Chemical composition and biological activity of propolis from Brazilian meliponinae

Twenty-one propolis samples produced by 12 different Meliponinae species were analyzed by GC-MS. Several chemical types of stingless bees' propolis could be grouped, according to the prevailing type of compounds like: 'gallic acid", "diterpenic" and "triterpenic" types. The results confirm that neither the bee species nor the geographical location determine the chemical composition of Meliponinae propolis and the choice of its plant source, respectively. This could be explained by the fact that Meliponinae forage over short distances (maximum 500 m) and thus use as propolis source the first plant exudate they encounter during their flights. The antibacterial, antifungal and cytotoxic activities of the samples were also investigated. Most samples had weak or no activity against E. coli, weak action against Candida albicans. Some of them showed significant activity against St. aureus., presumably connected to the high concentration of diterpenic acids. Samples rich in diterpenic acids possessed also high cytotoxic activity (Artemia salina test).     

Evaluation of phenolic compounds in Brazilian propolis from different geographic regions

Chemometrics has been shown quite efficient to uncover relationships between chemical composition of a sample and its geographical origin. Forty propolis samples originated from the the South and South East of Brazil were analyzed by HPLC and 18 compounds of interest were studied which included: caffeic, p-coumaric and ferulic acids, and some of their derivatives, pinobanksin, a derivative of kaempferol and five phenolic compounds (assigned as 3-prenyl4-hydroxycinnamic acid (PHCA); 2,2-dimethyl-6-carboxyethnyl-2H-1-benzopyran (DCBE); 3,5-diprenyl-4-hydroxycinnamic acid (DHCA); compound E (still unknown) and 6-propenoic-2,2-dimethyl-8-prenyl-2H-1-benzopyran acid (DPB). Principal Component Analysis (PCA) indicated three different groups of propolis samples, having the same typical chromatogram, evaluated by HPLC. Samples from the South East group were rich in derivatives of kaempferol. Samples from the South group I had a high content of DPB compound, but a low concentration of kaempferol derivatives and of DCBEN compound. Samples from the South group II were characterized by a high concentration of DCBEN, DHCA, p-coumaric and DPB compounds. Therefore, the identification of new compounds in Brazilian propolis can give, useful information about the plant sources of a given geographic region.     

Isolation, identification, synthesis and biological activity of volatile compounds from the heads of Atta ants

S-(+)-4-methyl-3-heptanone has been identified as the principal alarm pheromone of Atta texana and Atta cephalotes. Both enantiomers of 4-methyl-3-heptanone have been synthesized and their biological activities have been compared on both species of ants. Comparison of the geometric averages of response ratios, at threshold concentration levels on A. texana, showed S-(+)-4-methyl-3-heptanone to be about 100 times more active than the (?) enantiomer. A similar analysis also showed no inhibition of the activity of S-(+)-4-methyl-3-heptanone by the (?) enantiomer. A less rigorous study on A. cephalotes showed S-(+)-4-methyl-3-heptanone to be about 210 times more active than R-(?)-4-methyl-3-heptanone.Both ant species produce 3-octanone, possible trace amounts of 3-octanol, and both diastereomers of 4-methyl-3-heptanol. A. texana also produces (+)-2-heptanol, 2-heptanone, and 3-heptanol. A. cephalotes contains trace amounts of 2-heptanone.     

Identification of a naturally occurring retinoid X receptor agonist from Brazilian green propolis

Background

Brazilian green propolis (BGP), a resinous substance produced from Baccharis dracunculifolia by Africanized honey bees (Apis mellifera), is used as a folk medicine. Our present study explores the retinoid X receptor (RXR) agonistic activity of BGP and the identification of an RXR agonist in its extract.

Methods

RXRα agonistic activity was evaluated using a luciferase reporter gene assay. Isolation of the RXRα agonist from the ethanolic extract of BGP was performed using successive silica gel and a reversed phase column chromatography. The interaction between the isolated RXRα agonist and RXRα protein was predicted by a receptor–ligand docking simulation. The nuclear receptor (NR) cofactor assay was used to estimate whether the isolated RXRα agonist bound to various NRs, including RXRs and peroxisome proliferator-activated receptors (PPARs). We further examined its effect on adipogenesis in 3T3-L1 fibroblasts.

