10-Hydroxydecanoic Acid Potentially Elicits Antigen-Specific IgA Responses.

The effective antigen (Ag) uptake by microfold cells (M-cells) is important for the induction of an efficient mucosal immune responses. Here, we show that 10-hydroxydecanoic acid (10-HDAA) from royal jelly (RJ) potentially supports M-cell differentiation and induces effective antigen-specific mucosal immune responses in cynomolgus macaques. 10-HDAA increases the expression level of receptor activator of nuclear factor-kappaB (NF-κB) (RANK) in Caco-2 cells, which suggests that 10-HDAA potentially prompts the differentiation of Caco-2 cells into M-cells and increased transcytosis efficiency. This idea is supported by the following observations. Intranasal administration of 10-HDAA increased the number of M-cells in the epithelium overlying nasopharynx-associated lymphoid tissue (NALT) in macaques. Oral administration of 10-HDAA increased the number of M-cells in the follicle-associated epithelium (FAE) covering Peyer's patches (PPs) and significantly increased the antigen-specific immunoglobulin A (IgA) level in macaques. These findings suggest that the exogenous honeybee-derived medium-chain fatty acid 10-HDAA may effectively enhance antigen-specific immune responses.

Resveratrol derivative-rich melinjo (Gnetum gnemon L.) seed extract improves obesity and survival of C57BL/6 mice fed a high-fat diet.

Melinjo (Gnetum gnemon L.) seed extracts (MSEs) are rich in resveratrol dimers (gnemonoside A, C, D, gnetin C), trans-resveratrol, and other resveratrol derivatives. trans-Resveratrol is a widely studied caloric restriction mimetic. In mice fed a high-fat diet (HFD), trans-resveratrol protects against obesity, type 2 diabetes, and premature death. Here, treatment of HFD-fed mice with 2.0% MSE significantly reduced body weight gain (p < 0.001), blood insulin (p < 0.01), and HOMA-IR (p < 0.05) after 8 weeks compared with untreated HFD-fed mice. Additionally, 0.2% MSE treatment of HFD-fed mice significantly improved physiological activity (p < 0.05) at 18 months of age and reduced risk of death due to HFD by 25% (hazard ratio = 0.75, p = 0.036). These data show that MSE can improve several aspects of metabolic syndrome and survival in mice and may have health benefits as a dietary supplement.

trans-Resveratrol in Gnetum gnemon protects against oxidative-stress-induced endothelial senescence.

Gnetum gnemon is an arboreal dioecious plant that is cultivated in Indonesia. The seeds of this species mainly contain dimeric stilbenoid compounds [gnetin C (1), gnemonoside A (2), and gnemonoside D (3)] along with trans-resveratrol (4). trans-Resveratrol has been reported to have antiaging, anticancer, and antidiabetic effects, as well as being a calorie restriction mimetic. SIRT1 exerts a protective effect against vascular senescence. In this study, the effects of these four main stilbenoid derivatives of a G. gnemon seed endosperm ethanolic extract on endothelial senescence were investigated. In streptozotocin-induced diabetic mice, administration of the G. gnemon ethanolic extract increased SIRT1 and decreased endothelial senescence. The concentration of 1 in blood plasma was 6-fold higher than 4 in these mice. Next, the in vitro effects of the four main stilbenoid derivatives of G. gnemon seeds were investigated. Senescent human umbilical vein endothelial cells were induced by hydrogen peroxide. Endothelial senescence was inhibited by 4, which increased the expression of endothelial nitric oxide synthase and SIRT1, whereas 1-3 had no effect. These results indicated that the ethanolic extract of G. gnemon seeds inhibits endothelial senescence, suggesting that 4 plays a critical role in the prevention of endothelial senescence.

Hypoallergenicity and immunological characterization of enzyme-treated royal jelly from Apis mellifera.

Royal jelly (RJ), the exclusive food for queen bees, is taken as a dietary supplement because it is highly rich in nutrients. However, RJ is known to induce an anaphylactic response in some individuals. We evaluated in the present study the hypoallergenicity of alkaline protease-treated RJ in vitro and in vivo. We first confirmed that this treated RJ contained the same levels of vitamins, minerals and specific fatty acid as in untreated RJ. We then showed that the IgE-binding capacity of the treated RJ was very significantly reduced by conducting in vitro assays of the blood from RJ-sensitive patients. An in vivo skin-prick test on the RJ-sensitive patients also showed that, in the majority of the patients (3 out of 4 tested), the treated RJ did not evoke any allergenic response. It is thus advantageous to prepare hypoallergenic RJ by a protease enzyme treatment for its safe consumption.

