Design, Synthesis and Biological Evaluation of Novel N-Alkyl-4-Methyl-2,2- Dioxo-1H-2λ6,1-Benzothiazine-3-Carboxamides as Promising Analgesics.

INTRODUCTION: An analysis of the literature on the painkillers long used in traditional medicine, which are isolated from plant materials, has shown that many of them are alkylamides of various carboxylic acids. This fact served as the basis for the study of a large group of N-alkyl-4- methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamides as potential new analgesics. The objects of the study were synthesized in the traditional way involving the initial conversion of 4-methyl- 2,2-dioxo-1H-2λ6,1- benzothiazine-3-carboxylic acid to imidazolide, in which imidazolide was used as an acylating agent. The method is simple to implement and, as a rule, gives high yields of final alkylamides. However, in reaction with sterically hindered tert-butylamine, along with the "normal" product, an unexpected formation of N-tert-butyl-4-methyl-1-(4-methyl-2,2-dioxo-1H-2λ6,1- benzothiazine-3-carbonyl)-2,2-dioxo-2λ6,1-benzothiazine-3-carboxamide was observed, which was characterized by X-ray diffraction analysis as a monosolvate with N,N-dimethylformamide. These synthetic problems can be avoided using a more powerful acylating agent, 4-methyl-2,2-dioxo-1H- 2λ6,1- benzothiazine-3-carbonyl chloride. BACKGROUND: A large group of new N-alkyl-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3- carboxamides was synthesized. OBJECTIVE: On the basis of molecular docking, some derivatives of N-alkyl-4-methyl-2,2-dioxo-1H- 2λ6,1-benzothiazine-3-carboxamides have been designed. Their preliminary structure-activity relationships (SAR) have been studied. The most rational approaches to the synthesis of lead compounds have been developed. The most active compounds have shown high anti-inflammatory and analgesic activities. METHODS: The structure of all compounds prepared has been confirmed by the data of elemental analysis, 1H- and 13C NMR spectroscopy, and electrospray ionization liquid chromato-mass spectrometry. For rational drug design, optimization of further pharmacological screening and prediction of a possible mechanism of pharmacological action, molecular docking has been performed. For the determination of activity, pharmacological studies have been carried out. RESULTS: Pharmacological tests have determined that the transition from N-aryl(heteroaryl) alkylamides to "pure" N-alkylamides we carried out is accompanied by a significant reduction and even complete loss of anti-inflammatory effect with remaining analgesic activity. CONCLUSION: According to the studies, compounds from N-alkyl-4-methyl-2,2-dioxo-1H-2λ6,1- benzothiazine-3-carboxamides are potential anti-inflammatory and analgesic agents.

Novel pararosaniline based optical sensor for the determination of sulfite in food extracts.

Sulfite, which is a protective agent in various food industries, also is known as an allergen. Therefore, sulfite content in food must be monitored and controlled. In this context, a novel optical sensor is designed for simple, rapid and sensitive determination of the sulfite content in food samples. Acidified pararosaniline (PRA) hydrochloride reagent in cationic form was immobilized on the surface of the Nafion cation exchanger membrane by electrostatic interactions. In formaldehyde medium, the pale purple PRA-Nafion film was converted to rich purple due to the highly conjugated alkyl amino sulfonic acid formation in the presence of sulfite and the absorbance change at 588 nm was recorded. The proposed optical sensor gave a linear response in a wide concentration range for sulfite. The limit of detection (LOD) and the limit of quantification (LOQ) values obtained for sulfite were 0.084 and 0.280 ppm SO2 equivalent, respectively. The proposed optical sensor was validated in terms of linearity, additivity, precision and recovery parameters. The sulfite contents obtained for real food extracts were found to be comparable to the conventional iodometric titration results (with the exception of highly colored samples containing reducing agents, where iodometry was shown to exhibit a systematic error while the proposed sensor could measure the true value). The proposed optical sensor is insensitive to positive interferences from turbidity and colored components of the sample. Sulfite determination in a complex food matrix can be performed using the rapid, simple and sensitive PRA-based sensor without a need for pre-treatment.

Smart wound dressing based on κ-carrageenan/locust bean gum/cranberry extract for monitoring bacterial infections.

