Melon Extract Research Articles

Abstract LB-171: Dietary agent induced AMPK activation: Targeting stem cells for the prevention of colon cancer

Abstract Colorectal cancer (CRC) is a leading cause of cancer-related deaths in the United States. Although early diagnosis often leads to cure, most cases go undetected, thus needing other prevention-related strategies. AMP-activated protein kinase (AMPK) is an important regulator of cellular energy homeostasis. It has also been shown to control tumor progression by regulation of cell cycle, protein synthesis and cell growth and/or survival. Dietary agents can affect AMPK signaling thereby reducing risk of cancer progression. Bitter melon is recommended in ancient Indian/Chinese medicine for treatment of metabolic disorders. Bitter melon extracts (BME) have been shown to affect breast and prostate cancers, though the mechanism is not well known. Here, we report the inhibitory effect of BME on colon cancer stem cells via AMPK activation. We tested the effect of aqueous BME on two CRC lines HT-29 and SW480. It significantly inhibited the proliferation and clonogenic capacity of both the lines. In addition, cell cycle analysis showed a significant increase in cells arrested at the G2/M phase. Moreover, the whole extract was found to be more effective when compared to the skin extracts, suggesting that the active ingredient is present at higher levels in the flesh and seeds of the fruit than in the skin. We next determined the mechanism by which BME affects the growth of the colon cancer cells. Western blot analyses suggested that there was no caspase-3 activation. However, western blots demonstrated a significant induction in LC3B cleavage, which was further confirmed by immunocytochemistry. This suggests that the cells were undergoing autophagy. To confirm this, we performed Real Time RT-PCR and western blot analyses for autophagy markers along with flow based autophagy analyses. While BCL2 (pro-apoptotic marker) was reduced, there was an increase in Beclin-1 (marker for autophagy) in the two cell lines. Real Time RT-PCR analyses demonstrated increased levels of Atg5, 7 and 12 mRNA in BME treated cells when compared to control. Western blot analyses also showed enhanced phosphorylation of AMPK, and its downstream target proteins. BME were also found to reduce cellular ATP levels, which might be the cause of AMPK signaling. Cell proliferation was found to be reactivated in the presence of exogenous supply of ATP. To study the inhibitory effect of BME on colon cancer stem cells, spheroid formation assay was performed. There was a dose-dependent reduction in the number and size of the spheres. Furthermore, western blot analyses demonstrated a reduction in the expression of DCLK1, a quiescent stem cell marker which was further verified by immunocytochemistry and flow cytometry. Taken together, these results suggest that dietary agents such as BME can target colon cancer cells and mark them for autophagy. Further studies are warranted for developing bitter melon extract as an effective chemopreventive/therapeutic agent in CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-171. doi:1538-7445.AM2012-LB-171

Mechanisms Underlying Decreased Hepatic Triacylglycerol and Cholesterol by Dietary Bitter Melon Extract in the Rat

In these studies, we focused on finding the mechanism(s) underlying the bitter melon (Momordica charantia L.) methanol fraction (MF)-dependent reduction in the concentration of hepatic triacylglycerol (TAG) and cholesterol in the rat. Rats were fed diets containing low (5 %) fat for 2 weeks (experiment 1), or low (5 %) and high (15 %) fat for a longer period of 8 weeks (experiment 2). MF was supplemented at 1 % level in both experiments. After feeding, rats were sacrificed, and their livers were prepared as slices and hepatocytes, followed by incubation with [1(2)-¹⁴C] acetate or [1-¹⁴C] oleic acid (18:1 n-6). Under these conditions, we found that rats fed diets containing MF, as compared to those without MF, showed: (1) no adverse effects on food intake and growth, (2) a decreased hepatic TAG and total cholesterol, irrespective of the difference in dietary fat level or feeding period, and (3) a decreased incorporation of [1(2)-(¹⁴C] acetate and [1-¹⁴C] oleic acid into TAG of liver slices and hepatocytes. MF-supplemented rats also showed no altered incorporation of labeled acetate into cholesterol and cholesterol ester, an increased fecal excretion of neutral steroids, but not of acidic steroids, and an enhanced mRNA abundance of carnitine palmitoylacyltransferase I, which is the rate-limiting enzyme for fatty acid oxidation. These results suggest that dietary MF decreases hepatic TAG synthesis while enhancing fatty acid oxidation, thereby reducing the concentration of hepatic TAG. The liver cholesterol-lowering effect of MF, however, is probably mediated through an increased fecal excretion of neutral steroids, without an effect on cholesterogenesis.