Inhibitory effect of gnetin C, a resveratrol dimer from melinjo (Gnetum gnemon), on tyrosinase activity and melanin biosynthesis.

Tyrosinase is the key enzyme involved in melanogenesis. The aim of this study was to investigate the in vitro inhibitory effects of gnetin C, a resveratrol dimer isolated from melinjo (Gnetum gnemon) seeds, on tyrosinase activity and melanin biosynthesis in murine B16 cells. The inhibitory activities of gnetin C and resveratrol were shown to be almost equal against tyrosinase and melanin biosynthesis in the cells. The IC(50) values of gnetin C activity against tyrosinase and melanin biosynthesis were 7.0 and 7.6 µM, respectively, whereas resveratrol demonstrated IC(50) values of 7.2 and 7.3 µM, respectively. These results indicated that gnetin C inhibited melanogenesis, in a manner similar to that of resveratrol, by inhibiting tyrosinase and may therefore function as a new skin-whitening agent. However, the direct effects of gnetin C and resveratrol on murine tyrosinase activities were not equal. The IC(50) value of resveratrol was 10.1 µM, while gnetin C only exhibited a 25.2% enzyme inhibition at 16 µM. The IC(25) values for gnetin C and resveratrol were 15.5 and 4.0 µM, respectively. Therefore, it is suggested that the effects of gnetin C may be due to mechanisms other than the direct inhibition of tyrosinase activity.

Inhibitory activity of Brazilian green propolis components and their derivatives on the release of cys-leukotrienes.

The effects of Brazilian green propolis ethanol extract on Cry j1-induced cys-leukotrienes and histamine release from peripheral leukocytes of patients with allergic rhinitis were investigated. One of the key mechanisms for the anti-allergic properties of the extract was revealed to be the suppression of cys-LTs release. Furthermore, a series of propolis components and their phenethyl esters were synthesized and evaluated as inhibitors of cys-LTs release. Artepillin C, baccharin, and kaempferide were the major active components of the ethanol extract. The inhibitory activity of artepillin C phenethyl ester was comparable to that of existing LT synthesis inhibitors.

Artepillin C (ARC) in Brazilian green propolis selectively blocks oncogenic PAK1 signaling and suppresses the growth of NF tumors in mice.

There are mainly three types of propolis whose major anticancer ingredients are entirely different: (1) CAPE (caffeic acid phenethyl ester)-based propolis in Europe, Far East and New Zealand, (2) artepillin C (ARC)-based Brazilian green propolis and (3) Brazilian red propolis. It was shown previously that NF (neurofibromatosis)-associated tumors require the kinase PAK1 for their growth, and CAPE-based propolis extracts such as Bio 30 suppress completely the growth of NF tumors in vivo by blocking PAK1 signaling. Also it was demonstrated that ARC suppresses angiogenesis, suggesting the possibility that ARC also blocks oncogenic PAK1 signaling. Here it is shown for the first time that both ARC and green propolis extract (GPE) indeed block the PAK1 signaling selectively, without affecting another kinase known as AKT. Furthermore, it was confirmed that ARC as well as GPE suppress almost completely the growth of human NF tumor xenografts in mice, as does Bio 30. These results suggest that both CAPE-based and ARC-based propolis extracts are natural anti-PAK1 remedies and could be among the first effective NF therapeutics available on the market. Since more than 70% of human cancers such as breast and prostate cancers require the kinase PAK1 for their growth, it is quite possible that GPE could be potentially useful for the treatment of these cancers, as is Bio 30.

Metabolism and pharmacokinetics of medium chain fatty acids after oral administration of royal jelly to healthy subjects.