A smart wound dressing based on carrageenan (κC), locust bean gum (LBG), and cranberry extract (CB) for monitoring bacterial wound infections was developed and characterized using UV-vis spectroscopy, FT-IR, and SEM. The mechanical, swelling, cytotoxic and pH sensor properties were also investigated. UV-vis spectra demonstrated that the obtained κC:LBG:CB hydrogel film exhibited a visible change of colors as it was immersed in PBS solution pH 5.0, 7.3 and 9.0. The spectra of FT-IR suggested that chemical interactions had occurred between κC and CB extract. The obtained κC:LBG:CB hydrogel film exhibited adequate mechanical properties and a swelling behavior dependent on pH. Cytotoxicity tests indicated that κC:LBG:CB hydrogel film had dose-dependent cytotoxicity against NIH 3T3 fibroblast cells. The in vitro studies using Staphylococcus aureus and Pseudomonas aeruginosa demonstrated that the color changes of the κC:LBG:CB hydrogel film could be observed by naked eyes, confirming the potential use of the obtained hydrogel film as a visual system for monitoring bacterial wound infections.

Diketopyrrolopyrrole-Triphenylamine Organic Nanoparticles as Multifunctional Reagents for Photoacoustic Imaging-Guided Photodynamic/Photothermal Synergistic Tumor Therapy.

Herein, a donor-acceptor-donor (D-A-D) structured small molecule (DPP-TPA) is designed and synthesized for photoacoustic imaging (PAI) guided photodynamic/photothermal synergistic therapy. In the diketopyrrolopyrrole (DPP) molecule, a thiophene group is contained to increase the intersystem crossing (ISC) ability through the heavy atom effect. Simultaneously, triphenylamine (TPA) is introduced for bathochromic shift absorption as well as charge transport capacity enhancement. After formation of nanoparticles (NPs, ∼76 nm) by reprecipitation, the absorption of DPP-TPA NPs further displays obvious bathochromic-shift with the maximum absorption peak at 660 nm. What's more, the NPs architecture enhances the D-A-D structure, which greatly increases the charge transport capacity and impels the charge to generate heat by light. DPP-TPA NPs present high photothermal conversion efficiency (η = 34.5%) and excellent singlet oxygen (1O2) generation (ΦΔ = 33.6%) under 660 nm laser irradiation. PAI, with high spatial resolution and deep biotissue penetration, indicates DPP-TPA NPs can rapidly target the tumor sites within 2 h by the enhanced permeability and retention (EPR) effect. Importantly, DPP-TPA NPs could effectively hinder the tumor growth by photodynamic/photothermal synergistic therapy in vivo even at a low dosage (0.2 mg/kg) upon laser irradiation (660 nm 1.0 W/cm2). This study illuminates the photothermal conversion mechanism of small organic NPs and demonstrates the promising application of DPP-TPA NPs in PAI guided phototherapy.

Apple cider vinegar modulates serum lipid profile, erythrocyte, kidney, and liver membrane oxidative stress in ovariectomized mice fed high cholesterol.

The purpose of this study was to investigate the potentially beneficial effects of apple cider vinegar (ACV) supplementation on serum triglycerides, total cholesterol, liver and kidney membrane lipid peroxidation, and antioxidant levels in ovariectomized (OVX) mice fed high cholesterol. Four groups of ten female mice were treated as follows: Group I received no treatment and was used as control. Group II was OVX mice. Group III received ACV intragastrically (0.6% of feed), and group IV was OVX and was treated with ACV as described for group III. The treatment was continued for 28 days, during which the mice were fed a high-cholesterol diet. The lipid peroxidation levels in erythrocyte, liver and kidney, triglycerides, total, and VLDL cholesterol levels in serum were higher in the OVX group than in groups III and IV. The levels of vitamin E in liver, the kidney and erythrocyte glutathione peroxidase (GSH-Px), and erythrocyte-reduced glutathione (GSH) were decreased in group II. The GSH-Px, vitamin C, E, and ß-carotene, and the erythrocyte GSH and GSH-Px values were higher in kidney of groups III and IV, but in liver the vitamin E and ß-carotene concentrations were decreased. In conclusion, ACV induced a protective effect against erythrocyte, kidney, and liver oxidative injury, and lowered the serum lipid levels in mice fed high cholesterol, suggesting that it possesses oxidative stress scavenging effects, inhibits lipid peroxidation, and increases the levels of antioxidant enzymes and vitamin.

