Structure–activity relationships of acetylated flavone glycosides from Galeopsis ladanum L. (Lamiaceae)

Antioxidant activity, neuroprotective effect and acetylcholinesterase inhibitor activity were studied in acetylated flavones from Galeopsis ladanum L. (Lamiaceae), previously isolated and identified by UV-spectra, UPLC–MS/MS and ¹H-NMR spectroscopy. Structure–activity relationships of flavonoids have been determined in many antioxidant assays, generally focused on hydroxyl groups. In this study we have detected new interesting structure–activity relationships for the isolated flavonoids due to the acetylation of sugar moiety of these flavonoids. Methylation at 4′-OH and monoacetylation is beneficial to inhibiting AChE and shows a neouroprotective effect.     

Fractionation of lipids and purification of γ-linolenic acid (GLA) from Spirulinaplatensis

The microalgae, Spirulinaplatensis, is an excellent source of γ-linolenic acid (GLA), an essential polyunsaturated fatty acid and a potent nutraceutical. The fatty acid composition of S.platensis ARM 740 was determined after transmethylation by gas chromatography (GC). Lipid fractionation was achieved on silica gel column chromatography and preparative TLC. Neutral lipids, glycolipids and phospholipids accounted for 77.0%, 15.6% and 7.4%, respectively, of the total lipid fraction. S.platensis ARM 740 was found to contain 94% of the total GLA in the glycolipid fraction. Attempts were made to purify GLA methyl ester by using urea to form inclusion complexes with the saturated and the less unsaturated FAMEs (fatty acid methyl esters), which enhanced the purity of GLA methyl ester to 84%. A further approach to isolate GLA methyl ester with higher purity involved the use of argentated silica gel chromatography. An initial PUFA concentration step frequently adopted by most researchers to increase GLA purity was not necessary in the isolation of GLA from S.platensis. A GLA methyl ester with a purity of >96% and a recovery of 66% was obtained. Purity of the isolated GLA methyl ester was confirmed by GC and IR analysis with respect to authentic standard.     

Characterisation of aroma active compounds in black truffles (Tuber melanosporum) and summer truffles (Tuber aestivum) by gas chromatography–olfactometry

The aromatic composition of two different species of truffles (black and summer) was evaluated by gas chromatography–olfactometry (GC–O). Volatiles released by the truffles at 25 °C for 7.5 h were collected in a trapping system consisting of 400 mg of LiChrolut EN kept at 0 °C and further eluted with dichloromethane/methanol (95:5). The extract was analysed by two different GC–O strategies: (1) a semiquantitative GC–O study using a panel composed of nine individuals, (three of them truffle experts) and (2) an AEDA (aroma extract dilution analysis) experiment with a small panel of two judges. The results show that the aroma emitted by a typical black truffle is due to at least 17 different aroma molecules, six of which are reported for the first time: 1-hexen-3-one, 2-methyl-3-furanthiol, furaneol, 3-ethylphenol, 3-propylphenol and 5-methyl-2-propylphenol. The most important aroma compounds of black truffle aroma are 2,3-butanedione, dimethyl disulphide (DMDS), ethyl butyrate, dimethyl sulphide (DMS), 3-methyl-1-butanol and 3-ethyl-5-methylphenol. Quantitatively, black truffle emits mostly 3-ethyl-5-methylphenol (more than 50% of the total aroma molecules emitted), 5-methyl-2-propylphenol, β-phenylethanol and 3-ethylphenol. In the case of summer truffle, the most important aroma molecules are DMS, DMDS, methional, 3-methyl-1-butanol, 1-hexen-3-one and 3-ethylphenol. From the quantitative point of view, summer truffle emits mainly β-phenylethanol, DMS and 3-ethylphenol, but the emission is up to 100 times less than that of black truffles.     