Results

We identified drupanin as an RXRα agonist with an EC₅₀ value of 4.8 ± 1.0 μM. Drupanin activated three RXR subtypes by a similar amount and activated PPARγ moderately. Additionally, drupanin induced adipogenesis and elevated aP2 mRNA levels in 3T3-L1 fibroblasts.

Conclusions

Drupanin, a component of BGP, is a novel RXR agonist with slight PPARγ agonistic activity.

General significance

This study revealed for the first time that BGP activates RXR and drupanin is an RXR agonist in its extract.     

鹅源鸭疫里默氏杆菌的分离、鉴定与生物学特性研究

【目的】从疑似鸭疫里默氏杆菌病的病死雏鹅分离病原菌进行鉴定。【方法】根据细菌培养特性、生化特性、动物试验、血清型鉴定及分子生物学特性对分离菌株进行鉴定。【结果】分离菌株为革兰氏阴性菌,不发酵糖类和醇类,尿素酶试验和氧化酶还原试验为阳性,致病,不同分离株的16S rRNA基因经多重序列比对分析,结果显示鹅源分离株与鸭源鸭疫里默氏杆菌处于同一进化支上,与鸡源鸭疫里默氏杆菌进化关系稍远,血清型鉴定为1型。【结论】分离菌株为血清1型的鸭疫里默氏杆菌,对鸭和鹅均有高致病性,自家疫苗能够较好地保护雏鹅。     

Distribution of quinic acid derivatives and other phenolic compounds in Brazilian propolis

The quinic acid derivatives (including 4-feruoyl quinic and 5-ferruoyl quinic acids characterized for first time in propolis samples) and other phenolic compounds were quantified in thirteen Brazilian propolis samples by HPLC analysis. For chemometrical analysis, the distribution of quinic acid derivatives and other phenolic compounds were considered. The results suggest that the Brazilian propolis with floral origin from Citrus sp. have the highest concentration of the quinic acid derivatives (between 11.0 to 58.4 mg/mg of the dried crude hydroalcoholic extract) and therefore would probably show a more effective hepatoprotective activity.     

Isolation, identification, and biological evaluation of Nrf2-ARE activator from the leaves of green perilla (Perilla frutescens var. crispa f. viridis)

The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is a cellular defense system against oxidative stress. Activation of this pathway increases expression of antioxidant enzymes. Epidemiological studies have demonstrated that the consumption of fruits and vegetables is associated with reduced risk of contracting a variety of human diseases. The aim of this study is to find Nrf2-ARE activators in dietary fruits and vegetables. We first attempted to compare the potency of ARE activation in six fruit and six vegetables extracts. Green perilla (Perilla frutescens var. crispa f. viridis) extract exhibited high ARE activity. We isolated the active fraction from green perilla extract through bioactivity-guided fractionation. Based on nuclear magnetic resonance and mass spectrometric analysis, the active ingredient responsible for the ARE activity was identified as 2',3'-dihydroxy-4',6'-dimethoxychalcone (DDC). DDC induced the expression of antioxidant enzymes, such as γ-glutamylcysteine synthetase (γ-GCS), NAD(P)H: quinone oxidoreductase-1 (NQO1), and heme oxygenase-1. DDC inhibited the formation of intracellular reactive oxygen species and the cytotoxicity induced by 6-hydroxydopamine. Inhibition of the p38 mitogen-activated protein kinase pathway abolished ARE activation, the induction of γ-GCS and NQO1, and the cytoprotective effect brought about by DDC. Thus, this study demonstrated that DDC contained in green perilla enhanced cellular resistance to oxidative damage through activation of the Nrf2-ARE pathway.     

