Selective cytotoxic and anti-metastatic activity in DU-145 prostate cancer cells induced by Annona muricata L. bark extract and phytochemical, annonacin.

BACKGROUND: Annona muricata L. was identified as a popular medicinal plant in treatment regimens among cancer patients in Jamaica by a previously conducted structured questionnaire. Ethnomedically used plant parts, were examined in this study against human prostate cancer cells for the first time and mechanisms of action elucidated for the most potent of them, along with the active phytochemical, annonacin. METHODS: Nine extracts of varying polarity from the leaves and bark of A. muricata were assessed initially for cytotoxicity using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay on PC-3 prostate cancer cells and the ethyl acetate bark (EAB) extract was identified as the most potent. EAB extract was then standardized for annonacin content using High-performance Liquid Chromatography - Mass Spectrometry (HPLC-MS) and shown to be effective against a second prostate cancer cell line (DU-145) also. The mode of cell death in DU-145 cells were assessed via several apoptotic assays including induction of increased reactive oxygen species (ROS) production, reduction of mitochondrial membrane potential, activation of caspases and annexin V externalization combined with morphological observations using confocal microscopy. In addition, the potential to prevent metastasis was examined via inhibition of cell migration, vascular endothelial growth factor (VEGF) and angiogenesis using the chorioallantoic membrane assay (CAM). RESULTS: Annonacin and EAB extract displayed selective and potent cytotoxicity against the DU-145 prostate carcinoma cells with IC50 values of 0.1 ± 0.07 µM and 55.501 ± 0.55 µg/mL respectively, without impacting RWPE-1 normal prostate cells, in stark contrast to chemotherapeutic docetaxel which lacked such selectivity. Docetaxel's impact on the cancerous DU-145 was improved by 50% when used in combination with EAB extract. Insignificant levels of intracellular ROS content, depolarization of mitochondrial membrane, Caspase 3/7 activation, annexin V content, along with stained morphological evaluations, pointed to a non-apoptotic mode of cell death. The extract at 50 µg/mL deterred cell migration in the wound-healing assay, while inhibition of angiogenesis was displayed in the CAM and VEGF inhibition assays for both EAB (100 µg /mL) and annonacin (0.5 µM). CONCLUSIONS: Taken together, the standardized EAB extract and annonacin appear to induce selective and potent cell death via a necrotic pathway in DU-145 cells, while also preventing cell migration and angiogenesis, which warrant further examinations for mechanistic insights and validity in-vivo.

An Investigation of the Antioxidant Capacity in Extracts from Moringa oleifera Plants Grown in Jamaica.

Moringa oleifera trees grow well in Jamaica and their parts are popularly used locally for various purposes and ailments. Antioxidant activities in Moringa oleifera samples from different parts of the world have different ranges. This study was initiated to determine the antioxidant activity of Moringa oleifera grown in Jamaica. Dried and milled Moringa oleifera leaves were extracted with ethanol/water (4:1) followed by a series of liquid-liquid extractions. The antioxidant capacities of all fractions were tested using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. IC50 values (the amount of antioxidant needed to reduce 50% of DPPH) were then determined and values for the extracts ranged from 177 to 4458 µg/mL. Extracts prepared using polar solvents had significantly higher antioxidant capacities than others and may have clinical applications in any disease characterized by a chronic state of oxidative stress, such as sickle cell anemia. Further work will involve the assessment of these extracts in a sickle cell model of oxidative stress.

Renal and Hepatic Function in Hypercholesterolemic Rats Fed Jamaican Bitter Yam (Dioscorea polygonoides).

BACKGROUND: We reported that Jamaican bitter yam (Dioscorea polygonoides) has antilipemic potential in rats; however there is limited data on the toxicological profile of the yam. We therefore investigated the effects of bitter yam consumption for 6 or 12 weeks on renal and hepatic function in rats fed a high (4%) cholesterol diet. METHODS: Twenty four rats were divided into six groups (n = 4); three of which were used for each investigation (6 or 12 weeks). One group was administered 4% cholesterol diet, while the yam group had the cholesterol diet supplemented with 5% bitter yam. The control group was fed standard rat chow. Liver and kidney function tests were performed on serum, liver and kidney. Histological studies were conducted on liver samples. Acute toxicity tests were performed in rats and mice administered a single high dose of bitter yam (10 g/kg). RESULTS: Activities of liver and kidney AST and ALT differed (p ≤ .02) between control rats and those fed cholesterol with bitter yam for 12 weeks. Albumin to globulin ratio was reduced (p = .03) in rats fed cholesterol with bitter yam for 6 weeks as compared to the control group. Serum urea concentration was higher (p < .05) in rats fed bitter yam as compared to normal chow for 6 weeks. The cholesterol diet caused extensive fat deposition in liver cells; however this was inhibited by co-administration of bitter yam. CONCLUSION: Long-term administration of Jamaican bitter yam may induce slight changes in renal and hepatic functions.