Bitter Melon (Momordica charantia) Extract Suppresses Adrenocortical Cancer Cell Proliferation Through Modulation of the Apoptotic Pathway, Steroidogenesis, and Insulin-Like Growth Factor Type 1 Receptor/RAC-α Serine/Threonine-Protein Kinase Signaling

Adrenocortical carcinomas are rare but present with extremely poor prognosis. One of the approaches to control cancer progression and reduce cancer risk is prevention through diet. Bitter melon is widely consumed as a vegetable and especially as a traditional medicine in many countries. In this study, we have used human and mouse adrenocortical cancer cells as an in vitro model to assess the efficacy of bitter melon extract (BME) as an anticancer agent. The protein concentrations of BME and other extracts were measured before use. First, BME treatment of adrenocortical cancer cells resulted in a significantly dose-dependent decrease in cell proliferation. However, we did not observe an antiproliferative effect in adrenocortical cancer cells treated with extracts from blueberry, zucchini, and acorn squash. Second, apoptosis of adrenocortical cancer cells was accompanied by increased caspase-3 activation and poly(ADP-ribose) polymerase cleavage. BME treatment enhanced cellular tumor antigen p53, cyclin-dependent kinase inhibitor 1A (also called p21), and cyclic AMP-dependent transcription factor-3 levels and inhibited G1/S-specific cyclin D1, D2, and D3, and mitogen-activated protein kinase 8 (also called Janus kinase) expression, suggesting an additional mechanism involving cell cycle regulation and cell survival. Third, BME treatment decreased the key proteins involved in steroidogenesis in adrenocortical cancer cells. BME treatment decreased the level of phosphorylation of cyclin-dependent kinase 7, which is required, at least in part, for steroidogenic factor 1 activation. Finally, we observed that BME treatment significantly reduced the level of insulin-like growth factor 1 receptor and its downstream signaling pathway as evidenced by lower levels of phosphorylated RAC-α serine/threonine-protein kinase. Taken together, these data illustrate the inhibitory effect of bitter melon on cell proliferation of adrenocortical cancer through modulation of diverse mechanisms.

Bitter Melon Extract Impairs Prostate Cancer Cell-Cycle Progression and Delays Prostatic Intraepithelial Neoplasia in TRAMP Model

Prostate cancer remains the second leading cause of cancer deaths among American men. Earlier diagnosis increases survival rate in patients. However, treatments for advanced disease are limited to hormone ablation techniques and palliative care. Thus, new methods of treatment and prevention are necessary for inhibiting disease progression to a hormone refractory state. One of the approaches to control prostate cancer is prevention through diet, which inhibits one or more neoplastic events and reduces the cancer risk. For centuries, Ayurveda has recommended the use of bitter melon (Momordica charantia) as a functional food to prevent and treat human health related issues. In this study, we have initially used human prostate cancer cells, PC3 and LNCaP, as an in vitro model to assess the efficacy of bitter melon extract (BME) as an anticancer agent. We observed that prostate cancer cells treated with BME accumulate during the S phase of the cell cycle and modulate cyclin D1, cyclin E, and p21 expression. Treatment of prostate cancer cells with BME enhanced Bax expression and induced PARP cleavage. Oral gavage of BME, as a dietary compound, delayed the progression to high-grade prostatic intraepithelial neoplasia in TRAMP (transgenic adenocarcinoma of mouse prostate) mice (31%). Prostate tissue from BME-fed mice displayed approximately 51% reduction of proliferating cell nuclear antigen expression. Together, our results suggest for the first time that oral administration of BME inhibits prostate cancer progression in TRAMP mice by interfering cell-cycle progression and proliferation.

