Hidehiko Beppu

Determination of aloenin, barbaloin and isobarbaloin in Aloe species by micellar electrokinetic chromatography

Aloenin, barbaloin and isobarbaloin in JP Aloe, Aloe barbadensis (Aloe vera) and Aloe arborescens Miller var. natalensis Berger (Aloe arborescens Miller) were determined by micellar electrokinetic chromatography (MEKC) with 50 mM sodium dodecyl sulfate. Aloenin, barbaloin and isobarbaloin were well separated by MEKC and as little as 5.5 pg/11 nl of the three compounds could be detected. The determination took around 14 min.

Agaritine purified from Agaricus blazei Murrill exerts anti-tumor activity against leukemic cells.

BACKGROUND Mushrooms of the genus Agaricus are a common folk remedy against carcinoma. The active ingredients, polysaccharides and protein-polysaccharide complexes containing beta-glucan, have been isolated and shown to have indirect tumor-suppressing activity via an immunological activation. METHODS The diffusible fraction of a hot-water extract of Agaricus blazei Murrill (ABM) powder was fractionated by HPLC based on the anti-tumor activity against leukemic cells in vitro. The structure of the anti-tumor substance was determined by NMR and MS analyses. RESULTS We purified a tumorcidal substance from the diffusible fraction of ABM and identified it as agaritine, beta-N-(gamma-l(+)-glutamyl)-4-(hydroxymethyl) phenylhydrazine, having a molecular mass of 267 Da. This compound inhibited the proliferation of leukemic cell lines such as U937, MOLT4, HL60 and K562 with IC(50) values of 2.7, 9.4, 13.0, and 16.0 microg/mL, respectively, but showed no significant effect on normal lymphatic cells at concentrations up to 40 microg/mL. Although agaritine has been suspected of having genotoxic or carcinogenic properties, agaritine did not activate the umu gene of Salmonella, which reacts to carcinogens. GENERAL SIGNIFICANCE The results indicate that agaritine from ABM has direct anti-tumor activity against leukemic tumor cells in vitro. This is in contrast to the carcinogenic activity previously ascribed to this compound. Our results also show that this activity is distinct from that of beta-glucan, which indirectly suppresses proliferation of tumor cells.

Agaritine from Agaricus blazei Murrill induces apoptosis in the leukemic cell line U937.

BACKGROUND Agaricus blazei Murrill (ABM) has been shown to exhibit immunostimulatory and anti-cancer activities; however, its mechanism of action is poorly understood. We recently found that the diffusible fraction of hot-water extract of ABM exhibits anti-tumor activity toward leukemic cells, and identified it as agaritine, a hydrazine-containing compound. In the present study, we examined the morphological and cytochemical effects of agaritine on U937 cells to elucidate the tumoricidal mechanism of agaritine. METHODS Surface expression of phosphatidylserine (evaluated by annexin V binding), Fas antigen, DNA cleavage using TUNEL staining, changes in caspase activities and cytochrome c release, before and after treatment with agaritine, were examined using U937 cells. RESULTS Nuclear damage, DNA fragmentation, was observed by Wright-Giemsa, TUNEL staining and agarose gel electrophoresis when U937 cells were incubated with 10μg/mL of agaritine for 48h. Flow cytometric analysis indicated that agaritine augments the proportion of annexin V-positive U937 cells without significant change in Fas antigen expression. Activities of caspase-3, -8 and -9 were gradually increased after the addition of agaritine. In the presence of caspase-3 or granzyme B inhibitor, except for the caspase-8 inhibitor, annexin V expression was significantly decreased, suggesting that mainly caspase-3 and -9 participate in the apoptotic pathway. Furthermore, cytochrome c release was detected by western blotting analysis after agaritine treatment. CONCLUSIONS These results strongly suggest that the ABM constituent agaritine moderately induces apoptosis in U937 leukemic cells via caspase activation through cytochrome c release from mitochondria. GENERAL SIGNIFICANCE This is the first report suggesting that the anti-tumor effect of agaritine is mediated through apoptosis. The present results might provide helpful suggestions for the design of anti-tumor drugs toward leukemia patients.