Estrogenic effects of fusarielins in human breast cancer cell lines.

The fusarielins are a group of metabolites found in several Aspergillus and Fusarium species that have been reported to have with weak antifungal, antibiotic and cytotoxic effects. This study identifies fusarielin A, F, G and H isolated from Fusarium as mycoestrogens. Mycoestrogens are compounds from fungi that bind to the estrogen receptors and induce an estrogenic response in targeted cells. All four tested fusarielins stimulate MCF-7 cell proliferation with fusarielin H as the most potent, able to stimulate cell proliferation 4-fold in a resazurin metabolism assay at 25µM. MDA-MB-231 cells without the estrogen receptor-α and MCF-10a cells without estrogen receptors were not stimulated by fusarielins. Furthermore, the stimulation was prevented in MCF-7 cells when fusarielins were incubated in the presence of the estrogen receptor antagonist fulvestrant. These observations suggest that fusarielins bind to the estrogen receptor and act as weak mycoestrogens.

Novel molecular combination deriving from natural aminoacids and polyphenols: Design, synthesis and free-radical scavenging activities.

Following the recent output of scientific publications in the matter of synergic activity between different antioxidants, we have undertaken the present study with the aim to synthesize new molecules with radical-scavengers activity based on the conjugation of bioactive portions (i.e. phenols, cysteine, methionine or tyrosine), characterized by different structures and mechanisms of action, to promote the simultaneous quenching of different radical species in the site of the oxidative damage. In this context, derivatives of phenolic acid, aminoacids and dopamine have been also prepared. The newly synthesized compounds were evaluated in vitro applying specific and complementary antioxidant test such as DPPH assay and ORAC test. As emerged from the evaluation, prerequisites for the activity of the synthesized molecules were: i) the maintenance of at least two hydroxylic groups on the aromatic moiety of phenolic portion, ii) the presence of a spacer between the aromatic moiety and the carbonilic group.

Glutathione-dependent reductive stress triggers mitochondrial oxidation and cytotoxicity.

To investigate the effects of the predominant nonprotein thiol, glutathione (GSH), on redox homeostasis, we employed complementary pharmacological and genetic strategies to determine the consequences of both loss- and gain-of-function GSH content in vitro. We monitored the redox events in the cytosol and mitochondria using reduction-oxidation sensitive green fluorescent protein (roGFP) probes and the level of reduced/oxidized thioredoxins (Trxs). Either H(2)O(2) or the Trx reductase inhibitor 1-chloro-2,4-dinitrobenzene (DNCB), in embryonic rat heart (H9c2) cells, evoked 8 or 50 mV more oxidizing glutathione redox potential, E(hc) (GSSG/2GSH), respectively. In contrast, N-acetyl-L-cysteine (NAC) treatment in H9c2 cells, or overexpression of either the glutamate cysteine ligase (GCL) catalytic subunit (GCLC) or GCL modifier subunit (GCLM) in human embryonic kidney 293 T (HEK293T) cells, led to 3- to 4-fold increase of GSH and caused 7 or 12 mV more reducing E(hc), respectively. This condition paradoxically increased the level of mitochondrial oxidation, as demonstrated by redox shifts in mitochondrial roGFP and Trx2. Lastly, either NAC treatment (EC(50) 4 mM) or either GCLC or GCLM overexpression exhibited increased cytotoxicity and the susceptibility to the more reducing milieu was achieved at decreased levels of ROS. Taken together, our findings reveal a novel mechanism by which GSH-dependent reductive stress triggers mitochondrial oxidation and cytotoxicity.

Structure-based library design in efficient discovery of novel inhibitors.

Structure-based library design employs both structure-based drug design (SBDD) and combinatorial library design. Combinatorial library design concepts have evolved over the past decade, and this chapter covers several novel aspects of structure-based library design together with successful case studies in the anti-viral drug design HCV target area. Discussions include reagent selections, diversity library designs, virtual screening, scoring/ranking, and post-docking pose filtering, in addition to the considerations of chemistry synthesis. Validation criteria for a successful design include an X-ray co-crystal complex structure, in vitro biological data, and the number of compounds to be made, and these are addressed in this chapter as well.

