Brazilian propolis (AF-08) inhibits collagen-induced platelet aggregation without affecting blood coagulation.

Brazilian propolis (AF-08) is a dietary supplement containing a variety of flavonoids. It is used worldwide as a folk medicine. Flavonoids and a diet of fruits and vegetables containing them have been shown to reduce the risk of cardiovascular diseases (CVDs). Most of CVDs are caused by arterial thrombus formation. A thrombus is formed by the interaction between adhesion and aggregation of platelets to damaged blood vessels and blood coagulation consisting of extrisic and intrinsic pathways. Platelet aggregation and blood coagulation are closely linked to thrombosis. Therefore, we evaluated the effectiveness of AF-08 or its component flavonoids against thrombosis by examining their inhibition of platelet aggregation and blood coagulation. Human platelet-rich plasma was incubated with serial dilutions of AF-08 for 10 min to assess its inhibitory effect on platelet aggregation caused by collagen. The inhibitory effect of AF-08 on blood coagulation was evaluated by the prothrombin time (PT) and activated partial thromboplastin time (APTT), which reflect the coagulation function of extrinsic and intrinsic pathways, respectively. AF-08 significantly inhibited collagen-induced platelet aggregation but not PT and APTT, indicating that AF-08 inhibited platelet aggregation but not blood coagulation. Among three flavonoids contained in AF-08, apigenin and chrysin obviously inhibited platelet aggregation but the inhibitory effect of kaempferol was less effective. The three flavonoids did not affect PT and APTT. The inhibitory activity of AF-08 on human platelet aggregation without affecting blood coagulation was suggested to be partially due to apigenin and chrysin. AF-08 may be effective in suppressing platelet-based arterial thrombus formation and reducing the risk of CVDs.

Hyaluronic acid-green tea catechin conjugates as a potential therapeutic agent for rheumatoid arthritis.

Fibroblast-like synoviocytes are a key effector cell type involved in the pathogenesis of rheumatoid arthritis. The major green tea catechin, epigallocatechin-3-O-gallate (EGCG), has attracted significant interest for rheumatoid arthritis therapy because of its ability to suppress the proliferation and interleukin-6 secretion of synoviocytes. However, therapeutic efficacy of EGCG has been limited by a lack of target cell specificity. Herein we report hyaluronic acid-EGCG (HA-EGCG) conjugates as an anti-arthritic agent that is capable of targeting fibroblast-like synoviocytes via HA-CD44 interactions. These conjugates exhibited superior anti-proliferative and anti-inflammatory activities compared with EGCG under simulated physiological conditions. Near-infrared fluorescence imaging revealed preferential accumulation of the conjugates at inflamed joints in a collagen-induced arthritis rat model, and their anti-arthritic efficacy was investigated by measuring a change in the edema and histopathological scores. Our findings suggest the potential of HA-EGCG conjugates as an anti-arthritic agent for the treatment of rheumatoid arthritis.

Carrier-Enhanced Anticancer Efficacy of Sunitinib-Loaded Green Tea-Based Micellar Nanocomplex beyond Tumor-Targeted Delivery.

Although a few nanomedicines have been approved for clinical use in cancer treatment, that recognizes improved patient safety through targeted delivery, their improved efficacy over conventional drugs has remained marginal. One of the typical drawbacks of nanocarriers for cancer therapy is a low drug-loading capacity that leads to insufficient efficacy and requires an increase in dosage and/or frequency of administration, which in turn increases carrier toxicity. In contrast, elevating drug-loading would cause the risk of nanocarrier instability, resulting in low efficacy and off-target toxicity. This intractable drug-to-carrier ratio has imposed constraints on the design and development of nanocarriers. However, if the nanocarrier has intrinsic therapeutic effects, the efficacy would be synergistically augmented with less concern for the drug-to-carrier ratio. Sunitinib-loaded micellar nanocomplex (SU-MNC) was formed using poly(ethylene glycol)-conjugated epigallocatechin-3-O-gallate (PEG-EGCG) as such a carrier. SU-MNC specifically inhibited the vascular endothelial growth factor-induced proliferation of endothelial cells, exhibiting minimal cytotoxicity to normal renal cells. SU-MNC showed enhanced anticancer effects and less toxicity than SU administered orally/intravenously on human renal cell carcinoma-xenografted mice, demonstrating more efficient effects on anti-angiogenesis, apoptosis induction, and proliferation inhibition against tumors. In comparison, a conventional nanocarrier, SU-loaded polymeric micelle (SU-PM) comprised of PEG-b-poly(lactic acid) (PEG-PLA) copolymer, only reduced toxicity with no elevated efficacy, despite comparable drug-loading and tumor-targeting efficiency to SU-MNC. Improved efficacy of SU-MNC was ascribed to the carrier-drug synergies with the high-performance carrier of PEG-EGCG besides tumor-targeted delivery.

Hyaluronic acid-green tea catechin micellar nanocomplexes: Fail-safe cisplatin nanomedicine for the treatment of ovarian cancer without off-target toxicity.