Bitter Melon Extracts in Diabetic and Normal Rats Favorably Influence Blood Glucose and Blood Pressure Regulation

Bitter melon (BM) was tested in normal and streptozotocin (STZ)-induced diabetic rats. First, normal and diabetic Wistar rats were given four test extracts (EX-1-EX-4) of a wild-genotype BM or metformin by intubation. Second, normal Sprague-Dawley rats were divided into control and three test groups given for 52 days one of three BM preparations in food: Chinese or Indian commercial preparations or EX-4 from experiment I. In experiment I, extracts of BM administered at 50 mg/kg of body weight in normal rats reduced blood sugar for 4 hours without, unlike metformin, inducing hypoglycemia. In STZ-induced diabetic rats, two extracts administered at 250 mg/kg decreased glucose levels to values comparable to metformin at 150 mg/kg. At 4 hours, EX-1 and EX-4 significantly reduced blood glucose 67% and 63%, respectively, compared with metformin's 54%. In experiment II, all test groups had lowered systolic, but not diastolic, blood pressure. The China and EX-4 arms had significantly lowered serum glucose levels compared with the control. In the glucose tolerance test, only EX-4 had significantly lowered glucose levels. Only EX-4 had significantly lowered angiotensin converting enzyme (ACE) activity. All active arms showed significance in the losartan challenge (the renin-angiotensin system [RAS]), with the greatest effect in the EX-4 group. In the N(ω)-nitro-l-arginine-methyl ester challenge, only EX-4 exhibited a significant impact on the nitric oxide system, suggesting higher activity in this group. In the STZ-induced diabetic rat model, wild-type BM powerfully lowered glucose levels, and, in healthy adult rats, wild-type BM exhibited beneficial effects in the regulation of blood glucose, in RAS and ACE inhibition, and in nitric oxide generation.

Adenosine monophosphate kinase activator α-lipoic acid: A promising therapeutic agent for metabolic syndrome?

Adenosine monophosphate kinase activator α-lipoic acid: A promising therapeutic agent for metabolic syndrome?

Abstract 5587: Bitter melon (Momordica charantia) extract suppresses adrenocortical cancer cell proliferation and growth through modulation of the cell cycle pathway and steroidogenic activity

Abstract Malignant adrenal tumors or adrenocortical carcinomas are relatively rare; however, this form of cancer is highly malignant and presents with extremely poor prognosis as a consequence of metastasis or local invasion at the time of diagnosis. One of the approaches to control cancer progression and reduce cancer risk is prevention through diet. Bitter melon is widely consumed as a vegetable and especially as a traditional medicine in China, India, Japan, and Taiwan. In this study, we have used human and mouse adrenocortical cancer cells, H295R and Y1, respectively, as an in vitro model to assess the efficacy of bitter melon extract (BME) as an anticancer agent. First, we find that BME treatment of adrenocortical cancer cells results in a significant dose-dependent decrease in cell proliferation and growth. We also find that bitter melon extract has stronger anti-proliferation effect than blueberry extract on adrenocortical cancer cells. As expected, extracts from Acorn squash and Zucchini, two vegetables from the same Family of Cucurbitaceae as bitter melon, do not decrease cell proliferation in adrenocortical cancer cells. Second, apoptosis of adrenocortical cancer cells is accompanied by increased caspase activation and poly(ADP-ribose) polymerase cleavage. Further studies reveal that BME treatment enhances p53, p21, Mdm2, and ATF3 and inhibits cyclin D1, cyclin D2, cycline D3, mTOR, and JNK expression, suggesting an additional mechanism involving cell cycle regulation, cell motility, cell survival, and cell differentiation. Third, BME treatment also decreases steroidogenic activities in adrenocortical cancer cells by Western blotting and reporter gene assay. BME treatment of adrenocortical cancer cells results in a significant dose-dependent decrease in levels of NR5A1/SF1, MC2R, StAR proteins. Interestingly, we find that BME treatment decreases the level of phospho-CDK7, which is required, at least in part, for NR5A1/SF1 activation. Taken together, these data illustrate that inhibitory effect of bitter melon on the growth and proliferation of adrenocortical cancer cells through modulation of the cell cycle regulation and steroidogenic pathways. Therefore, BME can be used as a dietary supplement for prevention of adrenocortical cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5587. doi:10.1158/1538-7445.AM2011-5587

Abstract 4618: Oral administration of bitter melon extract prevents initiation of prostate cancer in TRAMP model

Abstract Prostate cancer remains the second leading cause of cancer deaths among American men. Earlier diagnosis increases survival rate in patients. However, treatments for advanced disease are limited to hormone ablation techniques and palliative care. Thus, new methods of treatment and prevention are necessary for inhibiting progression of the disease to a hormone refractory state. For centuries, Ayurveda has recommended the use of bitter melon (Momordica charantia) as a functional food to prevent and treat human health related issues. We have recently discovered an anti-proliferative effect of bitter melon extract (BME) in a number of cancer cell lines, including prostate, without affecting normal cell growth. Treatment of prostate cancer cells with bitter melon extract modulated Bax expression and induced poly(ADP-ribose) polymerase cleavage. TRAMP (transgenic adenocarcinoma of mouse prostate) male mice display prostatic intraepithelial neoplasia (PIN) at 10-12 weeks of age. We examined whether oral administration of BME as a dietary compound inhibits or delay the conversion of normal prostate to PIN in these mice. We have observed prevention of PIN in BME-fed mice as compared to untreated control. Further tissues analyses for mechanism of cancer prevention are in progress and the results will be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4618. doi:10.1158/1538-7445.AM2011-4618