Effects of Jamaican bitter yam (Dioscorea polygonoides) and diosgenin on blood and fecal cholesterol in rats.

A sapogenin-rich preparation from Jamaican bitter yam (Dioscorea polygonoides) has been shown to reduce blood cholesterol concentrations in hypercholesterolemic rats and mice. Also, diosgenin supplementation has been reported to have antilipemic effects in several animal species. We investigated potential mechanisms of the lipid-lowering actions of bitter yam and also whether the actions were mediated by diosgenin. Sprague-Dawley rats were fed a hypercholesterolemic diet (4% cholesterol) alone or with 5% bitter yam or 1% diosgenin supplementation for 6 weeks. The control group was fed normal rat chow. The serum lipid profile, fecal cholesterol concentration, and serum lipase activity were assessed at the end of the period. The induction of hypercholesterolemia was inhibited by coadministration of 5% bitter yam or 1% diosgenin in the diet. Serum lipid profiles were similar in rats fed bitter yam or diosgenin. The fecal cholesterol concentration was significantly (P < .01) higher in rats fed diosgenin compared to the cholesterol group. However, there was no corresponding elevation in the group fed bitter yam. Administration of bitter yam or diosgenin supplement significantly increased (P < .01) the serum lipase activity compared to the normal control and cholesterol groups. The cholesterol-supplemented diet inhibited normal gain in body weight over the period. This action was potentiated by diosgenin. The effects of the respective supplements on body weight were not completely explained by food consumption. Supplementation of the diet with Jamaican bitter yam may be therapeutically beneficial in the management of hypercholesterolemia.

Citrus peel polymethoxylated flavones extract modulates liver and heart function parameters in diet induced hypercholesterolemic rats.

The primary aim of this study was to investigate the effects of Ortanique peel polymethoxylated flavones extract (PMF(ort)) on organ function parameters in the serum of hypercholesterolemic and normal rats. Thirty Sprague-Dawley rats were fed high cholesterol diets supplemented with 1.5% PMF(ort) and niacin respectively for 49days. Hypercholesterolemic rats fed PMF(ort) had significant reductions in the activities of aspartate aminotransferase and alkaline phosphatase (69.12±3.34 and 87.22±8.42U/L respectively) compared to the untreated hypercholesterolemic group (118.61±4.85 and 132.62±10.62U/L respectively, p<0.05). Supplementation of the diet with niacin or PMF(ort) resulted in no significant differences in the serum levels of creatinine or urea in any of the groups. Total bilirubin was highest in the untreated hypercholesterolemic group. Supplementation of the diets of hypercholesterolemic rats with PMF(ort) resulted in significant reductions in the activities of serum creatine kinase and lactate dehydrogenase (119.3±25.3; 222.5±50.3U/L, p<0.05) respectively relative to the untreated hypercholesterolemic group (257.2±48.3; 648.8±103U/L, p<0.05). The results would suggest that PMF(ort) modulates hypercholesterolemia-associated organ injury in rats. PMF(ort) could therefore be a suitable candidate for prophylactic and therapeutic treatment of hypercholesterolemia-associated organ injury.

Comparative effects of dietary administered transgenic and conventional papaya on selected intestinal parameters in rat models.

Transformation of agricultural crops with novel genes has significantly advanced disease-resistance breeding, including virus resistance through the expression of virus sequences. In this study, the effects of long-term, repeated exposure to transgenic papayas carrying the coat protein gene of Papaya ringspot virus and conventional non-transgenic papaya on the histology and selected biochemical parameters of the intestinal tract were compared. For 3 months, male and female Wistar rats received diets containing transgenic or non-transgenic papaya at twice the equivalent of the average daily consumption of fresh papayas. Gross and macroscopic appearance of intestinal tissues, as well as stomach tissues, was comparable (P < 0.05) as were total intestinal bacterial counts and activities of beta-glucuronidase. Activities of disaccharidases were not affected, neither were those of amylase (P < 0.05). Although significant differences were noted in the activity of Ca(2+) and Na(+)/K(+) ATPase brush border enzymes, no morphological alteration in the integrity of the intestinal mucosa was found. Overall, negligible effects on feed intake, body weight, and fecal output were observed (P < 0.05). Taken together, long-term exposure to diets formulated with transgenic papaya did not result in biologically important unintended effects.