Chemopreventive effects of Aloe arborescens on N-nitrosobis(2-oxopropyl)amine-induced pancreatic carcinogenesis in hamsters

The modification effects of freeze-dried aloe (Aloe arborescens) whole leaf powder during the initiation phase of carcinogenesis were investigated in hamsters treated with N-nitrosobis(2-oxopropyl)amine (BOP). Female Syrian hamsters were given four weekly subcutaneous injections of BOP at a dose of 10mg/kg and then given 0, 1 or 5% aloe in their diet for 5 weeks. At week 54 of the experiment, all surviving animals were sacrificed and development of neoplastic and preneoplastic lesions was assessed histopathologically. The incidences of pancreatic adenocarcinomas, atypical hyperplasias or total atypical hyperplasias plus adenocarcinomas were significantly (P<0.05) decreased with BOP+5% aloe, and that of adenocarcinomas were also significantly (P<0.05) reduced in the BOP+1% aloe as compared to the BOP alone group. Multiplicities of pancreatic adenocarcinomas, atypical hyperplasias or total lesions were also significantly (P<0.01 or P<0.05) lower in the BOP+5% aloe group than with the BOP alone. Quantitative data for neoplastic lesions in the lung, liver, gall bladder, kidney and urinary bladder of hamsters were not significantly different among the three groups. In a satellite experiment, pretreatment with aloe significantly (P<0.01) reduced the formation of O6-methyldeoxyguanosine in epithelial cells of pancreatic ducts as compared to the BOP alone value. Our results thus indicate that aloe prevents BOP-induced pancreatic neoplasia in hamsters in relation to decreased DNA adduct formation in the target tissue.

In vivo effects of Aloe arborescens Miller var. natalensis Berger (Kidachi aloe) on experimental tinea pedis in guinea-pig feet

Trichophytosis was induced in guinea‐pigs and the antifungal effects of Aloe arborescens Miller var. natalensis Berger (Kidachi aloe) evaluated in comparison with lanoconazole, a commercially available antifungal agent. Trichophytosis was induced by inoculation of arthrospores of Trichophyton mentagrophytes cephalic strain SM‐110 (T. mentagrophytes SM‐110) onto the plantar part of guinea‐pig feet. Culture studies after application of 30% freeze‐dried Kidachi aloe for 10 days showed a 70% growth inhibition compared with the untreated animals.

Alteration of protein expression profile following voluntary exercise in the perilesional cortex of rats with focal cerebral infarction

Identification of functional molecules in the brain related to improvement of the degree of paralysis or increase of activities will contribute to establishing a new treatment strategy for stroke rehabilitation. Hence, protein expression changes in the cerebral cortex of rat groups with/without voluntary exercise using a running wheel after cerebral infarction were examined in this study. Motor performance measured by the accelerated rotarod test and alteration of protein expression using antibody microarray analysis comprised 725 different antibodies in the cerebral cortex adjacent to infarction area were examined. In behavioral evaluation, the mean latency until falling from the rotating rod in the group with voluntary exercise for five days was significantly longer than that in the group without voluntary exercise. In protein expression profile, fifteen proteins showed significant quantitative changes after voluntary exercise for five days compared to rats without exercise. Up-regulated proteins were involved in protein phosphorylation, stress response, cell structure and motility, DNA replication and neurogenesis (11 proteins). In contrast, down-regulated proteins were related to apoptosis, cell adhesion and proteolysis (4 proteins). Additional protein expression analysis showed that both growth-associated protein 43 (GAP43) and phosphorylated serine41 GAP43 (pSer41-GAP43) were significantly increased. These protein expression changes may be related to the underlying mechanisms of exercise-induced paralysis recovery, that is, neurite formation, and remodeling of synaptic connections may be through the interaction of NGF, calmodulin, PKC and GAP43. In the present study at least some of the participation of modulators associated with the improvement of paralysis might be detected.

Decreased levels of 2-amino-3-methylimidazo[4,5-f]quinoline-DNA adducts in rats treated with β-carotene, α-tocopherol and freeze-dried aloe

To assess mechanisms of chemoprevention of hepatocarcinogenesis by trans‐β‐carotene (β‐C), DL‐α‐tocopherol (α‐T), and freeze‐dried whole leaves of Kidachi aloe (Aloe), formation of 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ)‐DNA adducts was measured by 32P‐post‐labeling analysis, and CYP1A1 and CYP1A2 protein levels were analyzed by ELISA. Group 1 rats were fed diet containing 0.02%β‐C, 1.5%α‐T or 30% Aloe over an 8‐day period, while group 2 was given basal diet alone. On day 7, all animals were subjected to two‐thirds partial hepatectomy (PH). Twelve hours after PH, they received a single dose of the carcinogenic food pyrolysate IQ (100 mg/kg) intragastrically, to initiate hepatocarcinogenesis. Rats were killed 6, 12, 24 and 48 h after IQ administration. The levels of adducts, expressed as relative adduct labeling values in rats treated with β‐C, α‐T and Aloe, were decreased as compared with the control group at hour 24 (36 h after PH), with a significant difference in the case of the β‐C group (46.4% of the control value). Similarly, all showed a tendency for decrease at hour 48. Furthermore, the levels of CYP1A2, known to be responsible for activation of IQ, showed a significant reduction at hour 24. It is concluded that β‐C, and possibly also α‐T and Aloe, have the potential to reduce IQ‐DNA adduct formation, presumably as a result of decreased formation of active metabolites. The results may explain, at least in part, the previously observed inhibitory effects of these compounds on induction of preneoplastic hepatocellular lesions.

