Preventive Effects of Green Tea Extract against Obesity Development in Zebrafish.

Various natural products (NPs) have been used to treat obesity and related diseases. However, the best way to fight obesity is preventive, with accurate body weight management through exercise, diet, or bioactive NPs to avoid obesity development. We demonstrated that green tea extract (GTE) is an anti-obesity NP using a zebrafish obesity model. Based on a hypothesis that GTE can prevent obesity, the objective of this study was to assess GTE's ability to attenuate obesity development. Juvenile zebrafish were pretreated with GTE for seven days before obesity induction via a high-fat diet; adult zebrafish were pretreated with GTE for two weeks before obesity induction by overfeeding. As a preventive intervention, GTE significantly decreased visceral adipose tissue accumulation in juveniles and ameliorated visceral adiposity and plasma triglyceride levels in adult zebrafish obesity models. RNA sequencing analysis was performed using liver tissues from adult obese zebrafish, with or without GTE administration, to investigate the underlying molecular mechanism. Transcriptome analysis revealed that preventive GTE treatment affects several pathways associated with anti-obesity regulation, including activation of STAT and downregulation of CEBP signaling pathways. In conclusion, GTE could be used as a preventive agent against obesity.

Anti-Obesity Natural Products Tested in Juvenile Zebrafish Obesogenic Tests and Mouse 3T3-L1 Adipogenesis Assays.

(1) Background: The obesity epidemic has been drastically progressing in both children and adults worldwide. Pharmacotherapy is considered necessary for its treatment. However, many anti-obesity drugs have been withdrawn from the market due to their adverse effects. Instead, natural products (NPs) have been studied as a source for drug discovery for obesity, with the goal of limiting the adverse effects. Zebrafish are ideal model animals for in vivo testing of anti-obesity NPs, and disease models of several types of obesity have been developed. However, the evidence for zebrafish as an anti-obesity drug screening model are still limited. (2) Methods: We performed anti-adipogenic testing using the juvenile zebrafish obesogenic test (ZOT) and mouse 3T3-L1 preadipocytes using the focused NP library containing 38 NPs and compared their results. (3) Results: Seven and eleven NPs reduced lipid accumulation in zebrafish visceral fat tissues and mouse adipocytes, respectively. Of these, five NPs suppressed lipid accumulation in both zebrafish and 3T3-L1 adipocytes. We confirmed that these five NPs (globin-digested peptides, green tea extract, red pepper extract, nobiletin, and Moringa leaf powder) exerted anti-obesity effects in diet-induced obese adult zebrafish. (4) Conclusions: ZOT using juvenile fish can be a high-throughput alternative to ZOT using adult zebrafish and can be applied for in vivo screening to discover novel therapeutics for visceral obesity and potentially also other disorders.

RNA-seq Based Transcriptome Analysis of the Anti-Obesity Effect of Green Tea Extract Using Zebrafish Obesity Models.

Green tea is a popular beverage that is rich in polyphenolic compounds such as catechins. Its major content, (-)-epigallocatechin-3-gallate, has been shown to have beneficial effects on several diseases including cancer, metabolic syndrome, cardiovascular diseases, and neurodegenerative diseases. The aim of this study was to assess the anti-obesity effects and the underlying molecular mechanisms of green tea extract (GTE) using zebrafish larva and adult obesity models. We administered 100 µg/mL GTE to zebrafish larvae and performed a short-term obesogenic test. GTE significantly decreased the visceral adipose tissue volume induced by a high-fat diet. Oral administration (250 µg/g body weight/day) of GTE to adult diet-induced obese zebrafish also significantly reduced their visceral adipose tissue volume, with a reduction of plasma triglyceride and total cholesterol levels. To investigate the molecular mechanism underlying the GTE effects, we conducted RNA sequencing using liver tissues of adult zebrafish and found that GTE may ameliorate the obese phenotypes via the activation of Wnt/ß-catenin and adenosine monophosphate-activated protein kinase (AMPK) pathway signaling. In addition, the comparative transcriptome analysis revealed that zebrafish and mammals may share a common molecular response to GTE. Our findings suggest that daily consumption of green tea may be beneficial for the prevention and treatment of obesity.