Assessment of GH1, CAPN1 and CAST polymorphisms as markers of carcass and meat traits in Bos indicus and Bos taurus–Bos indicus cross beef cattle

The objective of this study was to investigate the effects of bovine GH1, CAPN1 and CAST gene polymorphisms on carcass and meat traits in Nellore and Nellore x Bos taurus beef cattle. Three hundred animals were genotyped for GH1/MspI (TC/G in intron 3), CAPN316 (AF_252504.2:g.5709C > G) and CAST/RsaI (AY_008267.1:g282C > G) and phenotyped for rib eye area, backfat thickness, intramuscular fat, shear force (SF), and myofibrillar fragmentation index (MFI). No significant associations were observed between the GH1/MspI and CAST/RsaI polymorphisms and phenotypes, although the relation between the CAST/RsaI genotypes and meat tenderness evaluated by MFI approached significant. The fact that the CAPN316 polymorphism did not show adequate segregation in Nellore cattle confirms the difficulty of using this marker in breeding programs of different Bos indicus breeds. However, the positive results of the association analysis obtained for Nellore x B. taurus crosses contributed to the validation of previous findings.     

Short communication: Effect of αS1-casein (CSN1S1) and κ-casein (CSN3) genotypes on milk composition in Murciano-Granadina goats

The effects of the caprine α_S1-casein (CSN1S1) polymorphisms on milk quality have been widely demonstrated. However, much less is known about the consequences of the κ-casein (CSN3) genotype on milk composition in goats. Moreover, the occurrence of interactions between CSN3 and CSN1S1 genotypes has not been investigated. In this study, an association analysis between CSN1S1 and CSN3 genotypes and milk quality traits was performed in 89 Murciano-Granadina goats. Total milk yield as well as total protein, fat, solids-not-fat, lactose, α_S1-casein (CSN1S1), and α_S2-casein (CSN1S2) contents were recorded every other month during a whole lactation (316 observations). Data analysis using a linear mixed model for repeated observations revealed no interaction between the CSN1S1 and CSN3 genotypes. With regard to the effect of the CSN3 locus, AB and BB genotypes were significantly associated with higher levels of total casein and protein content compared with the AA CSN3 genotype. In strong contrast with French breeds, the CSN1S1 genotype did not affect protein, casein, and fat concentrations in Murciano-Granadina goats. These results highlight the importance of taking into consideration the CSN3 genotype when performing selection for milk composition in dairy goats.     

Antioxidant potential of solvent extracts of Kappaphycus alvarezii (Doty) Doty – An edible seaweed

Various solvent extracts of Kappaphycus alvarezii, an edible red seaweed (family Solieriaceae) were screened for total phenol content and antioxidant activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferrous ion chelating activity, reducing power and antioxidant activity assays in a linoleic acid system with ferrothiocyanate reagent (FTC). The total phenol content of different extracts of K. alvarezii varied from 0.683 ± 0.040% to 2.05 ± 0.038%. The radical-scavenging activity of ethanol extract was, as IC₅₀ 3.03 mg ml⁻¹, whereas that of the water extract was IC₅₀ 4.76 mg ml⁻¹. Good chelating activity was recorded for methanol extract (IC₅₀ 3.08 mg ml⁻¹) wherein 67.0 ± 0.924% chelation was obtained using 5.0 mg ml⁻¹ of extract. The reducing power of the samples was in the following order: BHT > methanol > ethanol > ethyl acetate > water > hexane. But, in the linoleic acid system, the ethanol extract proved superior to the synthetic antioxidants butylated hydroxytoluene (BHT). Hence, these extracts could be considered as natural antioxidants and may be useful for curing diseases arising from oxidative deterioration.     