Spectroelectrochemical characterization of pain biomarkers.

This article reports the first electrochemical characterization of pain biomarkers that include arachidonic acid (AA), prostaglandin G(2) (PGG(2)), and cyclooxygenase 2 (COX-2). These biomarkers are mediators of pathophysiology of pain, inflammation, and cell proliferation in cancer. The article also reports the development of an electrochemical immunosensor for monitoring these pain biomarkers. The results revealed that direct electron transfer between AA metabolites and the electrode could be easily monitored and that an enzyme-modified electrode dramatically enhanced bioelectrocatalytic activity toward AA. Cyclic voltammetric analysis of AA revealed a concentration-dependent anodic current with a slope of 2.37 and a limit of detection (LOD) of 0.25nM. This unique AA/gold electrode electron transfer provides a good electrochemical sensing platform for prostaglandin H(2) (PGH(2)) as the basis for quantitation of pain. An amperometric signal intensity of a COX-2 antibody-modified gold electrode was linear with COX-2 concentration in the range of 0.1-0.5microg/ml and an LOD of 0.095microg/ml. The results also revealed a linear correlation of the concentration of PGG(2) with an LOD of 0.227microM.

Relationship between the physicochemical properties of starches and the glycemic indices of some Jamaican yams (Dioscorea spp.).

Starch granules from round leaf yellow yam (RY), Lucea yam (LY), white yam (WY), and Chinese yam (CY) grown in Jamaica were isolated and the relationship between starch amylose content, crystallinity, microscopic properties, in vitro digestibility, and the glycemic index (GI) of the tubers was investigated. The results indicate that RY had the highest amylose content (265.30 +/- 0.09 g/kg starch) while CY the lowest (111.44 +/- 0.03 g/kg starch). A corresponding variation in starch digestibility and GI was also observed, as CY which had the highest in vitro digestibility had the highest GI (21.27 +/- 0.01 and 97.42 +/- 0.62%, respectively), while RY, LY, and WY starches with low digestibility had lowest GI. Differences in the crystalline pattern of the different starches were observed, where RY, LY, and WY displayed the type B crystalline pattern while CY had the intermediate crystallite (type C).

The interplay between yam (Dioscorea sp.) starch botanical source, micromeritics and functionality in paracetamol granules for reconstitution.

A comparative investigation of the interplay between starch botanical source, micromeritics and their functionality in formulated paediatric paracetamol granules for reconstitution was conducted using starches extracted from five local yam (Dioscorea spp.) species. Significant differences were observed in the physicochemical properties of the different starches studied (p<0.05). The observed differences in paracetamol dissolution correlated well with the physicochemical properties of the starches. Granules formulated with Chinese yam and Bitter yam starches showed the fastest rate of paracetamol dissolution with T(80) of 2.2 and 2.6 min, respectively, at 2.5%w/w, and 3.75 and 4.00 min, respectively, at 10% w/w binder concentrations. Those formulated with Round leaf yellow yam starch had the slowest dissolution rate with T(80) of 4.5 and 8.75 min, respectively, at 2.5% and 10% binder concentrations. The results generally indicate a significant dependence of the rate of paracetamol dissolution from granules on starch surface tension, viscosity, swelling power and water binding capacity, particle size distribution, specific surface and porosity. These, in turn, were found to be starch botanic source-dependent suggesting careful consideration of botanic source when substituting one starch product for another in granule formulation.

Bone and faecal minerals and scanning electron microscopic assessments of femur in rats fed phytic acid extract from sweet potato (Ipomoea batatas).