蛹虫草子实体活性成分的分离鉴定

蛹虫草具有悠久的食药用历史,但关于其活性成分的种类仍知甚少。本文采用大孔树脂、硅胶柱、HPLC等色谱技术对蛹虫草Cordyceps militaris子实体粗提物进行分离纯化,从中得到3个化合物。通过波谱数据分析,化合物1–3分别被鉴定为：虫草素（3’-脱氧腺苷）（1）,色氨酸（2）和5,5’-dibuthoxy-2,2’-bifuran（3）,杂环化合物3为首次从蛹虫草子实体中获得。对化合物进行抗菌活性测定发现,虫草素和5,5’-dibuthoxy-2,2’-bifuran对枯草芽孢杆菌和大肠杆菌都有显著的抑菌活性,但是它们对酿酒酵母均未表现出明显抑菌活性。     

Isolation,identification and biological activity of chlamydosporol from Fusarium culmorum HM-8

An isolate of Fusarium culmorum (W.G. Smith), Sacc. HM-8 from a scabby wheat kernel sample from England produced a novel toxin when grown in culture on rice. This toxin, which was given the trivial name of HM-8, was purified, its toxic properties demonstrated and its structure determined by spectroscopic methods. Subsequent to preliminary reports of our findings, two other laboratories have independently isolated the same substance from F. chlamydosporum and F. acuminatum, and demonstrated the same structure by spectroscopic methods and X-ray crystallography. Identity of the metabolite from F. culmorum HM-8 with the published structure is based on (1) identical elemental composition derived from both elemental analysis and high resolution mass spectrometry in electron impact and fast atom bombardment modes, (2) comparable melting point and chromatographic properties, and (3) the presence of characteristic absorption bands in the infrared, ultraviolet, and nuclear magnetic resonance (proton and carbon-13) spectra. Because both laboratories which subsequently isolated this metabolite have used the name chlamydosporol, it is being adopted here. The mass spectral properties of chlamydosporol are described. The derivative 8-O-acetylchlamydosporol was prepared and characterized. Chlamydosporol caused food refusal and weight loss in rats, cytotoxic effects to cultured mouse and human fibroblast cells at concentrations of 2.5 g/ml and 7.5 g/ml, respectively, and mortality to chick embryos (10 to 70%) over a concentration range from 0.5 mg to 4 mg per egg.     

Isolation and identification of root-inhibiting compounds from corn gluten hydrolysate

Interest has centered on the use of plantderived compounds as natural herbicides, and they are considered to represent an environmentally sound approach to weed control. Corn gluten hydrolysate, found to have a growth-regulating effect on the root system of germinating grass seeds, has been suggested as a natural herbicide. A protocol was developed to extract, isolate, and identify the root-inhibiting compounds from corn gluten hydrolysate aqueous solution and a perennial ryegrass (Lolium perenne L.). A Petri dish bioassay was used to test the root-inhibiting activity. Five bioactive dipeptides were isolated by using Sephadex G-15 gel filtration, solid-phase extraction, and C18 reversed-phase high-performance liquid chromatography procedures. The five dipeptides were glutaminyl-glutamine, alaninyl-asparagine, alaninyl-glutamine, glycinyl-alanine, and alaninyl-alanine. Their root-inhibiting activity on perennial ryegrass was demonstrated in Petri dish bioassays.Journal Paper No. J-15597 of Iowa Agriculture and Home Economics Experiment Station, Ames, IA Project No. 3149     

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.     

大宝山矿区耐Cd2+细菌的分离鉴定及其生物学特性

【目的】从Cd2+污染土壤中分离获取强耐Cd2+微生物,研究其生物学特性。【方法】采用选择性培养基TSA对广东省大宝山重金属污染土壤中耐镉(Cd2+)细菌进行分离筛选,结合细菌形态观察、生理生化检测、16S rRNA基因序列分析等方法确定了它们的分类地位,并开展了细胞形态、Cd2+耐受性、Cd2+吸附性、对不同pH和盐度适应性、以及对抗生素的抗性等生物学特性的研究。【结果】从土壤中分离到8株具有强耐Cd2+能力的细菌(YN-6、YN-7、YN-8、YN-9、YN-16、YN-17、YN-18、YN-19),初步鉴定其分别属于Rhizobium、Roseateles、Cupriavidus、Methylobacterium、Variovorax、Rhizobium、Achromobacter和Leifsonia属。细菌生物学特性研究表明,8株耐Cd2+细菌的形态存在一定的差异,其中YN-6、YN-8、YN-9、YN-16、YN-17和YN-18菌体为短杆状,而YN-7和YN-19菌体为长杆状。它们对Cd2+的最低抑制浓度均在3 mmol/L以上,且每克细胞吸附1 mg以上的Cd2+。其中,菌株YN-8对Cd2+的耐受性最强(9 mmol/L),菌株YN-9对Cd2+的吸附能力最强(316.7 mg Cd2+/g cells)。不同pH、盐度和抗生素对8株细菌生长的影响存在较大差异。【结论】菌株YN-8和YN-9对环境的适应性较强,可以作为较好的试验材料应用于生物修复重金属Cd2+污染土壤的研究。     