Bio-active compounds of bitter melon genotypes (Momordica charantia L.) in relation to their physiological functions

Background: Bitter Melon (Momordica charantia L) is one of the most popular cooked vegetables in many Asian countries. Its experimental use in mice has indicated improvement in glucose tolerance against Type II diabetes and reduction in blood cholesterol. However, it has not been proven which alkaloids, polypeptides, or their combinations in the Bitter Melon extract are responsible for the medicinal effects. Green and white varieties of Bitter Melon differ strikingly in their bitter tastes, green being much more bitter than white. It is not yet known whether they are different in their special nutritional and hypoglycemic properties. Nutritional qualities of Bitter Melons such as protein, amino acids, minerals, and polyphenolics contents were determined using four selected varieties such as Indian Green [IG], Indian White [IW], Chinese Green [CG], and Chinese White [CW] grown at the University of Arkansas at Pine Bluff [UAPB] Agricultural Research Center. Results indicated that protein levels of IW were significantly higher than IG in both flesh and seed. Methods: Four Bitter Melon varieties, Indian Green [IG], Indian White [IW], Chinese Green [CG] and Chinese White [CW] were used for phytochemical analyses to determine protein contents, protein hydrolysis, amino acids contents, and their antioxidant and antimutagenic activities. All analyses were conducted following standard methods. Statistical analyses were conducted using JMP 5 software package [SAS]. The Tukey’s HSD procedure was used for the significance of differences at the 5% level. Results: Moisture contents across the four varieties of Bitter Melon flesh ranged between 92.4 and 93.5%, and that of seed ranged between 53.3 and 75.9%. Protein contents of the flesh were highest in IW [9.8%] and lowest in CG [8.4%]. Seed protein contents were the highest in IW [31.3%] and lowest in IG [27.0%]. Overall, white varieties had higher protein contents than the green varieties. Compared with soy protein, most of the essential amino acid contents of Bitter Melon were similar as in soy proteins. Some amino acids such as Alanine, Glycine, and Valanine were relatively higher in Bitter Melon flesh than in soy protein. Phenolics contents of the flesh, seed, and seed coat tissue [SCT] were significantly different [p<0.05] among the four varieties. The four varieties were similar in their antioxidant activities of the flesh tissues; however, they were significantly different in their antioxidant activities in the seed and seed coat tissues [SCT]. Bitter melon varieties IW and CG, tested for antimutagenic effects, both flesh and seed had considerably high activities against benzo[a]pyrene with Salmonella TA98 [92-100% inhibition] and Salmonella TA100 [79-86% inhibition].Conclusion: Based on these studies, Bitter Melon is a good source of phenolic compounds. All four varieties tested showed considerably high antioxidant and antimutagenic activities. Therefore, these natural plant phenolics can be a good source of biologically active compounds that may be applied in many food systems to enhance food values and special nutritional qualities. Further studies will be needed using more genetically diverse varieties to pin point the bioactive and functional compounds and their physiological properties. Key words: Momordica charantia, protein, polyphenolics, antioxidant, antimutagenicity

EVALUATION OF THE ANTIOXIDATIVE, ANTIDIABETIC AND ANTILIPIDEMIC EFFECT OF BITTER MEL ON SEEDS (CITRULLUS COLOCYNTHIS) ALCOHOLIC EXTRACT ON FEMALE RATS