The green tea catechin, (-)-epigallocatechin-3-O-gallate (EGCG), has gained significant attention as a potent adjuvant to enhance the antitumor efficacy of cisplatin while mitigating its harmful side effects. Herein we report the development of a fail-safe cisplatin nanomedicine constructed with hyaluronic acid-EGCG conjugate for ovarian cancer therapy. A simple mixing of this conjugate and cisplatin induces spontaneous self-assembly of micellar nanocomplexes having a spherical core-shell structure. The surface-exposed hyaluronic acid enables efficient delivery of cisplatin into CD44-overexpressing cancer cells via receptor-mediated endocytosis whereas the internally packed EGCG moieties offer an environment favorable for the encapsulation of cisplatin. In addition, the antioxidant effect of EGCG moieties ensures fail-safe protection against off-target organ toxicity originating from cisplatin-evoked oxidative stress. Pharmacokinetic and biodistribution studies reveal the prolonged blood circulation and preferential tumor accumulation of intravenously administered nanocomplexes. Moreover, the nanocomplexes exhibit superior antitumor efficacy over free cisplatin while displaying no toxicity in both a subcutaneous xenograft model and peritoneal metastatic model of human ovarian cancer. Our findings demonstrate proof of concept for the feasibility of green tea catechin-based micellar nanocomplexes as a safe and effective cisplatin nanomedicine for ovarian cancer treatment.

Enzymatic Analysis of Positional Fatty Acid Distributions in Triacylglycerols by 1(3)-Selective Transesterification with Candida antarctica Lipase B: a Collaborative Study.

The positional distributions of fatty acids (FAs) in fats and oils are principally analyzed by selectively transesterifying the target triacylglycerols (TAGs) at the 1(3) position using Pseudozyma (Candida) antarctica lipase, followed by recovering the resulting 2-monoacylglycerols (MAGs) by chromatography. FA compositions were measured by gas chromatography (GC) after methylating target TAGs and 2-MAGs. The method was collaboratively evaluated by 12 laboratories by analyzing the positional FA distributions in soybean, palm, and sardine oils. The maximum reproducibility relative standard deviations for the major FAs and those at the sn-2 positions of soybean, palm, and sardine oils were 4.41% and 3.92% (18:3n-3), 4.48% and 3.82% (18:0), and 8.93 and 8.24% (14:0), respectively. The values at the sn-2 position were always low. Therefore, these results indicated that the variations were mainly caused by the FA analysis procedure, i.e., the methylation and GC analyses, rather than the enzymatic transesterification and chromatography utilized to prepare 2-MAGs from the target oil.

Thirty-year follow-up of a TMD case treated based on the neuromuscular concept.

AIM: Occlusal therapy is employed to alleviate the symptoms of a temporomandibular disorder (TMD) at times. However, the long-term effect of occlusal therapy in the masticatory system is not well understood. This case study aims to present a 30-year follow-up of a TMD case. METHODOLOGY: The patient developed TMD with intermittent closed lock of the left temporomandibular joint (TMJ). Chief complaints included trismus, pain, and noise of the left TMJ during function. The patient's occlusal disharmony was assessed with use of electronic instruments and corrected based on the neuromuscular concept. A minimum-invasive and reversible approach using adhesive occlusal restorations was used. RESULTS: The jaw movement and masticatory muscle activity assessed at the 7- and 23-year follow-ups revealed that the established occlusion was well adapted, and re-established the patient's functional occlusion system. The patient has been free from TMD symptoms with the corrected occlusion for 30 years. CONCLUSIONS: Occlusal reconstruction based on the neuromuscular concept can be stably integrated into the patient's functional occlusion system.

Repressed ethylene production in the gynoecium of long-lasting flowers of the carnation 'White Candle': role of the gynoecium in carnation flower senescence.

Ethylene production and expression of ethylene biosynthetic genes was investigated in senescing flowers of carnation (Dianthus caryophyllus L.) cultivars 'White Candle (WC)' and 'Light Pink Barbara (LPB)', with long and short vase-lives, respectively. Ethylene production from the gynoecium and petals of senescing 'WC' flowers was below the limit of detection, in agreement with the repressed ethylene production from the whole flowers. However, exogenous ethylene treatment caused the accumulation of transcripts for DC-ACS1 and DC-ACO1 genes in both the gynoecium and petals, resulting in ethylene production from the flowers. Moreover, application of ABA or IAA, which are known to exhibit their action through the induction of ethylene synthesis in the gynoecium, to 'WC' flowers from their cut stem-end induced ethylene production and wilting in the flowers. These findings suggested that, in 'WC' flowers the mechanism of ethylene biosynthesis, i.e. the induction of expression of genes for ethylene biosynthesis and the action of resulting enzymes, was not defective, but that its function was repressed during natural senescence. Transcripts of DC-ACO1, DC-ACS3, and DC-ACS1 were present in the gynoecium of senescing 'LPB' flowers. In the gynoecium of senescing 'WC' flowers, however, the DC-ACO1 transcript was present, but the DC-ACS1 transcript was absent and the DC-ACS3 transcript was detected only in a small amount; the latter two were associated with the low rate of ethylene production in the gynoecium of 'WC' flowers. These findings indicated that the repressed ethylene production in 'WC' flowers during natural senescence is caused by the repressed ethylene production in the gynoecium, giving further support for the role of the gynoecium in regulating petal senescence in carnation flowers.