential of U. rufa on BPH using in vitro and in vivo models. MATERIALS AND METHODS: In vitro studies screened the efficacy of a 5α-reductase (5αR) inhibition and antioxidant activity of petroleum ether, ethyl acetate, ethanol and aqueous extracts from the stem of U. rufa. Phytochemical screening was performed to determine the active compound using high-performance liquid chromatography (HPLC). Ethyl acetate extract (UR-EtOAc) of U. rufa was used to evaluate the therapeutic efficacy in vivo models. BPH was induced by subcutaneous injection of testosterone propionate (3mg/kg) to male rats for 30 days. After 30 days of oral administration of UR-EtOAc at doses of 10 and 20mg/kg and finasteride at a dose of 1mg/kg, the prostate weight, prostate index (PI), testosterone and androgen receptor (AR) levels, and histopathological alteration of prostate gland were determined. Also, oxidative status and toxicity indices were assessed. RESULTS: UR-EtOAc exhibited the highest potency of inhibition of 5αR and possessed potent antioxidants rich in phenolics and flavonoids contents. The active compound analyzed by HPLC was ß-sitosterol. In vivo results show a significant reduction in prostate weight, PI, and AR in all treated groups when compared to the BPH model group (P<0.001). Also, the UR-EtOAc and finasteride treated groups had increased prostatic and serum testosterone levels when compared to the BPH model group. A histopathological investigation of the prostate glands supported the above results. UR-EtOAc elevated the antioxidant enzymes and reduced the malondialdehyde level in BPH-induced rats. Moreover, treatment of UR-EtOAc at all doses had no toxic effects on the vital organs and serum biochemical indices. CONCLUSIONS: UR-EtOAc from the stem of Uvaria rufa Blume appears to have the potential as a phytotherapeutic agent in the management of BPH, which provides the scientific evidence for traditional use.

Monascus purpureus-fermented Thai glutinous rice reduces blood and hepatic cholesterol and hepatic steatosis concentrations in diet-induced hypercholesterolemic rats.

BACKGROUND: Red yeast rice (RYR) is a fermented product used as a food supplement to promote blood circulation and lower blood cholesterol levels in eastern Asia. Interestingly, monacolin K is the most active compound in RYR that proved to inhibit HMG-CoA reductase in the cholesterol biosynthesis pathway. METHODS: The hypocholesterolemic effects of oral administration of Thai RYR, produced by fermentation of Thai glutinous rice (Oryza sativa L. var. Niaw San-pah-tawng) with Monascus purpureus CMU 002U, were determined in normal and hypercholesterolemic rats. The rats were divided into six groups, and fed two different kinds of diet. Groups I-II, normal rats fed with a normal diet (SP-diet), were treated with distilled water (SP-control) and 2.0 g/kg/day of RYR extract (SP-2 g). In Groups III-VI, the rats were rendered hypercholesterolemic by feeding them a high fat and cholesterol diet (HFC-diet), and were treated with distilled water (HFC-control), 1.0 g/kg/day (HFC-1 g), 2.0 g/kg/day (HFC-2 g) of RYR extract, and 5.0 mg/kg/day of rosuvastatin (HFC-rosuvastatin) for 30 days, respectively. RESULTS: The RYR extract significantly decreased the concentrations of serum total cholesterol and low density lipoprotein cholesterol (LDL-C), atherosclerotic index, LDL-C/HDL-C ratio and hepatic cholesterol levels in both HFC-1 g and HFC-2 g groups (p < 0.05) as compared with the HFC-control group, and with no significant change in high density lipoprotein cholesterol (HDL-C) concentrations among all six groups. The reduction of serum TC and LDL-C also paralleled the observed changes in mRNA expressions of the genes involved in cholesterol biosynthesis and homeostasis in the liver. The hypercholesterolemic rats treated with RYR extract were significantly higher in LDLR and HMGR expression, but lower in CYP7A1 expression when compared to the untreated hypercholesterolemic rats (HFC-control) (p < 0.05). The hepatic injuries in hypercholesterolemic rats were also obviously alleviated by RYR extract. CONCLUSIONS: The extract of Thai RYR possessed potent hypocholesterolemic and anti-atherogenic activities in diet-induced hypercholesterolemic rats. The possible mechanism involving cholesterol-lowering potential of the extract might contribute to its ability to increase LDL-C endocytosis in hepatocyte and to competitively inhibit HMG-CoA reductase, a key enzyme for cholesterol biosynthesis in liver.

Molecular analysis of echinostome metacercariae from their second intermediate host found in a localised geographic region reveals genetic heterogeneity and possible cryptic speciation.