Optimization of SC–CO2 extraction of zerumbone from Zingiber zerumbet (L) Smith

Response surface methodology (RSM) was applied to optimize the variables affecting the Supercritical carbon dioxide (SC–CO₂) extraction of non-polar compounds from Zingiber zerumbet using the Box-Behnken design (BBD). Dependent variables were the percentage of the chemical components in the ginger vis a vis α-caryophyllene (y₁), camphene (y₂), and zerumbone (2,6,10-cycloundecatrien-1-one, 2,6,9,9-tetramethyl-) (y₃). Pressure was the most significant parameter affecting the amount of each compound extracted. When temperature was kept constant and pressure was increased, all of the dependent variables increased concomitantly. Since pressure and temperature are two of the major influential factors in the extraction using SC–CO₂, any combination of these two parameters could be selected to ascertain the optimum combination for a particular compound in the extract. Extraction at 30 °C and 55 MPa with total amount of 30 g of CO₂ used was found to maximize all the responses.     

Determination of polyphenol levels variation in Capsicum annuum L. cv. Chelsea (yellow bell pepper) infected by anthracnose (Colletotrichum gloeosporioides) using liquid chromatography–tandem mass spectrometry

Healthy fruits of Capsicum annuum L. cv. Chelsea (yellow bell pepper) and one infected by Colletotrichum gloeosporioides were analysed for polyphenols via high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS). Among seven polyphenols characterized, four components in the C. annuum fruits were identified for the first time. To investigate the characteristics of the polyphenols as defence materials, the content change of the fruit polyphenols inoculated with C. gloeosporioides was monitored by HPLC. It was observed for the first time that de novo induced N-caffeoyl putrescine (1) and caffeoyl O-hexoside (2) appeared to act as a phytoalexin in the defence mechanism of the C. annuum fruits against C. gloeosporioides, and constitutively formed feruloyl O-glucoside (3), kaempferol O-pentosyldihexoside (4) and dihydroxyflavone O-hexoside (7) as a phytoanticipin in the diseased C. annuum fruits.     

Identification of buckwheat (Fagopyrum esculentum Moench) aroma compounds with GC–MS

Buckwheat has a strong characteristic aroma, but its phytochemical background has not yet been fully elucidated. The aims of this study were identification and quantification of individual compounds responsible for the buckwheat aroma. Volatiles from a freshly ground buckwheat flour were extracted by different methods: direct extraction with petroleum ether, pentane or methanol, distillation with Clevenger apparatus and a headspace solid-phase microextraction method. The extracts were analysed by GC–MS with electron ionisation. Compounds were identified by MS and by comparison of retention times with reference compounds. Direct extraction with methanol and distillation proved to be very efficient. In these extracts twenty-five and thirty-five compounds were identified, respectively. The first extract contained more hydrophilic compounds and the latter more volatile compounds. Most of the compounds were quantified and their odour activity value (OAV) calculated. Only two compounds (salicylaldehyde and phenylacetaldehyde) were found in both extracts. The compounds with the highest contribution to the buckwheat aroma were: 2,5-dimethyl-4-hydroxy-3(2H)-furanone, (E,E)-2,4-decadienal, phenylacetaldehyde, 2-methoxy-4-vinylphenol, (E)-2-nonenal, decanal, hexanal and salicylaldehyde (2-hydroxybenzaldehyde).     

Characterization of the components present in the active fractions of health gingers (Curcuma xanthorrhiza and Zingiber zerumbet) by HPLC–DAD–ESIMS

Curcuma xanthorrhiza and Zingiber zerumbet are two of the most commonly used ingredients in Indo-Malaysian traditional medicines, health supplements and tonics. Recently, a number of products derived from the aqueous extracts of these species have appeared in the market in the form of spray-dried powder packed in sachet or bottle. On-line high performance liquid chromatography, coupled with diode array detection and electrospray ion trap tandem mass spectroscopy (HPLC–DAD–ESI–MSⁿ), was used to analyze the components in the antioxidant-active fractions from the rhizomes of these species. Three components were identified from C. xanthorrhiza, including bisdemethoxycurcumin (1), demethoxycurcumin (2) and curcumin (3). The active fraction from Z. zerumbet consisted of five components, including kaempferol 3-O-rhamnoside (4), compound 5 [kaempferol 3-O-(2″-O-acetyl)rhamnoside (5a) or kaempferol 3-O-(3″-O-acetyl)rhamnoside (5b)], kaempferol 3-O-(4″-O-acetyl)rhamnoside (6), kaempferol 3-O-(3″,4″-O-diacetyl)rhamnoside (7) and kaempferol 3-O-(2″,4″-O-diacetyl)rhamnoside (8). To confirm their identities, the components from Z. zerumbet were isolated conventionally and were analyzed by spectroscopic techniques as well as by comparison with literature data.     