Post-meal perceivable satiety and subsequent energy intake with intake of partially hydrolysed guar gum.

Partially hydrolysed guar gum (PHGG), a soluble dietary fibre, has been shown to provide many health benefits. Previous studies had suggested that the combination of PHGG with protein provided a significant satiation effect on visual analogue scales (VAS). What was lacking was only the effect of administration of small doses of PHGG on post-meal satiation and subsequent energy intake. The objectives of the present investigations were to find the subjective perception of post-meal satiety with acute and long term administration of small amounts of PHGG alone with food, its effects on subsequent energy intake and the comparative effects among different types of soluble fibres. The following three separate studies were conducted: in study 1, healthy subjects (n 12) consumed PHGG along with breakfast, lunch and an evening snack; in study 2, healthy subjects (n 24) consumed 2 g of PHGG or dextrin along with yogurt as breakfast for 2 weeks; in study 3, healthy subjects (n 6) took 6 g each of either PHGG or indigestible dextrin or inulin along with lunch. In all the studies, various satiety parameters were measured on VAS before and after consumption of PHGG. The addition of PHGG showed significant (P < 0.05) acute (studies 1 and 3) and long-term (studies 1 and 2) satiety effects compared to the control and/or an equal amount of carbohydrate or other types of soluble fibre. Study 2 also indicated that the prolonged consumption of PHGG may significantly (P < 0.05) reduce energy intake from whole-day snacking. PHGG could be an ideal natural soluble fibre for delivering acute and long term satiety effects for comfortable appetite control.

Zebrafish obesogenic test identifies anti-adipogenic fraction in Moringa oreifera leaf extracts.

The zebrafish obesogenic test (ZOT) is a powerful tool for identifying anti-adipogenic compounds for in vivo screening. In our previous study, we found that Moringa oleifera (MO) leaf powder suppressed the accumulation of visceral adipose tissue (VAT) in ZOT. MO demonstrates a wide range of pharmacological effects; however, little is known about its functional constituents. To identify the anti-adipogenic components of MO leaves, we prepared extracts using different extraction methods and tested the obtained extracts and fractions using ZOT. We found that the dichloromethane extract and its hexane:EtOAc = 8:2 fraction reduced VAT accumulation in young zebrafish fed a high-fat diet. We also performed gene expression analysis in the zebrafish VAT and found that CCAAT/enhancer-binding protein beta and CCAAT/enhancer-binding protein delta (associated with early stages of adipogenesis) gene expression was downregulated after fraction 2 administration. We identified a new MO fraction that suppressed VAT accumulation by inhibiting early adipogenesis using the ZOT. Phenotype-driven zebrafish screening is a reasonable strategy for identifying bioactive components in natural products.

Globin Digest Improves Visceral Adiposity Through UCP1 Upregulation in Diet-Induced Obese Zebrafish and Mice.

Globin digest (GD), a bioactive oligopeptide derived from porcine hemoglobin proteins, has been demonstrated to have beneficial effects on improving postprandial hyperlipidemia, hyperglycemia, and liver injury. We previously reported the lipid-lowering effects of GD using a zebrafish obesogenic test. Here, we sought to evaluate the effect of GD on visceral adiposity and the underlying molecular mechanisms using zebrafish and mouse obesity models. GD ameliorated dyslipidemia and suppressed the accumulation of visceral adipose tissue (VAT) in adult obese zebrafish. Transcriptomic analysis by RNA sequencing of GD-treated adult zebrafish revealed that GD upregulated UCP1-related pathways. Further, we performed mouse experiments and found that GD intake (2 mg/g body weight/day) was associated with lowered plasma triglyceride and total cholesterol levels, decreased VAT accumulation, and improved adipocyte hypertrophy with the upregulation of Ucp1 expression in white adipose tissue at both the mRNA and protein levels. Taken together, these results indicate that GD improves visceral adiposity by upregulating UCP1 expression, providing a novel perspective on combating obesity.