鸡源奇异变形杆菌分离鉴定及生物学特性

【背景】奇异变形杆菌(Proteusmirabilis,PM)是人畜共患病原菌,在自然界分布广泛。近年来,随着畜禽奇异变形杆菌病发病率上升和耐药性增强,亟需开展对该菌的防控研究。【目的】分离鉴定鸡源奇异变形杆菌,鉴定其耐药性、致病性、生物被膜形成能力等生物学特性。【方法】采用聚合酶链式反应(Polymerase Chain Reaction,PCR)方法鉴定了从2019-2020年病鸡中分离的52株临床奇异变形杆菌。分别采用药敏试验、PCR、结晶紫染色法对临床菌株的耐药性、毒力基因及生物膜的形成进行研究。【结果】选择14株代表性菌株进行16SrRNA基因测序,结果表明所测分离菌株均为奇异变形杆菌。所有分离菌株均对克林霉素、阿奇霉素、红霉素、四环素和利福平耐药,对氯霉素和头孢曲松外的抗生素耐药性均高于50%。对分离的奇异变形杆菌的13个毒力基因检测表明,所有菌株均能检测到hpmA、hpmB、rpoA、mrpA、fliL、zapA、ureC、atfC、atfA、pmfA,而ucaA和rsbA检出率分别为19.23%(10/52)和48.08%(25/52),hlyA未检出。对分离株生物被膜形成能力检测结果表明,所有菌株均能形成生物被膜,19.23%(10/52)的分离菌株形成生物被膜能力强,而且25℃条件下成膜能力比37℃更强。【结论】鸡源奇异变形杆菌携带多种毒力基因,具有较强生物被膜形成能力,耐药模式多样且日趋严重,应进一步加强对奇异变形杆菌在动物致病性和耐药性方面的监控,以降低其感染风险。     

Cytotoxic constituents from Brazilian red propolis and their structure-activity relationship

Several classes of flavonoids [flavanoids (1-10), flavonol (11), isoflavones (12-18), isoflavanones (19-22), isoflavans (23-26), chalcones (27-30), auronol (31), pterocarpans (32-37), 2-arylbenzofuran (38), and neoflavonoid (39)] and lignans (40-42) isolated from the MeOH extract of Brazilian red propolis were investigated for their cytotoxic activity against a panel of six different cancer cell lines including murine colon 26-L5 carcinoma, murine B16-BL6 melanoma, murine Lewis lung carcinoma, human lung A549 adenocarcinoma, human cervix HeLa adenocarcinoma, and human HT-1080 fibrosarcoma cell lines. Based on the observed results, structure-activity relationships were discussed. Among the tested compounds, 7-hydroxy-6-methoxyflavanone (3) exhibited the most potent activity against B16-BL6 (IC(50), 6.66microM), LLC (IC(50), 9.29microM), A549 (IC(50), 8.63microM), and HT-1080 (IC(50), 7.94microM) cancer cell lines, and mucronulatol (26) against LLC (IC(50), 8.38microM) and A549 (IC(50), 9.9microM) cancer cell lines. These activity data were comparable to those of the clinically used anticancer drugs, 5-fluorouracil and doxorubicin, against the tested cell lines, suggesting that 3 and 26 are the good candidates for future anticancer drug development.     