Effects of β-κ-casein (CSN2-CSN3) haplotypes, β-lactoglobulin (BLG) genotypes, and detailed protein composition on coagulation properties of individual milk of Simmental cows

The aim of this study was to investigate the effects of CSN2-CSN3 (β-κ-casein) haplotypes, BLG (β-lactoglobulin) genotypes, content of milk protein fractions, and protein composition on coagulation properties of milk (MCP). Rennet coagulation time (RCT) and curd firmness (a₃₀) were measured using a computerized renneting meter, and the contents of major milk protein fractions were quantified by reversed-phase HPLC in individual milk samples of 2,167 Simmental cows. Cow genotypes at CSN2, CSN3, and BLG were ascertained by reversed-phase HPLC, and CSN2-CSN3 haplotype probabilities were estimated for each cow. Phenotypes for MCP were regressed on CSN2-CSN3 haplotype probabilities using linear models that also included the effects of herd-test-day, parity, days in milk, pH, somatic cell score, renneting meter sensor, sire of the cow, BLG genotype, and content of major protein fractions or, alternatively, protein composition. When the statistical model did not account for protein fraction contents or protein composition, haplotypes carrying CSN3 B were associated with shorter RCT and greater a₃₀ compared with those carrying CSN3 A. Haplotypes carrying CSN2 B had the effect of decreasing RCT and increasing a₃₀ relative to haplotype A²A. When effects of protein fractions content or protein composition were added to the model, no difference across haplotypes due to CSN3 and CSN2 alleles was observed for MCP, with the exception of the effect of CSN2 B on RCT, which remained markedly favorable. Hence, the effect of CSN3 B on MCP is related to a variation in protein composition caused by the allele-specific expression of κ-casein, rather than to a direct role of the protein variant on the coagulation process. In addition, the favorable effect exerted by CSN2 B on a₃₀ was caused by the increased β-casein content in milk. Conversely, CSN2 B is likely to exert a direct genetic effect on RCT, which does not depend upon variation of β-casein content associated with CSN2 B. Increased RCT was observed for milk yielded by BLG BB cows, even when models accounted for protein composition. Rennet clotting time was favorably affected by κ-casein content and percentage of κ-casein to total casein, whereas a₃₀ increased when contents and percentages of β-CN and κ-CN increased. Changes of milk protein composition and allele frequency at casein and whey protein genes affect variation of MCP.     

Effects of β-κ-casein (CSN2-CSN3) haplotypes and β-lactoglobulin (BLG) genotypes on milk production traits and detailed protein composition of individual milk of Simmental cows