Analysis of protein expression profile in the cerebellum of cerebral infarction rats after treadmill training.

Mizutani K, Sonoda S, Hayashi N, Takasaki A, Beppu H, Saitoh E, Shimpo K: Analysis of protein expression profile in the cerebellum of cerebral infarction rats after treadmill training. Objective:To investigate the relation between protein expression changes in the cerebellum and improvement of motor coordination in rats with cerebral infarction. Design:The rat group with treadmill training (n = 10) were compared with the rat group without treadmill training (n = 10) after 2.5 hrs of transient middle cerebral artery occlusion. Motor performance measured by the rotarod test and alteration of protein expression using two-dimensional electrophoresis based on proteomics in the cerebellum were examined. Results:In behavioral evaluation, the mean latency until falling from the rotating rod in the group with treadmill training was significantly longer (P < 0.01) than that in the group without treadmill training 24 days after surgery. As for protein expression, it was revealed by proteome analysis and Western blotting that the expression of the two protein spots, 25-kDa synaptosomal-associated protein and glial fibrillary acidic protein, were significantly enhanced in the cerebellum of rats with treadmill training than that in rats without a treadmill training. Conclusions:The 25-kDa synaptosomal-associated protein and glial fibrillary acidic protein may be related to the underlying mechanisms of improvement of motor coordination and exercise-induced angiogenesis, that is, remodeling of synaptic connections and proliferation of astroglial cells, respectively.

Effects of Aloe-emodin and Emodin on Proliferation of the MKN45 Human Gastric Cancer Cell Line

Aloe-emodin (1, 8-dihydroxy-3-hydroxyl-methylanthraquinone; AE) and emodin (1,3,8-trihydroxy-6- methylanthraquinone; EM) are anthraquinone derivatives that have been detected in some medical plants and share similar anthraquinone structures. AE and EM have been shown to exhibit anticancer activities in various cancer cell lines; however, the inhibitory effects of these derivatives on the growth of cancer cells were previously reported to be different. Gastric cancer is the second most common cause of cancer cell death worldwide. In the present study, we examined the inhibitory effects of 0.05 mM AE and 0.05 mM EM on the proliferation of the MKN45 human gastric cancer cell line. The proliferation of MKN45 cells was significantly inhibited in AE- and EM-treated groups 24 h and 48 h after treatment. Furthermore, the inhibitory effects of EM were stronger than those of AE. The cell cycle of MKN45 cells were arrested in G0/G1 phase or G0/G1 and G2/M phases by AE and EM, respectively. However, an analysis of intracellular polyamine levels and DNA fragmentation revealed that the mechanisms underlying cell death following cell arrest induced by AE and EM differed.

Changes in Serum Growth Factors in Stroke Rehabilitation Patients and Their Relation to Hemiparesis Improvement

Predicting recovery from hemiparesis after stroke is important for rehabilitation. A few recent studies reported that the levels of some growth factors shortly after stroke were positively correlated with the clinical outcomes during the chronic phase. The aim of this study was to examine the relationships between the serum levels of growth factors (vascular endothelial growth factor [VEGF], insulin-like growth factor-I [IGF-I], and hepatocyte growth factor [HGF]) and improvement in hemiparesis in stroke patients who received rehabilitation in a postacute rehabilitation hospital. Subjects were 32 stroke patients (cerebral infarction: 21 and intracerebral hemorrhage [ICH]: 11). We measured serum levels of VEGF, IGF-I, and HGF and 5 items of the Stroke Impairment Assessment Set (SIAS) for hemiparesis on admission and at discharge. Age-matched healthy subjects (n=15) served as controls. Serum levels of VEGF and HGF in cerebral infarct patients on admission were higher than those in control subjects, and the serum levels of IGF-I in stroke patients were lower than those in controls. The level of HGF in ICH patients on admission was negatively correlated with gains in SIAS, and higher outliers in HGF concentration were correlated with lower gains in SIAS. Focusing on the extremely high levels of these factors may be a predictor of the low recovery from hemiparesis after stroke.