Echinostome metacercariae are the infective stage for humans and animals. The identification of echinostomes has been based until recently on morphology but molecular techniques using sequences of ribosomal RNA and mitochondrial DNA have indicated major clades within the group. In this study we have used the ITS2 region of ribosomal RNA and the ND1 region of mitochondrial DNA to identify metacercariae from snails collected from eight well-separated sites from an area of 4000 km2 in Lamphun Province, Thailand. The derived sequences have been compared to those collected from elsewhere and have been deposited in the nucleotide databases. There were two aims of this study; firstly, to determine the species of echinostome present in an endemic area, and secondly, to assess the intra-specific genetic diversity, as this may be informative with regard to the potential for the development of anthelmintic resistance and with regard to the spread of infection by the definitive hosts. Our results indicate that the most prevalent species are most closely related to E. revolutum, E. trivolvis, E. robustum, E. malayanum and Euparyphium albuferensis. Some sites harbour several species and within a site there could be considerable intra-species genetic diversity. There is no significant geographical structuring within this area. Although the molecular techniques used in this study allowed the assignment of the samples to clades within defined species, however, within these groupings there were significant differences indicating that cryptic speciation may have occurred. The degree of genetic diversity present would suggest the use of targeted regimes designed to minimise the selection of anthelmintic resistance. The apparent lack of geographic structuring is consistent with the transmission of the parasites by the avian hosts.

Histopathological alterations of the gills, liver and kidneys in Anabas testudineus (Bloch) fish living in an unused lignite mine, Li District, Lamphun Province, Thailand.

The acidity of mine water generally makes it toxic to most organisms. The gills, kidneys and livers of Anabas testudineus Bloch fish inhabiting the acidic water (pH 2-4) of an unused lignite mine in Li District, Lamphun Province, Thailand were examined and compared to those of farmed fish. Tissue abnormalities were found in all investigated organs. Deterioration and telangiectasia of gill filaments were found. Liver tissue revealed hemorrhages, blood congestion and necrotic cells with mononuclear cell infiltration. In addition, hypertrophy of the epithelial cells of the renal tubules with reduced lumens, aneurisms of the renal tubules, and contractions of the glomeruli in the Bowman's capsule were observed. These histopathological findings suggest the acidic water in this habitat causes severe damage to the internal organs of fish and consequently alter their physiological status. Since the water in this pond is utilized by local people, these findings highlight the need for adequate water treatment.

Haplorchis taichui: worm recovery rate and immune responses in infected rats (Rattus norvegicus).

Worm recovery rate, mucosal mast cells (MMCs), eosinophils and serum IgE concentration in rats were investigated after orally feeding 300 Haplorchis taichui metacercariae to male rats. The duodenal, jejunal and ileal tissue sections were stained with 1% alcian blue and 0.5% safranin-O for MMC count. Eosinophil count and the serum IgE concentration assay were measured from cardiac puncture blood. The average worm recovery rates were 20.00%, 13.00%, 0.67%, 1.67% and 0.00% on day 3, 7, 14, 21 and 28 post-infection (PI), respectively. The number of MMCs in the infected rats were significantly higher than in the controls (P<0.01), reaching a peak on day 21 PI. They decreased thereafter, with the decline in worm recovery. Eosinophil count and Serum IgE concentration were also increased but not significantly higher than the controls. However, they showed a positive relationship to worm recovery. It could be concluded from the results that MMCs, eosinophils and IgE may play an important role in the expulsion of H. taichui from rat intestine. However, the mechanism by which the MMC result in the helminth expulsion still need to be understood, and it is recommended that other cells such as goblet cells be studied further.

Mucosal mast cell responses in rats (Rattus norvegicus) experimentally infected with Centrocestus caninus.

Mucosal mast cell (MMC) responses and worm recovery rates in rats experimentally infected with Centrocestus caninus were investigated. Metacercariae of C. caninus, procured from goldfish, Carassius auratus, were orally administered to twenty-five male rats (300 metacercariae each rat). The infected rats were sacrificed on days 3, 7, 14, 21 and 28 post-infection (PI) along with the control rats. Worm recovery was performed from each part of small intestine. To investigate MMC, duodenal, jejunal and ileal paraffinized-tissue sections were processed and stained with 1% alcian blue and 0.5% safranin-O. The average worm recovery rates were 42.8, 37.7, 21.2, 12.5 and 3.7% on days 3, 7, 14, 21 and 28 PI, respectively. The majority of the worms (98.9%) were collected from the duodenum and jejunum. The MMC numbers in the infected rats were significantly higher than those of the controls (p<0.05). A peak level was observed on days 14 PI and the numbers gradually decreased thereafter. The results reveal that MMC plays an important role in the expulsion of C. caninus from the host intestine. A more precise description of the role the MMC plays in helminth expulsion is still needed to understand the mechanism of host defense against intestinal helminthic infection, along with other effector cells, such as goblet cells.