Phytic acid was extracted from sweet potato (Ipomoea batatas) and fed to Wistar rats with or without zinc for 3 weeks. Animals were then sacrificed and bone and faecal minerals were assessed. The ultra-structure of the bones was examined via scanning electron microscopy. Phytic acid extract or commercial phytic acid supplemented diets (D + Zn + PE or D + PE) displayed reduced bone calcium levels (101.27 +/- 59.11 and 119.27 +/- 45.36 g/kg) compared to the other test groups. Similarly, reduced calcium were observed in the control groups (D + Zn and D) fed formulated diets with or without zinc supplementation (213.14 +/- 15.31 and 210 +/- 6.88 g/kg) compared to the other test groups. The group fed supplemented commercial phytic acid diet (D + CP) demonstrated the lowest femur magnesium (3.72 +/- 0.13 g/kg) while the group fed phytic acid extract supplementation (D + PE) recorded the highest level (4.84 +/- 0.26 g/kg) amongst the groups. Femur iron was highest in the group fed commercial phytic acid supplemented diet (D + CP -115.74 +/- 2.41 g/kg) compared to the other groups. Faecal magnesium levels were significantly higher in the two test groups fed phytic acid extract with or without zinc (D + Zn + PE or D + PE) compared to all other groups. All the groups which had phytic acid supplemented diets had significantly thinner bone in the trabecular region, compared to the groups fed formulated diet or zinc supplemented formulated diet (D or D + Zn). These observations suggest that the consumption of foods high in phytic acid may contribute to a reduction in the minerals available for essential metabolic processes in rats.

In vitro availability of some essential minerals in commonly eaten processed and unprocessed Caribbean tuber crops.

The levels of three essential minerals Ca, Fe and Mg and the extent of their availability were assessed in four commonly eaten Caribbean tuber crops [dasheen (Xanthosoma spp.), Irish potato (Solanum tuberosum), sweet potato (Ipomoea batatas) and yellow yam (Dioscorea cayenensis)] in their processed and unprocessed states. Calcium was highest in cooked dasheen (5150+/-50 mg/kg) while Magnesium was highest in uncooked Irish potato (3600+/-200 mg/kg). There was no significant loss of calcium from the food samples upon cooking. All the uncooked food samples displayed higher levels minerals assessed compared to the cooked samples except for cooked Irish potato that recorded the level of iron (182.25+/-8.75 mg/kg). Availability of these minerals in the cooked and uncooked tubers crops upon digestion also showed a similar pattern. In conclusion, the consumption of these tuber crops in the Caribbean may not be responsible for the reported cases of iron deficiency in the region. However, the availability of minerals from these tuber crops when consumed with other foods (the usual practice in the Caribbean) needs further investigation.

Determination of polymethoxylated flavones in peels of selected Jamaican and Mexican citrus (Citrus spp.) cultivars by high-performance liquid chromatography.

The concentrations of the polymethoxylated flavones (PMFs) in peels of selected citrus cultivars grown in Jamaica and Mexico were determined. The PMFs were extracted from sun-dried citrus peels with reagent-grade methanol. Analyses were carried out by reverse-phase HPLC and UV detection. The column used was a C(18) 5 microm (150 x 4.6 mm) Discovery column. Elution was in the gradient mode, using a ternary mobile phase. The results showed that all the citrus cultivars used contained at least three of the six major PMFs quantified. Ortanique peel contained the highest quantity of PMFs (34,393 +/- 272 ppm), followed by tangerine (28,389 +/- 343 ppm) and Mexican sweet orange (sample 1; 21,627 +/- 494 ppm). The major PMFs, i.e. sinensetin, nobiletin, tangeretin, heptamethoxyflavone, tetramethylscutellarein and hexamethyl-o-quercetagetin, present in the peels of 20 citrus cultivars, was quantified. The results were compared with those of Florida citrus peels. A large amount of citrus peels and byproducts are produced in the Caribbean which could provide a cheap and convenient source of PMFs.

Intestinal lipids and minerals in streptozotocin-induced diabetic rats fed bitter yam (Dioscorea polygonoides) sapogenin extract.

Yam is the leading form of staple for millions of people in the tropical and subtropical countries. They are good sources of carbohydrate. However, the protein content of yam is low. The effect of bitter yam sapogenin extract or commercial diosgenin on faecal minerals and intestinal lipids in streptozotocin-induced diabetic rats was studied. Sapogenin extract or commercial diosgenin (1%) supplemented diets were fed to diabetic male Wistar rats for three weeks. Bitter yam sapogenin extract or commercial diosgenin did not significantly alter faecal magnesium, calcium, and zinc excretion but significantly decreased faecal sodium and potassium excretion. The absorption of iron was impaired by bitter yam sapogenin extract or commercial diosgenin during the first week of feeding. Bitter yam sapogenin extract or commercial diosgenin supplements significantly decreased intestinal lipids towards normal. Faecal lipids excreted was significantly higher in diabetic rats fed bitter yam sapogenin extract or commercial diosgenin for the three weeks period compared to the diabetic control group. These results show that bitter yam sapogenin extract or commercial diosgenin does not have the same effects on mineral excretion in diabetes. There was no direct correlation between the decrease in excretion of mono-valent cations and the activity of intestinal Na+/K+ATPase.