Attitudes Toward Psychological Disorders and Alternative Medicine in Saudi Participants.

Background: This study was designed to investigate Saudis' attitudes toward mental distress and psychotropic medication, attribution of causes, expected side effects, and to analyze participants' expectations toward alternative or complementary medicine using aromatic and medicinal plants, through a survey. Method: The study included 674 participants (citizens and residents in Saudi Arabia) who were randomly contacted via email and social media and gave their consent to complete a questionnaire dealing with 39 items that can be clustered in six parts. Descriptive statistics and Chi-square for cross-tabulation were generated using SPSS. Results: Among the 664 participants, 73.4% believed that there are some positive and negative outcomes of psychotropic medication. Participants (72.0%) think that the most important reason leading to psychological disorders is mainly due to the loss of a relative or beloved person, and 73.9% considered psychic session as one of the possible treatments of psychological disorders. Surprisingly, only 18.8% of the participants agreed that medicinal and aromatic plants could be a possible treatment of the psychological disorder. Participants (82%) consider that physicians are the most trustful and preferred source of information about alternative and complementary medicine.

The protective role of amla (Emblica officinalis Gaertn.) against fructose-induced metabolic syndrome in a rat model.

We investigated the effects of amla (Emblica officinalis Gaertn.) on fructose-induced metabolic syndrome using a rat model. Male Wistar rats were fed a high-fructose (65 %) diet or standard chow for 1 week, and treated with an ethyl acetate (EtOAc) extract of amla, a polyphenol-rich fraction, at 10 or 20 mg/kg body weight per d, or vehicle, for 2 weeks. Serum glucose, TAG, total cholesterol and blood pressure levels of the high-fructose diet-fed rats were increased compared with those of the normal rats (P < 0.001). However, the EtOAc extract of amla ameliorated the high fructose-induced metabolic syndrome, including hypertriacylglycerolaemia and hypercholesterolaemia. Also, the elevated levels of hepatic TAG and total cholesterol in rats given the high-fructose diet were significantly reduced by 33.8 and 24.6 %, respectively (P < 0.001), on the administration of the EtOAc extract of amla at the dose of 20 mg/kg with the regulation of sterol regulatory element-binding protein (SREBP)-1 expression. The protein levels of PPARalpha and SREBP-2 were not affected by the feeding of the high-fructose diet or EtOAc extract of amla. In addition, oral administration of the amla extract at the dose of 20 mg/kg significantly inhibited the increased serum and hepatic mitochondrial thiobarbituric acid-reactive substance levels (21.1 and 43.1 %, respectively; P < 0.001). Furthermore, the amla extract inhibited the increase of cyclo-oxygenase-2 with the regulation of NF-kappaB and bcl-2 proteins in the liver, while the elevated expression level of bax was significantly decreased by 8.5 and 10.2 % at the doses of 10 and 20 mg/kg body weight per d, respectively. These findings suggest that fructose-induced metabolic syndrome is attenuated by the polyphenol-rich fraction of amla.

Characteristic Analysis of Trigonelline Contained in Raphanus sativus Cv. Sakurajima Daikon and Results from the First Trial Examining Its Vasodilator Properties in Humans.

Vascular disease poses a major public health problem worldwide. Trigonelline isolated from Raphanus sativus cv. Sakurajima Daikon (Sakurajima radish) induces nitric oxide production from vascular endothelial cells and enhances vascular function. Here, we investigated the characteristics of trigonelline and its effects on endothelial function after consumption of Sakurajima radish by humans. Our results show that Sakurajima radish contains approximately 60 times more trigonelline than other radishes and squashes. Additionally, no significant differences were observed between varieties of Sakurajima radish, suggesting that any type of Sakurajima radish can be ingested for trigonelline supplementation. The effects of cooking and processing Sakurajima radish were also evaluated, as were the effects of freezing, and changes in osmotic pressure and pH. A first-in-human trial using Sakurajima radish showed that ingestion of 170 g/day of Sakurajima radish for ten days increased blood trigonelline concentrations and significantly improved flow-mediated dilation, which is a measure of vascular endothelial function. Overall, our findings suggest that the trigonelline contained in Sakurajima radish may contribute to improved human vascular endothelial function. Hence, Sakurajima radish may enhance vascular endothelial function as a functional food.