Background: Diabetes mellitus and hyperlipidemia are well-known risk factors for several illnesses including atherosclerosis, heart and vascular diseases and stroke. Herbal medicine is still the mainstay of about 75–80% of the world population, mainly in the developing countries, for primary health care because of better cultural acceptability, better compatibility with the human body and lesser side effects. Herbal extracts are introduced to the mankind since many centuries, several herbal extracts in different oral formulas have been recommended for diabetes mellitus and hyperlipidemia treatment all over the world. Objective: The present study was carried out to investigate the hypoglycemic, hypolipidemic and antioxidative effects of the extract of Bitter melon (Citrullus colocynthis) in young adult Sprague Dawley female rats for eight weeks. Materials and Methods: Forty female young adult Sprague Dawley female rats were divided into two sections. Section I (Normal animals) was S.C. injection with a vehicle (0.9% NaCl) and divided into two groups (10 rats / group), one of these served as control group and the second group was received extract of Bitter melon (Citrullus colocynthis) seed 50 mg/kg/day for 8 weeks and served as normal treated group. Group II was S.C. injection with Alloxan (diabetic rats) and divided into two group (10 rats each), one of these served as diabetic control group while the second group was received extract of Citrullus colocynthis (C-colocynthis) seed (50mg/kg/day) orally for 8 weeks and served as diabetic treated group. After 8-weeks experiment was terminated and animals were sacrificed, heart blood was drawn and sera were separated for assessment of blood glucose, lipid profile, and lipid peroxidation value (MDA), reduced glutathione (GSH), lactate dehydrogenase (LDH), alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) as well as total and direct bilirubin. Results: Both normal and diabetic rats showed a significant decrease in blood glucose, lipid peroxidation value (MDA), total cholesterol (TC), triglycerides (TG) as well as total and direct bilirubin. While, levels of reduced glutathione (GSH), lactate dehydrogenase (LDH), alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and total lipid were significantly increased in both normoglycemic and hyperglycaemic rats except ALT, AST and ALP in hyperglycaemic rats had no changes. Conclusions: The present study shed more of the light on the effect of Bitter melon (Citrullus colocynthis) extract and proves that this extract has antioxidant, hypoglycemic and hypolipidemic properties in the rats and may be used for treating diabetes mellitus.

Bitter melon (Momordica charantia) triterpenoid extract reduces preadipocyte viability, lipid accumulation and adiponectin expression in 3T3-L1 cells

Bitter melon (Momordica charantia) contains a variety of potentially bioactive compounds which includes two classes of saponins known as cucurbitane and oleanane-type triterpenoids. A bitter melon (BM) extract was investigated for the potential to reduce cell viability, reduce lipid accumulation in differentiating 3T3-L1 cells and affect subsequent adiponectin expression. BM extract contained at least five different triterpenoids and reduced preadipocyte viability with an LC50 concentration after 24h determined to be 0.402±0.04mg/mL, 0.314±0.01mg/mL for 48h and 0.310±0.01mg/mL for 72h. BM treatment increased the release of lactate dehydrogenase (LDH) from cells and significantly (p<0.05) increased cells in the G2/M while reducing cells in the G1 phase of the cell cycle. BM treatment of 3T3-L1 cells resulted in a decreased in lipid accumulation and significantly (p<0.05) decreased intracellular triglyceride amount compared to untreated control cells. PPARγ expression was significantly (p<0.05) down-regulated. Adiponectin expression was significantly (p<0.05) decreased after 12 and 24h of treatment and was increased in response to troglitazone, a positive control. BM extract reduced preadipocyte proliferation by a G2/M arrest of the cell cycle and reduced lipid accumulation of the adipocyte.

Bitter Melon: Adding Zing to Breast Cancer Treatment

Extracts of a bitter melon reduce tumor growth in breast cancer cells.

Extraction, Quantification, and Antioxidant Activities of Phenolics from Pericarp and Seeds of Bitter Melons (Momordica charantia) Harvested at Three Maturity Stages (Immature, Mature, and Ripe)

Bitter melon (Momordica charantia) is an exotic vegetable used for consumption and medicinal purposes mainly throughout Asia. Phenolics were extracted from pericarp (fleshy portion) and seeds of bitter melons harvested at three maturation stages (immature, mature, and ripe) using ethanol and water solvent systems. Total phenolic assessment demonstrated 80% of ethanol to be the optimal solvent level to extract phenolics either from pericarp or seed. Main phenolic constituents in the extracts were catechin, gallic acid, gentisic acid, chlorogenic acid, and epicatechin. Free radical scavenging assay using 2,2-diphenyl-1-picrylhydrazyl (DPPH) demonstrated the bitter melon extracts as slow rate free radical scavenging agents. There were low correlations between the total phenolic contents and antiradical power values of the extracts, suggesting a possible interaction among the phenolic constituents occurred. Bitter melon phenolic extracts contain natural antioxidant substances, and could be used as antioxidant agents in suitable food products.

