Shigenori Kumazawa

ANTIOXIDANT ACTIVITY OF PROPOLIS OF VARIOUS GEOGRAPHIC ORIGINS

Abstract Propolis is a resinous substance collected by honeybees from various plant sources. The antioxidant activities of propolis of various geographic origins, i.e., Argentina, Australia, Brazil, Bulgaria, Chile, China (Hebei, Hubei, and Zhejiang), Hungary, New Zealand, South Africa, Thailand, Ukraine, Uruguay, United States, and Uzbekistan were compared. Ethanol extracts of propolis (EEP) were prepared and evaluated for antioxidant activities of EEP samples by the β-carotene bleaching and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay systems. Major constituents were identified in EEP by HPLC analysis with photo-diode array (PDA) and mass spectrometric (MS) detection, and quantitatively analyzed. EEP from Argentina, Australia, China, Hungary and New Zealand had relatively strong antioxidant activities, and were also correlated with the total polyphenol and flavonoid contents. Propolis with strong antioxidant activity contained antioxidative compounds such as kaempferol and phenethyl caffeate.

Antioxidant activity and constituents of propolis collected in various areas of China

Abstract Propolis is a resinous substance collected by honeybees from various plant sources. The composition of propolis depends on time, vegetation, and the area of collection. This study examined the antioxidant activity of propolis from various areas of China: Heilongjiang, Neimongol, Hebei, Shandong, Shanxi, Gansu, Henan, Hubei, Sichuan, Hunan, Yunnan and Hainan. Ethanol extracts of propolis (EEP) were prepared and evaluated for their antioxidant activities by β-carotene bleaching, 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging, and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation decolorization assays. Furthermore, the major constituents in EEP were identified by high-performance liquid chromatography (HPLC) analysis with a photodiode array (PDA) and mass spectrometric (MS) detection, and each component was quantitatively analyzed. All propolis samples except that from Yunnan had relatively strong antioxidant activity accompanied by high total polyphenol contents. Propolis with strong antioxidant activity contained large amounts of antioxidative compounds, such as caffeic acid, ferulic acid and caffeic acid phenethyl ester. On the other hand, propolis from Yunnan and Hainan had compounds not present in propolis from other areas.

Covalent modification of proteins by green tea polyphenol (-)-epigallocatechin-3-gallate through autoxidation.

Green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) has various beneficial properties including chemopreventive, anticarcinogenic, and antioxidant actions. The interaction with proteins known as EGCG-binding targets may be related to the anticancer effects. However, the binding mechanisms for this activity remain poorly understood. Using mass spectrometry and chemical detection methods, we found that EGCG forms covalent adducts with cysteinyl thiol residues in proteins through autoxidation. To investigate the functional modulation caused by binding of EGCG, we examined the interaction between EGCG and a thiol enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Concentration-dependent covalent binding of EGCG to GAPDH was found to be coupled to the irreversible inhibition of GAPDH activity. Mutation experiments revealed that EGCG is primarily bound to the cysteinyl thiol group of the active center, indicating that the irreversible inhibition of GAPDH is due to the covalent attachment of EGCG to the active-center cysteine. Moreover, using EGCG-treated cancer cells, we identified GAPDH as a target of EGCG covalent binding through specific interactions between catechols and aminophenyl boronate agarose resin. Based on these findings, we propose that the covalent modification of proteins by EGCG may be a novel pathway related to the biological activity of EGCG.

Correlation between antiangiogenic activity and antioxidant activity of various components from propolis

Propolis possesses various physiological activities. In this study, we examined the antiangiogenic and antioxidant activities of various components from propolis: acacetin, apigenin, artepillin C, caffeic acid phenethyl ester, chrysin, p-coumaric acid, galangin, kaempferol, pinocembrin, and quercetin. The effects of these components were tested on in vitro models of angiogenesis, tube formation and growth of human umbilical vein endothelial cells (HUVECs). Furthermore, these components were evaluated for their antioxidant activities by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging and ferric reducing/antioxidant power (FRAP) assays. Two propolis components, caffeic acid phenethyl ester, and quercetin, possessed strong inhibitory effects on tube formation and on endothelial cell proliferation and, coincidentally, showed strong antioxidant activity. Artepillin C, galangin, and kaempferol also possessed strong antiangiogenic and antioxidant activities to a slightly less degree. In contrast, acacetin, apigenin, and pinocembrin possessed a considerable degree of antiangiogenic activities, although they showed very low antioxidant activities. From these results, we propose that components from propolis such as artepillin C, caffeic acid phenethyl ester, galangin, kaempferol, and quercetin might represent a new class of dietary-derived antioxidative compounds with antiangiogenic activities. These propolis components may have the potential to be developed into pharmaceutical drugs for the treatment of angiogenesis-dependent human diseases such as tumors.