Effects of theanine, a unique amino acid in tea leaves, on memory in a rat behavioral test.

We identified an effect of theanine on memory functions in a novel object test. Rats were fed theanine for 3 weeks ad libitum, and then they performed the object test. The theanine-fed group performed search behavior for the novel object in the test session. The results suggest that theanine-fed rats showed improved recognition, and that theanine affected learning and memory.

Administration of theanine, a unique amino acid in tea leaves, changed feeding-relating components in serum and feeding behavior in rats.

We identified an effect of gamma-glutamylethylamide (theanine) on feeding in a rat study. Oral theanine suppressed the food intake of rats. The serum glucose level did not differ from the control, but the insulin concentration was reduced and the corticosterone concentration was increased by theanine. We suggest that the effect of theanine on feeding involved hormones.

Possible carcinogenic risks of copper gluconate and their prevention by co-administered green tea catechins evaluated by a rat medium-term multi-organ carcinogenicity bioassay protocol.

Carcinogenic risks of copper gluconate, green tea catechins and their combined exposure were evaluated using a rat medium-term multi-organ carcinogenicity bioassay protocol. Male BrlHan:WIST@Jcl (GALAS) rats were given N-nitrosodiethylamine (DEN), N-methylnitrosourea (MNU), 1,2-dimethylhydrazine (DMH), N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) and 2,2'-dihydroxy-di-n-propylnitrosamine (DHPN) for a total multiple initiation period of 4 weeks (DMBDD treatment). Rats were then given a diet containing copper gluconate at a concentration of 0, 10, 300, 3000 or 6000 ppm with or without a co-administration of catechins starting 1 week later by admixing in the drinking water at a concentration of 5000 ppm. All survivors were sacrificed at the end of week 29. Number of putatively preneoplastic, glutathione S-transferase placental form-positive, liver lesions significantly increased by copper gluconate of 300 ppm or greater. In addition, both incidence and grade of hyperplasia in the forestomach significantly increased by copper gluconate of 6000 ppm. Catechins, exerting no effects by themselves, inhibited these effects of copper gluconate. The present results indicate that copper gluconate may possess carcinogenic risks for the liver and forestomach at its high dose level, and that co-administered green tea catechins may exert preventive effects.

Theanine, r-glutamylethylamide, increases neurotransmission concentrations and neurotrophin mRNA levels in the brain during lactation.

Theanine (r-glutamylethylamide) is one of the major amino acid components in green tea. Recent studies suggest that theanine affects neurotransmission, especially inhibitory neurotransmission. In this study, we investigated whether theanine affects brain development in infant rats, because inhibitory neurotransmission is required for mature brain function. Mother rats were fed theanine ad libitum after confinement. The body weight gain rate of infants was not different from control infants. We detected theanine in the infant serum and measured neurotransmitter concentration and nerve growth factor (NGF) mRNA level in the infant rat brain. Some neurotransmitters, including dopamine, serotonin, glycine and GABA concentration, increased in the infant brain and NGF mRNA level increased in the cerebral cortex and hippocampus. However, these differences were lost by the end of nerve maturity. These results suggest that theanine enhanced synthesis of nerve growth factor and neurotransmitters during a nerve maturing period and promoted central nerve system maturation (CNS). Thus, theanine accelerated maturation. In conclusion, theanine may assist in healthy brain function development.

L-Theanine reduces psychological and physiological stress responses.