Bitter Melon: Antagonist to Cancer

The incidence of cancer is increasing worldwide, in spite of substantial progress in the development of anti-cancer therapies. One approach to control cancer could be its prevention by diet, which inhibits one or more neoplastic events and reduces cancer risk. Dietary compounds offer great potential in the fight against cancer by inhibiting the carcinogenesis process through the regulation of cell homeostasis and cell-death machineries. For centuries, Ayurveda (Indian traditional medicine) has recommended the use of bitter melon (Momordica charantia) as a functional food to prevent and treat diabetes and associated complications. It is noteworthy to mention that bitter melon extract has no-to-low side effects in animals as well as in humans. The anti-tumor activity of bitter melon has recently begun to emerge. This review focuses on recent advancements in cancer chemopreventive and anti-cancer efficacy of bitter melon and its active constituents. Several groups of investigators have reported that treatment of bitter-melon-related products in a number of cancer cell lines induces cell cycle arrest and apoptosis without affecting normal cell growth. Therefore, the effect of bitter melon should be beneficial for health, and use of the non-modified dietary product is cost effective.

Bitter Melon (Momordica charantia) Extract Inhibits Breast Cancer Cell Proliferation by Modulating Cell Cycle Regulatory Genes and Promotes Apoptosis

Breast cancer is one of the most common cancers among women in the United States. Although there are effective drugs for treating advanced stages of breast cancers, women eventually develop resistance. One of the approaches to control breast cancer is prevention through diet, which inhibits one or more neoplastic events and reduces cancer risk. In this study, we have used human breast cancer cells, MCF-7 and MDA-MB-231, and primary human mammary epithelial cells as an in vitro model to assess the efficacy of bitter melon (Momordica charantia) extract (BME) as an anticancer agent. BME treatment of breast cancer cells resulted in a significant decrease in cell proliferation and induced apoptotic cell death. Apoptosis of breast cancer cells was accompanied by increased poly(ADP-ribose) polymerase cleavage and caspase activation. Subsequent studies showed that BME treatment of breast cancer cells inhibited survivin and claspin expression. Fluorescence-activated cell sorting analysis suggested that MCF-7 cells treated with BME accumulated during the G2-M phase of the cell cycle. Further studies revealed that BME treatment enhanced p53, p21, and pChk1/2 and inhibited cyclin B1 and cyclin D1 expression, suggesting an additional mechanism involving cell cycle regulation. Together, these results show that BME modulates signal transduction pathways for inhibition of breast cancer cell growth and can be used as a dietary supplement for prevention of breast cancer.

Molecular Cloning and Immunochemical Characterization of a New Japanese Cedar Pollen Allergen Homologous to Plant Subtilisin-Like Serine Protease

Protease activities in allergen sources are thought to be involved in triggering allergic inflammation through the disruption of epithelial barrier or the induction of proinflammatory cytokines. Protease allergens may also work as type 2 helper T cell (TH2) adjuvants through the cleavage of cell surface receptors. Here, we report molecular cloning and immunochemical characterization of a new Japanese cedar (Cryptomeria japonica) pollen allergen (CPA9) homologous to serine protease, which is initially found as a high IgE-binding spot on our two-dimensional (2-D) IgE immunoblotting map. The cpa9 cDNA encoded a 757 amino acid polypeptide showing a significant sequence identity with plant subtilisin-like serine protease family members including melon major allergen Cuc m 1. We found that native CPA9 purified from C. japonica pollen showed a high IgE-binding frequency and IgE cross-reactivity with melon extract.

NsLTP and profilin are allergens in mustard seeds: cloning, sequencing and recombinant production of Sin a 3 and Sin a 4

Patients allergic to mustard are frequently sensitized to peach. To identify and analyse new yellow mustard allergens that could be involved in IgE cross-reactivity. Sera from mustard-allergic patients with symptoms to peach were studied. Mass spectrometry analyses provided sequences of IgE-reactive proteins. cDNAs encoding Sin a 3 and Sin a 4 were amplified by polymerase chain reaction, cloned and sequenced. The recombinant allergens were obtained in Pichia pastoris and Escherichia coli, respectively, and used for ELISA, immunoblotting and inhibition experiments. Sequence alignment was used to identify common IgE epitopes. Sin a 3- and Sin a 4-specific cDNAs encode for mature proteins of 92 and 131 amino acids that belong to nsLTP and profilin protein families, respectively. Sin a 3 and Sin a 4 showed 54% and 80% identity with allergenic nsLTP from peach and profilin from melon, respectively. Both recombinant allergens were IgE-reactive in ELISA and immunoblotting. Peach pulp and peel, and melon extracts nearly abolished the IgE binding to recombinant Sin a 3 or recombinant Sin a 4 in immunoblotting. Sin a 3 (nsLTP) and Sin a 4 (profilin) were identified as new mustard allergens and showed IgE cross-reactivity with fruits such as peach or melon, respectively. The knowledge of these two allergens will contribute towards better understand with cross-reactivity between mustard and other plant food allergens, and their availability will provide physicians with useful tools for molecular diagnosis.