The unique fatty acids in royal jelly (RJ), 10-hydroxy-2-decenoic acid and 10-hydroxydecanoic acid are expected to be associated with many health benefits, but little is known on the pharmacokinetics and metabolism. The aim of this study is to confirm the metabolism and pharmacokinetics of RJ fatty acids in humans. Twelve volunteers received RJ capsules or enzyme treated RJ (ETRJ) capsules (800 mg). The other group received two doses of ETRJ tablets (800 mg and 1600 mg). Plasma samples were collected up to 12 h after the RJ intake and urine samples were collected within 24 h after ETRJ tablet consumption. The samples were analyzed by LC/MS/MS. A multivariate analysis of the RJ dose plasma samples detected 2-decenedioic acid (2-DA), sebacic acid (SA), and 3-hydroxysebacic acid (3-HSA) with significantly different intensities (P < 0.05) before and after RJ intake. The area under the concentration (AUC) of 2-DA, SA, and 3-HSA was 2500.05 ± 569.58, 322.57 ± 137.36, and 242.98 ± 58.36 ng h mL-1, respectively. By enzyme treatment, the AUC of 2-DA, SA, and 3-HSA was significantly increased (P < 0.05). The values of AUC and urinary excretion of these metabolites were dose-dependent. The major RJ fatty acids were metabolized to dicarboxylate, absorbed into the circulation and their absorption increased by enzyme treatment. This study provides useful information that will support studies aimed at clarifying the identity of bioactive RJ constituents and their biological effect, and further the development of RJ.

Royal Jelly Supplementation Improves Menopausal Symptoms Such as Backache, Low Back Pain, and Anxiety in Postmenopausal Japanese Women.

OBJECTIVES: This study aimed to evaluate the effect of Royal Jelly (RJ) at a dose of 800 mg/day on menopausal symptoms in healthy Japanese postmenopausal women with placebo-controlled design. MATERIAL AND METHODS: A total of 42 healthy Japanese postmenopausal women have been recruited for this study. The subjects were randomized to oral treatment with either 800 mg of protease-digested lyophilized powder of RJ (enzyme-treated RJ) or placebo (800 mg of dextrin) daily for 12 weeks. The level of menopausal symptoms has been evaluated every 4 weeks, using menopausal symptoms questionnaire of Japanese women. Independent t-test was used to evaluate statistical significance of the treatment effects between the two groups. RESULTS AND CONCLUSION: All of the 42 women have completed the trial. There were significant differences related to the anxiety score (P = 0.046) and backache and low back pain score (P = 0.040) between 800 mg/day enzyme-treated RJ and placebo-treated groups after 12 weeks of administration, and no significant differences were found between the two groups in 4 weeks after intervention. No side effects were observed in either group. This study demonstrates that enzyme-treated RJ supplementation with doses of 800 mg/day is effective in relieving menopausal symptoms such as anxiety, backache, and low back pain in Japanese postmenopausal women.

Royal Jelly Delays Motor Functional Impairment During Aging in Genetically Heterogeneous Male Mice.

Aging is associated with motor disorders that decrease the quality of life (QOL). Royal jelly (RJ), used as a dietary supplement, has shown various health benefits and, therefore, it has the potential to improve the QOL during aging. We have previously developed protease enzyme-treated RJ to avoid the anaphylactic response induced by RJ supplementation. However, the effects of a lifelong treatment with RJ on normal aging have not been fully clarified. In this study, we investigated the effects of enzyme-untreated RJ (NRJ) and enzyme-treated RJ (ERJ) on the aging process focusing on motor functions, by using a genetically heterogeneous (HET) mouse model experimentally endowed with genetic diversity. We performed four different physical performance tests (grip strength, wire hang, horizontal bar, and rotarod). We showed that the age-related impairment of the motor functions was significantly delayed in RJ-treated mice. Both NRJ and ERJ were similarly effective against these types of aging-associated declines. Histological analyses revealed that the RJ treatment affected the muscle fiber size at an advanced age. We also demonstrated that age-related changes in muscle satellite cell markers and catabolic genes were affected in RJ-treated mice. These results suggest that non-protein components of RJ improved the motor function in aging mice. These findings indicate that RJ has the potential to change the QOL during aging by regulating the motor function.

Oral administration of heat-killed Lactobacillus kunkeei YB38 improves murine influenza pneumonia by enhancing IgA production.