L-Theanine is an amino acid contained in green tea leaves which is known to block the binding of L-glutamic acid to glutamate receptors in the brain. Because the characteristics of L-Theanine suggest that it may influence psychological and physiological states under stress, the present study examined these possible effects in a laboratory setting using a mental arithmetic task as an acute stressor. Twelve participants underwent four separate trials: one in which they took L-Theanine at the start of an experimental procedure, one in which they took L-Theanine midway, and two control trials in which they either took a placebo or nothing. The experimental sessions were performed by double-blind, and the order of them was counterbalanced. The results showed that L-Theanine intake resulted in a reduction in the heart rate (HR) and salivary immunoglobulin A (s-IgA) responses to an acute stress task relative to the placebo control condition. Moreover, analyses of heart rate variability indicated that the reductions in HR and s-IgA were likely attributable to an attenuation of sympathetic nervous activation. Thus, it was suggested that the oral intake of L-Theanine could cause anti-stress effects via the inhibition of cortical neuron excitation.

Amla (Emblica officinalis Gaertn.) attenuates age-related renal dysfunction by oxidative stress.

To investigate the effects of amla on renal dysfunction involved in oxidative stress during the aging process, we employed young (2 months old) and aged (13 months old) male rats and administered SunAmla (Taiyo Kagaku Co., Ltd., Japan) or an ethyl acetate (EtOAc) extract of amla, a polyphenol-rich fraction, at a dose of 40 or 10 mg/kg body weight/day for 100 days. The administration of SunAmla or EtOAc extract of amla reduced the elevated levels of serum creatinine and urea nitrogen in the aged rats. In addition, the tail arterial blood pressure was markedly elevated in aged control rats as compared with young rats, while the systolic blood pressure was significantly decreased by the administration of SunAmla or EtOAc extract of amla. Furthermore, the oral administration of SunAmla or EtOAc extract of amla significantly reduced thiobarbituric acid-reactive substance levels of serum, renal homogenate, and mitochondria in aged rats, suggesting that amla would ameliorate oxidative stress under aging. The increases of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expression in the aorta of aging rats were also significantly suppressed by SunAmla extract or EtOAc extract of amla, respectively. Moreover, the elevated expression level of bax, a proapoptotic protein, was significantly decreased after oral administration of SunAmla or EtOAc extract of amla. However, the level of bcl-2, an antiapoptotic protein, did not show any difference among the groups. The expressions of renal nuclear factor-kappaB (NF-kappaB), inhibitory kappaB in cytoplasm, iNOS, and COX-2 protein levels were also increased with aging. However, SunAmla or EtOAc extract of amla reduced the iNOS and COX-2 expression levels by inhibiting NF-kappaB activation in the aged rats. These results indicate that amla would be a very useful antioxidant for the prevention of age-related renal disease.

Antioxidant properties of casein calcium peptides and their effects on lipid oxidation in beef homogenates.

The antioxidant activity of casein calcium peptides in several in vitro assay systems was investigated. Casein calcium peptides were prepared by the microbial enzymic hydrolysis of casein calcium. The main peak of the molecular mass distribution of the peptides was about 3 kDa. Casein calcium peptides showed strong antioxidant activity with the beta-carotene bleaching method, and they also showed scavenging activity against radicals such as superoxide radicals, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, and hydroxyl radicals. Antioxidant activity was increased with an increasing peptide concentration. Casein calcium peptides also showed strong antioxidant activity against lipid oxidation in ground beef homogenates. These results suggest that casein calcium peptides are a suitable natural antioxidant that prevents the lipid oxidation of meat and related food ingredients.

Green tea polyphenols and dietary fibre protect against kidney damage in rats with diabetic nephropathy.

In this study we examined the effect of green tea polyphenols (GTP) and partially hydrolysed guar gum (PHGG) as dietary fibre on diabetic nephropathy, using rats that had been subjected to subtotal nephrectomy and injection of streptozotocin. The subtotally nephrectomized rats were subjected to resection of three-quarters of the kidney. Rats with diabetic nephropathy were divided into four groups: untreated controls, and animals that received GTP (100 mg kg-1 body weight day-1), PHGG (100 mg kg-1 body weight day-1) and GTP plus PHGG (50 mg kg-1 body weight day-1 plus 50 mg kg-1 body weight day-1). After 50 days of administration, attenuation of urinary protein excretion and the morphological changes peculiar to diabetic nephropathy were observed in all three treated groups. Furthermore, the group treated with GTP plus PHGG showed an improvement of kidney weight and serum levels of urea nitrogen, creatinine and creatinine clearance. Hyperglycaemia, as assessed in terms of blood glucose and glycosylated protein levels, was also improved by administration of GTP plus PHGG. On the other hand, GTP administration increased the activity of superoxide dismutase in the kidney to a significant extent. A significant reduction in the total cholesterol concentration was also observed in the PHGG-treated group. These results suggest that GTP and PHGG could be beneficial as additional therapy in the management of diabetic nephropathy.