Aluminium-induced DNA damage and adaptive response to genotoxic stress in plant cells are mediated through reactive oxygen intermediates.

Experiments employing growing root cells of Allium cepa were conducted with a view to elucidate the role of reactive oxygen intermediates (ROI) in aluminium (Al)-induced DNA damage, cell death and adaptive response to genotoxic challenge imposed by ethyl methanesulphonate (EMS) or methyl mercuric chloride (MMCl). In a first set of experiments, root cells in planta were treated with Al at high concentrations (200-800 microM) for 3 h without or with pre-treatments of dihydroxybenzene disulphonic acid (Tiron) and dimethylthiourea (DMTU) for 2 h that trap O(2)(.-)and hydrogen peroxide (H(2)O(2)), respectively. At the end of treatments, generation of O(2)(.-) and H(2)O(2), cell death and DNA damage were determined. In a second set of experiments, root cells in planta were conditioned by Al at low concentrations (5 or 10 microM) for 2 h and after a 2 h intertreatment interval challenged by MMCl or EMS for 3 h without or with a pre-treatment of Tiron or DMTU. Conditioning treatments, in addition, included two oxidative agents viz rose bengal and H(2)O(2) for comparison. Following treatments, root cells in planta were allowed to recover in tap water. Genotoxicity and DNA damage were evaluated by micronucleus (MN), chromosome aberration (CA) or spindle aberration (SA) and comet assays at different hours (0-30 h) of recovery. The results demonstrated that whereas Al at high concentrations induced DNA damage and cell death, in low concentrations induced adaptive response conferring genomic protection from genotoxic challenge imposed by MMCl, EMS and Al. Pre-treatments of Tiron and DMTU prevented Al-induced DNA damage, cell death, as well as genotoxic adaptation to MMCl and EMS, significantly. The findings underscored the biphasic (hormetic) mode of action of Al that at high doses induced DNA damage and at low non-toxic doses conferred genomic protection, both of which were mediated through ROI but perhaps involving different networks.

Xylanase production by Penicillium canescens on soya oil cake in solid-state fermentation.

There is an increasing interest for the organic residues from various sectors of agriculture and industries over the past few decades. Their application in the field of fermentation technology has resulted in the production of bulk chemicals and value-added products such as amino acid, enzymes, mushroom, organic acids, single-cell protein, biologically active secondary metabolites, etc. (Ramachandran et al., Bioresource Technology 98:2000-2009, 2007). In this work, the production of extracellular xylanase by the fungus Penicillium canescens was investigated in solid-state fermentation using five agro-industrial substrates (soya oil cake, soya meal, wheat bran, whole wheat bran, and pulp beet). The best substrate was the soya oil cake. In order to optimize the production, the most effective cultivation conditions were investigated in Erlenmeyer flasks and in plastic bags with 5 and 100 g of soya oil cake, respectively. The initial moisture content, initial pH, and temperature of the culture affected the xylanase synthesis. The optimal fermentation medium was composed by soya oil cake crushed to 5 mm supplemented with 3% and 4% (w/w) of casein peptone and Na(2)HPO(4) x 2H(2)O. After 7 days of incubation at 30 degrees C and under 80% of initial moisture, a xylanase production level of 18,895 +/- 778 U/g (Erlenmeyer flasks) and 9,300 +/- 589 U/g (plastic bags) was reached. The partially purified enzyme recovered by ammonium sulfate fractionation was completely stable at freezing and refrigeration temperatures up to 6 months and reasonably stable at room temperature for more than 3 months.

Effects of shear stress on intracellular calcium change and histamine release in rat basophilic leukemia (RBL-2H3) cells.

Massage, one form of physical therapy, is widely used for a large number of musculoskeletal disorders, but its exact mechanism still remains to be elucidated. One hypothesis is that the shear stress caused by massage may induce cutaneous mast cells to release histamine, thereby improving the local tissue microcirculation of blood. In the present work, a mast cell line (rat basophilic leukemia cells, RBL-2H3) was used in vitro to study cellular responses to the stimulus of shear stress generated by a rotating rotor in a cell dish. The intracellular calcium ([Ca2+]c) was studied by confocal fluorescence microscopy with Fluo-3/AM staining and the released histamine was measured with a fluorescence spectrometer using o-phthalaldehyde (OPA) staining. An elevation of [Ca2+]c occurred immediately after the shear stress, followed by histamine release. However, both [Ca2+]c increase and histamine release disappeared when a Ca2+-free saline was used, indicating that the rise in the [Ca2+]c is due to a Ca2+ influx from the extracellular buffer. Furthermore, Ruthenium red, a transient receptor potential vanilloid (TRPV) inhibitor, could effectively block the shear stressinduced histamine release, suggesting that TRPV membrane proteins are the likely targets of the shear stress. Because histamine is a well-known mediator of microvascular tissue dilation, these results may have an important impact on understanding the mechanism involved in massage therapy.