Influenza is one of the important respiratory tract infections that require special attention for maintaining health and hygiene. The removal of influenza virus (IFV) by secretory IgA produced by the respiratory epithelium has been reported to be a critical host defense mechanism. Therefore, we isolated Lactobacillus kunkeei YB38 (YB38), the promoter of the salivary IgA secretion in humans, from honeybee pollen and studied the effect of heat-killed YB38 treatment for preventing IFV infection in a mouse model. Female BALB/c mice received YB38 orally for 21 consecutive days and were then inoculated nasally with IFV. The YB38-treated group with a daily dose of 100 mg/kg showed an increased survival rate after IFV infection relative to the control. IgA secretion in the respiratory epithelium in the YB38-treated group (100 mg/kg) was significantly increased after 6 days of infection, while IL-6 production in the same respiratory site and the number of cells infiltrating into alveoli were significantly decreased. Moreover, lung tissue damage that appeared after IFV infection was reduced. These results suggested that the YB38 dose induced early and local IgA secretion at the infection site, inhibited persistent IFV infection, and prevented the infiltration of inflammatory immune cells or production of excessive IL-6, resulting in less damage to lung tissues.

Positions of Hydroxyl Groups in Chrysin are Critical for Inhibiting Plasminogen Activator Inhibitor-1 Release from Human Umbilical Vein Endothelial Cells.

Chrysin suppresses the TNFα-induced increase in the secretion of plasma plasminogen activator inhibitor 1 (PAl-1), a risk factor for thrombotic diseases, from human umbilical vein endothelial cells (HUVECs). The present study aimed to determine the association between the location of the hydroxyl groups in chrysin.to levels of-PAI-1. in the medium of HUVEC stimulated with TNFα. We cultured HUVEC for 3 h in medium containing chrysin or various flavonoids and then stimulated them with TNFα (10 ng/mL) for 12 h. Levels of PAI-1 antigen measured using ELISA showed that chrysin significantly inhibited the PAl- I increase with an IC50 of 15.6 µM. The flavones, galangin, baicalein, 5-hydroxyflavone, 6-hydroxyflavone, 7-hydroxyflavone and quercetin did not significantly inhibit the PAI- increase. Apigenin and luteolin were cytotoxic and thus their ability to inhibit PAI production could not be evaluated. Chrysin also inhibited PAI- mRNA expression whereas the other compounds did not. Hydroxyl groups located in the A-5 and A-7 positions were essential for the inhibitoryactivity, which along with cytotoxicity, was significantly influenced by adding a third hydroxyl group.

Effects of protease-treated royal jelly on muscle strength in elderly nursing home residents: A randomized, double-blind, placebo-controlled, dose-response study.

Although we have found that protease-treated royal jelly (pRJ) benefit for the skeletal muscle mass and strength in the aged animals, the potential beneficial effects have not been evaluated in humans. The aim of this study was to determine whether pRJ intake had beneficial effects on muscle strength in elderly nursing home residents. One hundred and ninety-four subjects enrolled into this multicenter, randomized, double-blind, placebo-controlled study. Subjects received either placebo(Group 1), pRJ 1.2 g/d(Group 2), or 4.8 g/d(Group 3). Data through 1 year are reported for 163 subjects. The primary outcome measure is handgrip strength. Secondary outcomes include several physical performance tests (six-minute walk test, timed up and go test, and standing on one leg with eyes closed). The dropout rate was 16.0%. The means (95% confidence interval) of change in handgrip strength for placebo, low-dose, and high-dose groups are -0.98(-2.04,0.08), 0.50(-0.65,1.65) and 1.03(-0.37,2.44) kg (P = 0.06, P for trend = 0.02), respectively. No significant effects of the interventions were observed for physical performances. These findings suggest that pRJ treatment might not improve, but rather attenuate the progression of decrease in muscle strength in elderly people. In addition, we have not found that pRJ intervention can achieve improvement or attenuating the decrease in physical performance.

Effect of AgK114 on picryl chloride-induced chronic contact hypersensitivity responses.