Effects of External Factors on the Interaction of Tea Catechins with Lipid Bilayers

Green tea contains a high concentration of such catechins as (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECg), and (−)-epigallocatechin gallate (EGCg). Their biological activities have been evaluated by in vitro experiments using cultured cells or bacteria, but the order of activity of the various catechins differed with the study. We have been studying the interaction of tea catechins with lipid bilayers, and clarified that the number of hydroxyl groups on the B-ring, the presence of the galloyl moiety, and the stereochemical structure of each catechin govern their affinity for lipid bilayers. We investigated in this present study the effects of various external factors on the affinity of tea catechins for lipid bilayers by using liposomes as model membranes. The amount of tea catechins incorporated into the lipid bilayers increased with increasing salt concentration in an aqueous medium and decreased with increasing negative electric charge of the lipid bilayers. Furthermore, the amount of EGCg or ECg incorporated into the lipid bilayers increased with increasing EC concentration. These results reveal that the salt concentration in an aqueous medium, the electric charge of the membrane, and the presence of other catechins governed the affinity of tea catechins for the lipid bilayers.

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. Copyright

Direct evidence of interaction of a green tea polyphenol, epigallocatechin gallate, with lipid bilayers by solid-state Nuclear Magnetic Resonance.

The interaction of a tea catechin, epigallocatechin gallate (EGCg), with the model membrane of dimyristoylphosphatidylcholine (DMPC) was studied by solid-state 31P and 2H NMR. The 31P chemical shift anisotropy of the DMPC phosphate group decreased on addition of EGCg. The 2H NMR spectrum of [4-2H]EGCg, which is deuterated at the 4-position, in the DMPC liposomes gave deuterium nuclei with much smaller quadrupole splittings than those in the solid phase. These 31P and 2H NMR observations provide direct experimental evidence that the EGCg molecule interacts with the lipid bilayers.

A new prenylated flavonoid from propolis collected in Okinawa, Japan

The new prenylflavonoid, isonymphaeol-B (1), together with three known compounds, nymphaeol-A (2), nymphaeol-B (3), and nymphaeol-C (4), were isolated from propolis collected in Okinawa, the southern-most prefecture of Japan. The structure of each compound was determined by spectral methods, including mass spectrometry and 2D NMR. Each compound had 1,1-diphenyl-2-picryl-hydrazyl radical-scavenging activity.

Distribution and excretion of bilberry anthocyanines in mice.

The physiology and tissue distribution of bilberry anthocyanins were studied in mice. After oral administration of bilberry extract (100 mg/kg body weight), both unmodified and methylated anthocyanins appeared in the plasma. The plasma concentration of total anthocyanins reached a maximum of 1.18 +/- 0.3 microM after 15 min and then sharply decreased. Their urinary excretion was highest between 0 and 6 h after administration and had ceased by 24 h. The total quantities of bilberry anthocyanins excreted into urine represented 1.88% (range, 0.62% to 2.45%) of consumed anthocyanins. Thirteen anthocyanins were identified in bilberry extracts. Of these, malvidin-3-glucoside and -3-galactoside were the principal anthocyanins in the plasma 60 min after administration. When mice were maintained for 2 weeks on a diet containing 0.5% of bilberry extracts, the plasma concentration of anthocyanins reached a maximum of 0.26 muM. Anthocyanins were detected only in the liver, kidney, testes, and lung, with maximum tissue concentrations of 605, 207, 149, and 116 pmol/g, respectively. In these organs, malvidin-3-glucoside and -3-galactoside were the predominant anthocyanins. Anthocyanins were not detectable in the spleen, thymus, heart, muscle, brain, white fat, or eyes. We conclude that bilberry anthocyanins were absorbed into the body and distributed in specific organs, particularly the liver, kidney, and testis. The most common anthocyanins in tissues were malvidin glycosides.

Fingerprinting of propolis by easy ambient sonic-spray ionization mass spectrometry.

Chemical profiles of a representative set of 49 propolis ethanolic extracts collected worldwide (North and South America, Europe, Asia and Oceania) were obtained via easy ambient sonic-spray ionization mass spectrometry (EASI-MS). This simple and easily implemented fingerprinting technique analyses directly (without any pre-separation or sample manipulation) a tiny droplet of the ethanolic extract placed on a inert surface under ambient conditions. Data acquisition took about a minute per sample with no substantial sample carry-over. Extraction of propolis with ethanol by using an ultrasonic bath method gave EASI-MS data similar to the traditional maceration method, reducing total analysis time for the crude propolis resin from a week to just ca 1h. Principal component analysis of the EASI-MS data is shown to group samples according to the plant sources of their resins, which characterizes their geographical origin.