Intestinal disaccharidases and some renal enzymes in streptozotocin-induced diabetic rats fed sapogenin extract from bitter yam (Dioscorea polygonoides).

In this study, the effects of bitter yam sapogenin extract or commercial diosgenin on intestinal disaccharidases and some renal enzymes in diabetic rats were investigated. Diabetic male Wistar rats were fed diets supplemented with 1% sapogenin extract or commercial diosgenin for 3 weeks. Plasma glucose, intestinal disaccharidases and the activities of transaminases, acid phosphatase, glucose-6-phosphatase, ATP citrate lyase, glucose-6-phosphate dehydrogenase and pyruvate kinase were assessed for the level of metabolic changes in the kidney of diabetic rats. Sapogenin extract or commercial diosgenin supplementation resulted in a significant decrease in lactase and maltase activities in all three regions of the intestine compared to the diabetic control group. However, the test diets significantly reduced intestinal sucrase activity in the proximal and mid regions. Test diets supplementation resulted in a significant decrease in the activities of the transaminases compared to the normal and diabetic control groups. The activity of glucose-6-phosphatase was significantly increased while the activities of ATP citrate lyase, pyruvate kinase and glucose-6-phosphate dehydrogenase were significantly reduced in the kidney of the diabetic control rats compared to the normal group. Test diets supplementation did not significantly alter glucose-6-phosphatase, ATP citrate lyase and pyruvate kinase activities compared to the diabetic control. However, there was a significant increase in glucose-6-phosphate dehydrogenase activity toward the normal group. In conclusion, the consumption of bitter yam sapogenin extract or commercial diosgenin demonstrated hypoglycemic properties, which are beneficial in diabetes by reducing intestinal disaccharidases activities; however, bitter yam sapogenin extract may adversely affect the integrity of kidney membrane.

Proximate analysis and some antinutritional factor constituents in selected varieties of Jamaican yams (Dioscorea and Rajana spp.).

Two wild (Dioscorea polygonoides and Rajana cordata) and seven cultivated varieties of Jamaican yams (Dioscorea spp.) were analyzed for their proximate composition and the levels of antinutritional factors. The protein level range was 47.8 +/- 2.6 to 88.0 +/- 2.5 g/kg dry weight. The lowest level was seen in D. cayenensis. The range for the dietary fiber content in the tubers was 16.3 +/- 0.7 to 63.5 +/- 0.4 g/kg dry weight. The wild yam varieties recorded higher levels. Saponins level was <600 mg/kg dry weight in all the tubers analyzed except for bitter yam (2962.5 +/- 60.5 mg/kg dry weight). Total phenol content ranged from 1.3 +/- 0.1 to 79.3 +/- 6.1 g/kg while total condensed tannin content ranged from 0.1 +/- 0.0 to 26.7 +/- 3.8 g/kg dry weight. Samples that showed high levels of phenols also had high levels of condensed tannins. All the samples analyzed contained low levels of lectins and no alkaloids were detected. The levels of antinutritional factors did not clearly delineate the wild varieties from the edible varieties.

Hypoglycemic effects of steroidal sapogenins isolated from Jamaican bitter yam, Dioscorea polygonoides.

In this study, three steroidal sapogenins (Delta3 diosgenin, diosgenin, and pennogenin) and the phytosterols, stigmasterol and beta-sitosterol were isolated from Jamaican bitter yam, Dioscorea polygonoides. Their effects on fasting blood glucose and intestinal amylase and ATPases in streptozotocin-induced diabetic rats were studied. The diabetic rats (fed supplemented and unsupplemented diets) lost weight significantly compared to the normal group. There was a significant increase in the activity of alpha-amylase in the proximal region of the small intestinal mucosa of diabetic rats fed sapogenin extract or commercial diosgenin. However, this did not result in increased fasting blood glucose. Instead, supplementation of the diet with bitter yam sapogenin extract significantly decreased fasting blood glucose compared to the diabetic group. Supplementation of the diet with bitter yam sapogenin extract or commercial diosgenin significantly reduced Na+-K+-ATPase activity in all three regions compared to the diabetic control group. Commercial diosgenin supplementation resulted in a significant increase in Ca2+ ATPase activity in proximal region compared to the diabetic control and bitter yam sapogenin extract groups. The effect of bitter yam sapogenin extract or commercial diosgenin on intestinal Na+-K+-ATPase activity could account for their hypoglycemic properties. However, there was adverse effect on the body weight.