Matrix effects observed during pesticides residue analysis in fruits by GC

The influence of the sample matrix in the GC-electron-capture detection analysis of the pesticides dimethoate, diazinon, chlorothalonil, parathion methyl and fenitrothion in fruits samples has been studied. Experiments have been carried out where the pesticide responses in standard solutions prepared in selected solvent were compared with their response when present in apple, mango, papaya, banana, pineapple and melon extracts. The presence of matrix effects (MEs) and their extent were shown to be simultaneously influenced by several factors (matrix concentration, matrix type, pesticide concentration, analytical range). Pronounced MEs were observed particularly for dimethoate and diazinon in all matrices tested; in lower concentrations, all pesticides presented significant ME. The other pesticides presented variable ME. Higher ME enhancement was detected at lower pesticide concentration levels of and/or at higher matrix concentration solutions. The ME detected for fenitrothion, in the analytical range evaluated, were dependent on matrix type. For each pesticide, solvent and matrix-matched calibrations were compared for all fruit samples, and it could be concluded that quantitation based on standard solutions prepared in blank matrix extract (matrix-matched calibration) should be used to compensate the MEs and to obtain more accurate results for the pesticides studied.

An SOD rich melon extract Extramel ® prevents aortic lipids and liver steatosis in diet-induced model of atherosclerosis

Background and aims Oxidative stress has been involved in the early steps of atherosclerosis and previous studies on hypercholesterolemic hamsters have shown that non-enzymatic antioxidant could prevent fatty streak formation. Therefore, we investigated whether a melon juice extract (Extramel ®) rich in superoxide dismutase (SOD) would prevent the development of early atherosclerosis. Methods and results The effects of Extramel ® on plasma cholesterol, aortic fatty streak formation, hepatic steatosis, superoxide anion tissue production and NAD(P)H oxidase expression were studied in hamsters fed with an atherogenic diet (HF), receiving by gavage either water or Extramel ® at 0.7, 2.8 or 5.6 mg/d. After 12 weeks of oral administration, Extramel ® lowered plasma cholesterol and non-HDL cholesterol and induced blood and liver SOD activities. It also strongly reduced the area of aortic fatty streak by 49–85%, cardiac (45%) and liver (67%) production of superoxide anion and liver p22 phox subunit of NAD(P)H oxidase expression by 66%, and attenuated the development of hepatic steatosis. Conclusion These findings support the view that chronic consumption of melon juice extract rich in SOD has potential beneficial effects with respect to the development of atherosclerosis and liver steatosis, emphasizing its use as potential dietary therapy.

Preventive Effect of a Melon Extract Rich in Superoxide Scavenging Activity on Abdominal and Liver Fat and Adipokine Imbalance in High-Fat-Fed Hamsters

Studies showed that dietary antioxidants could be a therapy against obesity that is associated with a state of oxidative stress. Thus, this paper investigates whether a dietary ingredient, a melon juice extract rich in superoxide dismutase, would prevent the development of such obesity in hamsters. Five groups received a standard diet or a high-fat diet (HF) plus a daily gavage with water (control) or extract at 0.7, 2.8, or 5.6 mg/day. After 84 days, the higher dose lowered triglyceridemia (68%), production of liver superoxide anion (12%), mitochondrial cytochrome c oxidase activity (40%), lipid and protein oxidation products (35 and 35%, respectively), and leptinemia (99%) and increased adiponectinemia (29%), leading to a concomitant reduction in insulinemia (39%), insulin resistance (41%), and abdominal lipids (25%). The extract triggered a remarkable decrease of liver lipids (73%) and fully prevented the steatohepatitis induced by the HF diet. Chronic consumption of this melon extract may represent a new alternative to reduce obesity induced by a high-fat diet.