BACKGROUND: Mouse AgK114 (a glycosylphosphatidylinositol (GPI) anchored membrane-associated protein) expression is found in somatotrophs of the pituitary gland in correlation with the expression of growth hormone. In this study, the effects of AgK114 on the systemic immune response were examined in contact hypersensitivity (CHS) model mice. MATERIALS AND METHODS: AgK114 was intraperitoneally injected into BALB/c mice that were sensitized and challenged with picryl chloride (PiCl). Serum IgE levels and the antigen-specific cytokine production by lymph node (LN) cells were examined. RESULTS: The serum IgE levels in the CHS mice treated with 10 microg/head of AgK114 during the repeated challenge with PiCl were significantly decreased compared with those of the control mice. Moreover, IL-4 production by LN cells in response to 2,4,6-trinitrobenzene-sulfonic acid sodium salt-treated splenocytes was decreased in the AgK114-treated CHS mice compared with that of the control mice. CONCLUSION: Our results suggest that systemic administration of AgK114 exerted immunoregulatory functions on the allergic responses, resulting in the inhibition of IgE production.

mAgK114 suppresses lymphocyte infiltration into epidermis in the picryl chloride-induced atopic dermatitis-like skin lesions of NC/Nga mice.

BACKGROUND: We have previously shown that when mouse AgK114 (mAgK114, a glycosylphosphatidylinositol anchored membrane-associated protein) is applied to the wound area, the inflammatory responses in the early recovery phase of damaged tissue are enhanced and wound closure is accelerated. This suggests that mAgK114 has an important effect on skin wound repairing. MATERIALS AND METHODS: Whether mAgK114 supresses the development, in NC/Nga mice, of atopic dermatitis (AD)-like skin lesions induced by repeated application of 2,4,6-trinitrochlorobenzene (picryl chloride, PiCl) was examined under specific pathogen-free conditions. RESULTS: Histopathologically, the application of mAgK114-ointment to the PiCl-treated NC/Nga mice remarkably suppressed severe lymphocytic infiltration into the epidermis, although total skin severity scores, histological changes in hypertrophy, erosion and infiltration of inflammatory cells into the corium and subcutaneous tissues were comparable between the mAgK114-treated group of mice and the control group. CONCLUSION: Our results suggest that mAgK114 would be beneficial for the treatment of atopic dermatitis by suppressing severe lymphocytic infiltration into the epidermis.

Effects of a diet containing Brazilian propolis on lipopolysaccharide-induced increases in plasma plasminogen activator inhibitor-1 levels in mice.

BACKGROUND: Brazilian propolis has many biological activities including the ability to help prevent thrombotic diseases, but this particular effect has not been proven. Plasma levels of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, increase under inflammatory conditions such as infection, obesity and atherosclerosis and such elevated levels predispose individuals to a risk of developing thrombotic diseases. AIM: This study aimed to determine the effects of a diet containing Brazilian propolis on lipopolysaccharide (LPS)-induced increases in plasma PAI-1 levels. MATERIALS AND METHODS: Mice were fed with a diet containing 0.5% (w/w) Brazilian propolis for 8 weeks. Thereafter, the mice were subcutaneously injected with saline containing 0.015 mg/kg of LPS and sacrificed 4 h later. RESULTS: Orally administered Brazilian propolis significantly suppressed the LPS-induced increase in PAI-1 antigen and its activity in mouse plasma. CONCLUSION: This study indicated that Brazilian propolis contains natural products that can decrease thrombotic tendencies in mice.

Trans-Resveratrol Enhances the Anticoagulant Activity of Warfarin in a Mouse Model.

AIM: Resveratrol is a popular ingredient in dietary supplements. Some patients concomitantly use dietary supplements and medicines in Japan. In the present study, we determined whether trans-resveratrol and melinjo (Gnetum gnemon L.) seed extract (MSE), which contains resveratrol dimers, interacted with drugs using a mouse model. METHODS: Male C57BL/6J mice were fed experimental diets containing 0.005%, 0.05%, or 0.5% (w/w) trans-resveratrol or MSE for 1 or 12 weeks. The expression of liver cytochrome P-450 (CYP) mRNA and activity of liver microsomal CYP were measured. To determine the influence of resveratrol or MSE on drug efficacy, the anticoagulant activity of warfarin was examined in mice that were fed diets containing trans-resveratrol or MSE for 12 weeks. RESULTS: When the mice were fed experimental diets for 1 week, none of the doses of trans-resveratrol and MSE affected body weight, liver weight, or plasma AST and ALT levels. Trans-resveratrol also did not affect CYP1A1, CYP1A2, CYP2C, or CYP3A activities. In contrast, 0.5% MSE slightly increased CYP1A1 activity. When the mice were fed experimental diets for 12 weeks, 0.05% trans-resveratrol increased CYP1A1, CYP2C, and CYP3A activities, whereas 0.5% MSE suppressed CYP3A activity. Under these conditions, 0.5% trans-resveratrol enhanced the anticoagulant activity of warfarin, although CYP2C activity increased. However, MSE did not affect the anticoagulant activity of warfarin. CONCLUSION: The 0.05% trans-resveratrol did not interact with warfarin in a mouse model, whereas 0.5% trans-resveratrol may have enhanced the anticoagulant activity of warfarin.