The aim of this study was to investigate the effects of CSN2-CSN3 (β-κ-casein) haplotypes and BLG (β-lactoglobulin) genotypes on milk production traits, content of protein fractions, and detailed protein composition of individual milk of Simmental cows. Content of the major protein fractions was measured by reversed-phase HPLC in individual milk samples of 2,167 cows. Protein composition was measured as percentage of each casein (CN) fraction to total CN and as percentage of β-lactoglobulin (β-LG) to total whey protein. Genotypes at CSN2, CSN3, and BLG were ascertained by reversed-phase HPLC, and CSN2-CSN3 haplotype probabilities were estimated for each cow. Traits were analyzed by using a linear model including the fixed effects of herd-test-day, parity, days in milk, and somatic cell score class, linear regressions on haplotype probabilities, class of BLG genotype, and the random effect of the sire of the cow. Effects of haplotypes and BLG genotypes on yields were weak or trivial. Genotype BB at BLG and haplotypes carrying CSN2 B and CSN3 B were associated with increased CN content and CN number. Haplotypes including CSN3 B were associated with increased κ-CN content and percentage of κ-CN to total CN and with decreased percentages of α_S1- and γ-CN to total CN. Allele CSN2 B had the effect of increasing β-CN content and decreasing content of α_S1-CN. Haplotypes including allele CSN2 A¹ exhibited decreased β-, α_S2-, and γ-CN concentrations and increased α_S1- and κ-CN contents, whereas CSN2 I had positive effects on β-CN concentration and trivial effects on content of other protein fractions. Effects of haplotypes on CN composition were similar to those exerted on content of CN fractions. Allele BLG A was associated with increased β-LG concentration and percentage of β-LG to total whey protein and with decreased content of other milk proteins, namely β-CN and α_S1-CN. Estimated additive genetic variance for investigated traits ranged from 14 to 39% of total variance. Increasing the frequency of specific genotypes or haplotypes by selective breeding might be an effective way to change milk protein composition.     

Acidification,crushing and thermal treatments can influence the profile and stability of folate poly-γ-glutamates in broccoli (Brassica oleracea L. var. italica)

The influence of different treatments, i.e., crushing, high temperature short time (90 °C/4 min) (HTST) and low temperature long time (60 °C/40 min) (LTLT) blanching, acidification (pH 4.3), and sequences of these treatments on the folate poly-γ-glutamate profile and stability were investigated. In this study, broccoli was used as a case study. Regarding the folate poly-γ-glutamate profile, endogenous folate poly-γ-glutamates in broccoli florets were found predominantly as hepta- and hexa-γ-glutamates. Crushing raw broccoli, acidification and LTLT blanching enhanced folate deconjugation resulting in monoglutamate, di- and tri-γ-glutamates. Compared to other treatments, HTST blanching preformed prior to crushing resulted in the highest concentration of long chain poly-γ-glutamates. Regarding folate poly-γ-glutamates stability, acidification combined with LTLT blanching decreased folate stability whereas HTST blanching combined with different sequences of blanching and crushing did not affect folate poly-γ-glutamates stability. It was concluded that crushing (prior to heating), acidification and blanching could be strategically applied to increase the folate monoglutamate content of broccoli.     

Volatiles of the Balkan endemic Daucus guttatus ssp. zahariadii and cultivated and wild-growing D. carota – A comparison study

The hydrodistilled essential oil obtained from the aerial parts of Daucus guttatus Sibth. & Sm. ssp. zahariadii Heywood, an endemic plant species of the Balkan Peninsula, as well as solvent extracts and essential oils from different parts of Daucus carota L. have been analysed by GC and GC–MS and screened for antimicrobial activity against 12 bacterial and two fungal strains. The volatiles of the two plant taxa differed significantly in both their chemical identity and antimicrobial effect. The dominant constituent of D. guttatus oil was apiol (43.3%), which was absent from all samples of D. carota. The diethyl ether extract of D. carota inhibited the growth of the yeast Candida albicans while the oil of D. guttatus at 25 mg/ml had no effect on the growth of the fungal organisms tested. Additionally, the oil of D. guttatus showed prominent antibacterial activity against a pathogenic Corynebacterium pyogenes.     

Effect of hydroxypropyl-β-cyclodextrin on the aggregation of (E)-resveratrol in different protonation states of the guest molecule

The aggregation state of (E)-resveratrol was studied in the presence and absence of hydroxypropyl-β-cyclodextrin (HP-β-CD) using absorption and steady-state fluorescence at different pH values. The results revealed that this potent antioxidant shows a monomer/aggregate equilibrium which is dependent on the protonation state of (E)-resveratrol. This equilibrium can be modified by the presence of HP-β-CD, which reduces the aggregation of the (E)-resveratrol molecules, producing individual molecules of the solute and preventing side effects due to aggregation phenomena. At pH values higher than the pK_a1 of (E)-resveratrol, the coexistence of different protonated/deprotonated forms of this antioxidant does not permit the fluorimetric determination of the complexation constant (K_F) of (E)-resveratrol with HP-β-CD. However, when the Hildebrand–Benesi equation was used to calculate this constant at physiological pH, this problem was resolved, with a K_F value of 14490 ± 723 M⁻¹ and the stoichiometry of the complexation process 1:1 for all the cases tested. The results obtained in this paper resolve the contradictory data published about the complexation process of (E)-resveratrol by cyclodextrins.     