Chronological observations of intestinal histopathology in rats (Rattus norvegicus) infected with Centrocestus caninus.

Intestinal pathological enzyme activity changes were studied chronologically in rats after Centrocestus caninus infection. A single inoculation of 300 metacercariae isolated from the gills of goldfish (Carassius auratus), was orally administered to male rats (n = 15). Uninfected animals were used as controls (n = 5). At days 3, 7, 14, 21, and 28 post-infection (PI), three infected rats, and one from each control group, were sacrificed. The duodenum, jejunum, and ileum were removed separately and fixed in 10% formalin and 10% cold formal calcium solution for histopathological and alkaline phosphatase activity investigations, respectively. The worms were found intruded into the intervillous space of the mucosa and the mucosa showed villous atrophy, crypt hyperplasia and stromal inflammation with inflammatory cell accumulations. Alkaline phosphatase (ALP) activity also showed retardation. However, it seemed that these phenomena would return to normal at the end of the experiment. It can be concluded, from our data, that C. caninus could cause mild histopathological alterations and reduce ALP activity in the small intestines.

Safety evaluation of aqueous extracts from Aegle marmelos and Stevia rebaudiana on reproduction of female rats.

The purpose of this study was to evaluate the safety of a Thai medicinal plant, Aegle marmelos, and a non-caloric sweetener, Stevia rebaudiana, on the reproduction of female rats. Female rats were treated orally with aqueous extract of A. marmelos (6%) and S. rebaudiana at various concentrations (0, 0.2, 1, or 10%) for 60 days (1 ml/day) before mating. The control rats received only distilled water. At the end of the treatment period, treated females were mated with untreated males and the effects on reproduction were examined at day 14 of pregnancy. No notable abnormalities were observed in any of the pregnant rats. The number of corpus lutea, implanted and dead fetuses, as well as the sizes of the fetuses in the treated rats were not significantly different from those of the controls. Based on these results, it may be concluded that aqueous extracts of A. marmelos and S. rebaudiana at the concentrations used in this study do not alter the reproduction of female rats.

Effect of Kaempferia parviflora Wall. ex. Baker on sexual activity of male rats and its toxicity.

Kaempferia parviflora Wall. Ex. Baker (Krachaidum) has long been used among Thai men for sexual enhancement. The aim of this study was to determine the effect of K. parviflora ethanolic extract on the sexual behavior of male rats and its toxicity. The experiment was divided into three groups of rats given K. parviflora extract at doses of 60, 120, and 240 mg/kg BW for 60 days, whilst a control group received distilled water at 1 ml/day per oral. The results showed that all groups of male rats had significantly higher courtship behavior during the first 10-minute period of observation than in the 2nd and 3rd 10-minute periods, except those receiving the highest dose of K. parviflora. They revealed the same amount of courtship behavior throughout a whole 30-minute period, which was significantly lower than the control group. There was no significant difference between treated and control groups in other sexual behaviors; mount frequency (MF), intromission frequency (IF), mount latency (ML), or intromission latency (IL). Toxicological study revealed no significant difference of hemoglobin, WBC or differential cell count. All dosages had no effect on kidney and liver function, according to the normal values of blood urea nitrogen (BUN), creatinine (Crea), aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Nevertheless, the histopathological study showed a morphological change in the liver. It was concluded that K. parviflora extract at 240 mg/kg BW reduced the time in the first 10 mintues of rat courtship behavior and the use of high and chronic doses of K. parviflora in humans should be considered inadvisable.

Susceptibility of rodents to Stellantchasmus falcatus infection.

Two rodent hosts, rats (Rattus norvegicus) and mice (Mus musculus), were examined for their susceptibility to Stellantchasmus falcatus infection. The metacercariae were recovered from half-beaked fish, Dermogenus pusillus, collected from Hang Dong District, Chiang Mai Province. A single inoculation of three hundred metacercariae was orally conducted with male rats (n=15) and mice (n=15). Uninfected animals were used as controls (n=5). At days 3, 7, 14, 21 and 28, post-infection (PI), three rats and mice, also one from each control group, were sacrificed for adult worm recovery, and the blood was drawn by heart puncture and prepared for white blood cell and eosinophil counts. The results showed that adult worm recovery gradually decreased after day 21 PI in the rats, whereas, reduction in the mice was found after day 7 PI and the adult worms completely disappeared at day 28 PI. Hematologically, total white blood cell counts in two kinds of infected hosts were not statistically different from those of controls. However, eosinophil counts of infected rats slightly increased during days 7-14 PI, and started to decrease at day 21 PI. A similar finding was observed in the infected mice. From these results, it can be concluded that the rat is a more suitable host for S. falcatus than the mouse. To understand the mechanisms implicated in determining host susceptibility, intensive studies are required.