An orally effective dihydropyrimidone (DHPM) analogue induces apoptosis-like cell death in clinical isolates of Leishmania donovani overexpressing pteridine reductase 1.

The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis. The enzyme pteridine reductase 1 (PTR1) of L. donovani acts as a metabolic bypass for drugs targeting dihydrofolate reductase (DHFR); therefore, for successful antifolate chemotherapy to be developed against Leishmania, it must target both enzyme activities. Leishmania cells overexpressing PTR1 tagged at the N-terminal with green fluorescent protein were established to screen for proprietary dihydropyrimidone (DHPM) derivatives of DHFR specificity synthesised in our laboratory. A cell-permeable molecule with impressive antileishmanial in vitro and in vivo oral activity was identified. Structure activity relationship based on homology model drawn on our recombinant enzyme established the highly selective inhibition of the enzyme by this analogue. It was seen that the leishmanicidal effect of this analogue is triggered by programmed cell death mediated by the loss of plasma membrane integrity as detected by binding of annexin V and propidium iodide (PI), loss of mitochondrial membrane potential culminating in cell cycle arrest at the sub-G0/G1 phase and oligonucleosomal DNA fragmentation. Hence, this DHPM analogue [(4-fluoro-phenyl)-6-methyl-2-thioxo-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylic acid ethyl ester] is a potent antileishmanial agent that merits further pharmacological investigation.

A newly isolated Paecilomyces sp. WSH-L07 for laccase production: isolation, identification, and production enhancement by complex inducement.

Laccase can catalyze the oxidation of a wide range of organic and inorganic substrates. In this study, an easily detectable method was employed for screening laccase-producing microorganisms by using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as laccase-secretion indicator. A novel laccase-producing strain was isolated and identified as Paecilomyces sp. WSH-L07 according to the morphological characteristics and the comparison of internal transcribed spacer (ITS) ribosomal DNA (rDNA) gene sequences. In further investigation, the production of laccase by Paecilomyces sp. WSH-L07 was greatly enhanced by the nontoxic inducers of copper sulphate and methylene blue. Under the induction of 50 microM copper sulphate and 20 microM methylene blue, the maximum laccase production was obtained. When these inducers were added into cultivation medium at 24 h and 12 h, respectively, an increment of about 100 times of laccase activity compared with that of in inducer-free medium and about two times of that of in single copper-supplemented medium was observed. Compared with other Paecilomyces species, Paecilomyces sp. WSH-L07 exhibit the better laccase-producing characteristics with an activity of 1,650 U/l on the eighth day, suggesting its potential ability for industrial application.

Kinetics and thermodynamics of basic dye sorption on phosphoric acid esterifying soybean hull with solid phase preparation technique.

In this paper, the solid phase preparation method of a cationic sorbent, which bears hydroxyl groups of phosphoric acid derived from esterified soybean hull (ESH), was reported. The sorption kinetics and thermodynamics of two basic dyes, acridine orange (AO) and malachite green (MG), from aqueous solution onto ESH were investigated with a batch system. The isothermal data of dye sorptions followed the Langmuir model better than the Freundlich model. The maximum sorption capacity (Q(m)) of ESH for AO and MG was 238.1 mg/g and 178.57 mg/g, respectively. The dye sorption processes could be described by the pseudo-second-order kinetic model. The thermodynamic study indicated that the dye sorptions were spontaneous and exothermic. Lower temperatures were favorable for the sorption processes.

A study on medicinal plants from Malaysia focused on Acalypha siamensis Oliv. ex Gage. isolation and structure of a new tetraterpene, acalyphaser A.