Identification and quantification of constituents of Gardenia jasminoides Ellis (Zhizi) by HPLC-DAD–ESI–MS

A simple, rapid and specific HPLC method was carried out for the analysis of characteristic constituents in Gardenia jasminoides Ellis (Zhizi), namely iridoids, caffeoyl quinic acid derivatives and crocins. The separation was successfully obtained using a C₁₈ column by gradient elution with mixtures of methanol and water as mobile phases; detection wavelength was set at 240 nm for iridoid glycosides, 315 nm for quinic acid derivatives and 438 nm for crocins.     

α-Amylase inhibitory effect of 3β-olean-12-en-3-yl (9Z)-hexadec-9-enoate isolated from Spondias mombin leaf

Spondias mombin is a traditional herb used in the treatment of diabetes mellitus by traditional healers in southwest Nigeria. In this study, we investigated the antidiabetic activity using α-amylase inhibitory assay and isolated an active compound. The bioactivity assay-guided study demonstrated the presence of an α-amylase inhibitory fraction from S. mombin leaf. An active compound, 3β-olean-12-en-3-yl (9Z)-hexadec-9-enoate was also studied. This is reported, from this plant, for the first time. The methanol extract, diethyl ether fraction and the isolated compound exhibited significant enzyme inhibitory activity against Aspergillus oryzae α-amylase. Our study revealed, for the first time, the isolation and α-amylase inhibitory activity of 3β-olean-12-en-3-yl (9Z)-hexadec-9-enoate from S. mombin leaf.     

In vitro and in vivo assessment of cytochrome P450-mediated herb–drug interaction of Ssang-hwa-tang

We have evaluated the herb–drug interaction potential of Ssang-hwa-tang (SHT) mediated by cytochrome P450 (CYP) inhibition/induction. Further, the effects of fermentation on the CYP-mediated herb–drug interaction potential were determined. SHT showed inhibitory activity toward CYP1A2, but not 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4 in human liver microsomes. The results of the enzyme kinetic study suggested that the SHT-induced CYP1A2 inhibition is mixed reversible inhibition. The hepatic CYP expression and activity in rats treated with SHT were examined. The expression/activity of CYP2E1 increased as a result of SHT extract treatment (P < 0.005 or P < 0.001, respectively), which raises the possibility that SHT may increase the toxicity of environmental toxicants through the elevation of CYP2E1-mediated metabolic activation. SHT fermentation using Lactobacillus fermentum or Lactobacillus gasseri resulted in attenuation of the SHT-induced CYP1A2 inhibition, but not CYP2E1 induction, suggesting that changes in the chemical composition of SHT through fermentation can affect the inhibition of CYP1A2 activity.     

烟草耐黄瓜花叶病(Cucumber Mosaic Virus)育种研究

当今世界尚未发现烟草CMV抗源,据此,笔者经多年在烟草苗床和大田加压选择,育成三个高耐CMV品系:C151、C152和C212。在自然感染和田间接毒测定中,其抗性均占显著优势,平均病情指数比台湾育成的TT6、TT7低,室内接种鉴定对大陆各烟区CMV病毒株系的耐力远比TT6、TT7强,对各地CMV流行株系表现明显的抗性,达显著和极显著水平。尤其C212,其病情指数为28.88,是三品系中最低者,抗(耐)力居首位。TMV接毒鉴定,C151为过敏枯斑反应,是当前对CMV和TMV兼抗性能的较好桥梁品系。