Comparison of different fibers for in vitro production of short chain fatty acids by intestinal microflora.

The fermentation of dietary fiber in the large intestine and the by-products of this fermentation are thought to protect against colonic diseases. As it is difficult to measure the fermentation of dietary fiber in an intact animal, in vitro techniques have been developed to compare the fermentability of various dietary fibers. The objective of this project was to compare short chain fatty acid (SCFA) production with different fibers in an in vitro fermentation model. A wide range of commercially available dietary fiber sources was compared for SCFA production. Fibers were fermented with a fecal innoculum for 0, 2, 4, 8, 12, and 24 hours. SCFAs were measured by gas chromatography. SCFA production varied among the fiber sources. Hydrolyzed guar gum and galactomannan produced the greatest amounts of total SCFAs. Butyrate production was higher with the fiber sources than the glucose control. Acetate production was less for psyllium than the other fibers. Thus, different dietary fiber sources are more readily fermented by fecal microflora. These differences most likely affect the physiological differences seen among dietary fiber sources.

Influence of amla (Emblica officinalis Gaertn.) on hypercholesterolemia and lipid peroxidation in cholesterol-fed rats.

The effects of amla on low-density lipoprotein (LDL) oxidation and cholesterol levels were investigated in vitro and in vivo using Cu(2+)-induced LDL oxidation and cholesterol-fed rats. SunAmla and ethyl acetate (EtOAc) extract of amla significantly inhibited thiobarbituric acid (TBA)-reactive substance level in the Cu(2+)-induced LDL oxidation and the effects were stronger than those of probucol. In addition, the administration of SunAmla (at a dose of 20 or 40 mg/kg body weight/d) or EtOAc extract of amla (at a dose of 10 or 20 mg/kg body weight/d) for 20 d to rats fed 1% cholesterol diet significantly reduced total, free and LDL-cholesterol levels in a dose-dependent manner, and EtOAc extract of amla exhibited more potent serum cholesterol-lowering effect than SunAmla in the same amount. Furthermore, the oxidized LDL level in serum was markedly elevated in cholesterol-fed control rats as compared with normal rats, while it was significantly decreased by the administration of SunAmla or EtOAc extract of amla. Moreover, the serum TBA-reactive substance level was also significantly decreased after oral administration of SunAmla or EtOAc extract of amla. These results suggest that amla may be effective for hypercholesterolemia and prevention of atherosclerosis.

The nontoxic mushroom Auricularia auricula contains a polysaccharide with anticoagulant activity mediated by antithrombin.

An acidic polysaccharide with anticoagulant activity was isolated from the edible mushroom Auricularia auricula using water, alkali or acid extracts. The alkali extract showed the highest anticoagulant activity and was thereby further purified using gel filtration chromatography. Specific anticoagulant activity of the purified polysaccharide was 2 IU/mg and its average mass was approximately 160 kDa. The polysaccharide from this species of mushroom contains mainly mannose, glucose, glucuronic acid and xylose but no sulfate esters. Its anticoagulant activity was due to catalysis of thrombin inhibition by antithrombin but not by heparin cofactor II. Inhibition of Factor Xa by antithrombin was not catalyzed by the polysaccharide. The glucuronic acid residues were essential for the anticoagulant action of the mushroom polysaccharide since the activity disappeared after reduction of its carboxyl groups. In ex vivo tests using rats orally fed with the polysaccharide, we observed an inhibitory effect on platelet aggregation as observed with aspirin, a well-known antiplatelet agent. The polysaccharides from these mushrooms may constitute a new source of compounds with action on coagulation, platelet aggregation and, perhaps, on thrombosis.