Distribution of a novel protein AgK114 expression in the normal tissues of adult mice: dual expression of AgK114 and growth hormone in anterior pituitary cells.

The novel antigen K114 (AgK114) has been previously identified in normal hamster skin, and its expression has been up-regulated accompanying tissue damages of the skin, although there is no information on its biological functions. To determine the physiological role of AgK114, we prepared anti-mouse AgK114 monoclonal antibody and studied its tissue distribution in healthy adult mice by immunocytochemistry. A widespread and unique expression of AgK114 peptide was found in the selected organs of various systems (hair follicle cells and sebaceous gland of skin, ciliated epithelial cells of trachea and bronchial tube, striated portion of submandibular gland, distal convoluted tubule cells of kidney, ciliated epithelial cells of oviduct, medulla of adrenal gland and anterior lobe of pituitary gland). Interestingly, dual expression of AgK114 peptide and growth hormone in somatotrophs was found in anterior lobe of pituitary gland by double immunocytochemistry. AgK114 peptide was expressed widely in many regionally well-defined cellular systems in various peripheral tissues, suggesting that AgK114 peptide may have some roles of physiological functions in these organs. The data from our current study have provided a rationale for further studies of functional roles of AgK114 peptide in a variety of organs or tissues under physiological conditions.

Safety assessment of melinjo (Gnetum gnemon L.) seed extract: acute and subchronic toxicity studies.

Melinjo (Gnetum gnemon L.) is widely cultivated in Southeast Asia. Its fruit and seeds are common ingredients in Indonesian foods. The seeds are very rich in resveratrol dimers such as gnetin C and its glucosides, gnemonoside A and gnemonoside D, and also contain trans-resveratrol and its glucoside, trans-piceid. The safety of melinjo seeds is assured, since people in Southeast Asia have consumed them for a long time; however, their safety has not been scientifically verified. In this study, the safety of melinjo seed extract (MSE) powder was assessed in an acute oral toxicity study, a 4-week repeated dose toxicity study, and in a micronucleus test in rats. In the acute and subchronic toxicity studies, the group administered the powder did not show any toxicologically significant MSE-related changes, compared with the control group. The no observed adverse effect level (NOAEL) was determined as 1000 mg/kg/day. A genotoxicity test (rat bone marrow micronucleus test) was negative for MSE powder at levels up to 4000 mg/kg/day. These results might provide supportive evidence of safety of melinjo seeds, which has been used as food ingredients for a long time.

Pharmacokinetics and safety of resveratrol derivatives in humans after oral administration of melinjo (Gnetum gnemon L.) seed extract powder.

Fruits and seeds of melinjo (Gnetum gnemon L.) are resveratrol derivative-rich materials. Pharmacokinetics of resveratrol derivatives in healthy volunteers after oral administration of 1000 mg of melinjo seed extract (MSE) powder were assessed and compared with those after oral dosing of trans-resveratrol (tRV) powder containing 4.8 mg of tRV only, equivalent to the content in 1000 mg MSE powder. Plasma tRV concentrations with enzymatic hydrolysis were maintained over 24 h, with a tmax of 12 h and a mean residence time (MRT) of 14 h, 5 and 2 times higher than those for tRV powder intake, respectively. Gnetin C, a resveratrol dimer, with hydrolysis was maintained in plasma for >96 h with a 36 h MRT. With repeated doses once daily for 28 days, plasma tRV and gnetin C concentrations with hydrolysis were in good agreement with the theoretical curves. MSE powder was well tolerated up to the oral dosing of 5000 mg with no serious adverse events.