嗜水气单胞菌RB5-M1的分离鉴定及脱色条件研究

【目的】筛选具有高效脱色活性黑5能力的菌株。【方法】利用梯度浓度驯化法,从上海松江污水处理厂的活性污泥中分离获得了一株具有良好脱色能力的细菌RB5-M1。【结果】经形态学观察、生理生化特性鉴定以及16S rRNA基因序列分析,发现该菌株与嗜水气单胞菌(Aeromonas hydrophila)的同源性达到99.86%。【结论】菌株RB5-M1在35°C、pH 8.0的条件下,厌氧条件培养(氮气85%、二氧化碳6%)24 h,测定的平均脱色率可达94.1%,最高脱色率为99.8%。     

HPTLC-MS as an efficient hyphenated technique for the rapid identification of antimicrobial compounds from propolis

The well-known anti-infective properties of propolis are determined by its chemical composition, which in turn is influenced by geographical factors and reflects the botanical diversity in the vicinity of the beehive. Although there are several reports on the anti-infective properties of crude propolis, few are aimed at identifying specific compound(s) responsible for the observed activities. Using South African propolis as an example, the application of high performance thin layer chromatography-bioautography in tandem with mass spectrometry was investigated for the rapid identification of antimicrobial and anti-quorum sensing (anti-QS) compounds. Pinocembrin was found to be responsible for the observed antifungal activity of the propolis against Candida albicans. Three compounds were found to be active against all of the evaluated Gram-positive and Gram-negative bacteria. The identity of the first was confirmed as pinobanksin, one remains unidentified, while the third corresponds to either pinobanksin 3-O-pentanoate or 2-methylbutyrate. The identification of caffeic acid as the anti-QS component was confirmed quantitatively using the violacein inhibitory assay.     

麂子源蜂房哈夫尼亚菌的分离鉴定及生物学特性

【背景】蜂房哈夫尼亚菌是革兰阴性杆菌,是一种机会致病菌、腐生菌,常见于人和动物肠道、污水、土壤和乳制品中,能引起人和动物败血症,而且具有潜在的致腹泻作用。【目的】为对昆明轿子雪山自然保护区内死亡麂子体内潜在的致病菌进行分离鉴定及生物学特性分析。【方法】无菌采集死亡麂子的部分肠道组织进行细菌的分离培养和鉴定,并对分离获得的菌株进行药物敏感性试验及动物回归试验。【结果】鉴定分离菌株为蜂房哈夫尼亚菌,编号KMJZXS0312。药敏试验结果表明,该菌对青霉素、头孢噻吩等7种抗生素耐药,对氟苯尼考、卡那霉素、呋喃唑酮、阿莫西林中介,对恩诺沙星、复方新诺明等13种抗生素敏感。动物回归试验表明,该菌能致小鼠死亡,引起小鼠胃和肠道胀气,肠道薄而透亮,肝脏点状出血,肺脏有针尖大小出血点,肝脏病理切片显示,肝细胞轻度水样变性,肝细胞肿胀,胞质疏松淡染。【结论】本实验从麂子肠组织分离到一株具有致病性的蜂房哈夫尼亚菌,对其致病机制进行了分析,并进行了药物敏感试验,提供了菌株新的生物学信息,具有重要的公共卫生学意义。     

Hepatoprotective and anti-Helicobacter pylori activities of constituents from Brazilian propolis.

Propolis is a resinous hive product collected by honeybees from various plant sources. It is extensively used in food, beverage and in folk medicine for treating various ailments and reported to have broad spectrum of biological activities. The hepatoprotective activity of propolis and constituents from its MeOH extract belonging to various classes were tested on D-galactosamine (D-GalN)/tumor necrosis factor-alpha (TNF-alpha)-induced cell death in primary cultured mouse hepatocytes. The result indicated that hepatoprotective activity of alcoholic extract of tropical Brazilian propolis is mainly due to phenolic compounds including flavonoids. All the four isolated flavonoids possessed stronger inhibitory activity (IC50, < 25 microM) than silibinin (IC50, 39.6 microM) on TNF-alpha-induced cell death. The labdane-type diterpenes isolated from the MeOH extract also exhibited significant hepatoprotective activity in the same experimental model. Moreover, the labdane-type diterpenes and some of the prenylated phenolic compounds possessed antibacterial activity against Helicobacter pylori.     

Isolation,structural identification and biological characterization of two conopeptides from the Conus pennaceus venom

A novel anti-mollusk conopeptide pn4c was isolated from the Conus pennaceus venom by repeated HPLC fractionation based on the activity against freshwater snails. The primary structure of pn4c was determined by the mass spectrometric de novo sequencing analysis. In addition, pn3a was isolated from the same fraction containing pn4c, as a peptide with unknown functions.