As a part of our chemical studies on Malaysian medicinal plants, five Malaysian plant species were evaluated by cytotoxicity assays using P388 murine leukemia cells. Since Acalypha siamensis exhibited the strongest growth inhibition, its constituents were studied as the object of search for bioactive materials. A novel tetraterpene, acalyphaser A (1), was isolated in the course of the purification. Its structure was elucidated on the basis of 1D- and 2D-NMR techniques, and mass spectrometry.

[Study of local immunostimulating effect of the use of different pharmaceutical forms of the remedy Traumeel S in treatment of inflammatory parodontal diseases].

In order to optimize the way of the remedy "Traumeel S" administration there was studied by the luminol-depending chemiluminescent method immunostimulating effect of its different pharmaceutical forms on functional status of gingival blood granulocytes of patients with inflammatory periodontal diseases. The immunostimulating effect of Traumeel S depended upon only the inflammatory periodontal disease severity and not the ways of its introduction. But the use of the preparation in the pellet form gave some additional advantage: simplicity of use, painlessness, dosage accuracy and possibility not to attend doctor.

Carboxy-terminal truncations modify the outer pore vestibule of muscle chloride channels.

Mammalian ClC-type chloride channels have large cytoplasmic carboxy-terminal domains whose function is still insufficiently understood. We investigated the role of the distal part of the carboxy-terminus of the muscle isoform ClC-1 by constructing and functionally evaluating two truncation mutants, R894X and K875X. Truncated channels exhibit normal unitary conductances and anion selectivities but altered apparent anion binding affinities in the open and in the closed state. Since voltage-dependent gating is strictly coupled to ion permeation in ClC-1 channels, the changed pore properties result in different fast and slow gating. Full length and truncated channels also differed in methanethiosulphonate (MTS) modification rate constants of an engineered cysteine at position 231 near the selectivity filter. Our data demonstrate that the carboxy-terminus of ClC channels modifies the conformation of the outer pore vestibule.

Development of a new hybrid gel phantom using carrageenan and gellan gum for visualizing three-dimensional temperature distribution during hyperthermia and radiofrequency ablation.

We developed a new hybrid gel phantom using carrageenan and gellan gum for the purpose of visualizing three-dimensional temperature distribution. The phantom, which contains carrageenan, gellan gum, non-ionic surface active agent, potassium chloride, n-butanol, sodium azide, and water, shows good transparency at room temperature, and has the advantage that the heated region becomes white and opaque due to segregation of the surface active agent. Carrageenan and gellan gum were added to improve the transparency and fragility of the hybrid gel. Potassium chloride was used to adjust the electrical conductivity of the gel to a range of 5-130 MHz, so that it would be equivalent to that of muscle tissue for each frequency used by electromagnetic heating devices. N-butanol was used to adjust the clouding temperature to a range between 45 and 55 degrees C. In the present study we clarified the important properties of the new phantom, and developed formulae for easy determination of the amounts of ingredients necessary for the desired clouding temperature and electric conductivity. The characteristics of this phantom are: a) a solid form to avoid convection by heat conduction; b) sufficient strength without fragility to form a torso without the use of a reinforcing agent; c) high transparency at room temperature and visualization of the heating area as a white turbidity; d) time-lapse and accurate visualization of the changing temperature area without thermal hysteresis; e) electrical properties similar to those of human tissues; f) ease of production; and g) low cost and good safety. This phantom might assist oncologists in their routine checking and study of the performance of electromagnetic heating devices for hyperthermia and radiofrequency ablation.

Antioxidative ability in a linoleic acid oxidation system and scavenging abilities against active oxygen species of enzymatic hydrolysates from pollen Cistus ladaniferus.

The enzymatic hydrolysates from pollen Cistus ladaniferus were digested and prepared using three kinds of enzymes (pepsin, trypsin, and papain) and the antioxidative properties were investigated. The yields, total phenolic contents, and protein contents of these hydrolysates were as follows: yields (about 21-45%), total phenolics (10.39-14.33 microg/mg sample powder), and proteins (129.62-137.35 microg/mg sample powder), respectively. The hydrolysates possessed strongly antioxidative and scavenging abilities against reactive oxygen species. The present studies revealed that hydrolysates from honeybee-collected pollen are of benefit not only to the materials of health food diets, but also to patients with various diseases such as cancer, cardiovascular diseases, and diabetes.