Observation and quantification of self-associated adenosine extracted from royal jelly products purchased in USA by HPLC

The concentration of adenosine in pure royal jelly creams and dietary supplements purchased in the United States was determined by reversed phase high performance liquid chromatography (HPLC). Preliminary studies revealed the existence of several forms of adenosine via self-association and base-pairing in solutions. Therefore, proper optimisation of pH and compositions of extraction solvents and mobile phases was critical for successful separation and quantification. In this work, adenosine in samples was extracted by sonication in a mixture consisting of 5% ethanol and 95% deionized water and separated using a Zorbax Eclipse XDB-C18 column (150 × 4.6 mm) and a mobile phase of 93% deionized water and 7% acetonitrile at 25 °C. The flow rate of a mobile phase was set to 1.0 ml/min and the UV detection was performed at 260 nm. The average recovery rate of adenosine was 92.8–99.2% with the relative standard deviation (RSD) of 0.1–1.3% over concentrations ranging from 5 to 160 μg/ml. The limit of detection (LOD) and limit of quantification (LOQ) were found to be ∼0.01 and ∼0.05 μg/ml, respectively. Quantification was carried out using calibration curves constructed by both internal standard (ISTD) and external standard (ESTD) methods. Our results show that the concentration of adenosine lies between ∼27 and 50 μg/g for pure royal jelly creams and between ∼2 and ∼173 μg/g for royal jelly supplements.     

Estimation and characterisation of major royal jelly proteins obtained from the honeybee Apis merifera

Royal jelly (RJ) contains many components, including proteins. We focused on major royal jelly proteins (MRJPs) under natural conditions, and attempted to determine the content ratios and molecular forms of MRJPs by size-exclusion HPLC, SDS–PAGE, 2-DE and MALDI TOF/TOF MS. Soluble RJ proteins were extracted by dialysis followed by several centrifugation techniques. Soluble RJ proteins were universally separated into five peaks (640 kDa, 280 kDa, 100 kDa, 72 kDa and 4.5 kDa) by size-exclusion HPLC on a Superose 12 column. Among these peaks, both the 280 kDa and 72 kDa peaks were major, but the intensity of the 280 kDa peak differed markedly among original RJ samples (n = 70). The main 280 kDa protein was separated into a 55 kDa band by reducing and non-reducing SDS–PAGE. This protein was also separated into multiple spots ranging from pH 4.2 to 6.5 by 2-DE. These spots were identified as MRJP 1 by MALDI TOF/TOF MS. From these results, MRJP 1 was thought to comprise an oligomer complex linked by non-covalent bonds under natural conditions. Another major protein, the 72 kDa peak on Superose 12 HPLC, was identified as MRJP 2.     

A rapid method to isolate soluble royal jelly proteins

Soluble royal jelly (RJ) proteins (SRJPs) include the major RJ protein (MRJP) family, which contribute to the physiological actions of RJ. Although SRJPs are prepared using conventional methods involving dialysis and centrifugation, dialysis is a time-consuming process. We have therefore developed a simple method to isolate SRJPs from RJ. This new method produces 20-fold higher levels of SRJPs than that of the conventional procedure; hence, the levels obtained by the new and existing methods were compared. A 1-h ultracentrifugation separated SRJPs in the supernatant into upper, middle and lower layers. Each layer was analyzed by size-exclusion HPLC, SDS–PAGE and 2-DE. The upper and middle layers contained MRJP2 (52 kDa) and MRJP3 (60–70 kDa), while the lower layer contained MRJP1 (290 kDa). In nature, MRJP1 is a monomer and/or oligomer. When the lower layer was analyzed by Superose 12 HPLC, MRJP1 was predominantly an oligomer. Our MRJP isolation method reduces the procedure time by using ultracentrifugation without dialysis to obtain SRJPs and produces layers containing MRJP1 oligomers, MRJP2 and MRJP3.     

Determination of cis- and trans- α- and β-carotenoids in Taiwanese sweet potatoes (Ipomoea batatas (L.) Lam.) harvested at various times

A HPLC method was improved to determine sweet potato carotenoids rapidly with good separation efficiency. A C30 column and a gradient solvent system consisting of methanol–acetonitrile–water (84/14/2, v/v/v, solvent A) and dichloromethane (solvent B) (a mixture of 80% A and 20% B was used initially, and then the mixing was programmed to 55% B within 15 min and kept to the end) were used for analysis. The flow rate was 1 ml/min and detection was at 450 nm. A total of 11 all-trans and cis forms of α- and β-carotene in Taiwanese sweet potato (Ipomoea batatas (L.) Lam.) could be resolved within 16 min. The orange-fleshed sweet potato (Tainung 66) had higher total carotenoid content than the yellow-fleshed one (Tainung 57) at the same harvest time. The total carotenoid levels in both crops harvested at various times were in the order: October > July > April > January.     

Characterization of honey from different floral sources. Its functional properties and effects of honey species on storage of meat

The antioxidative effects of honey species and their related products were evaluated using a lipid peroxidation model system. The antioxidant activities of honey species gradually decreased with passage of time. Buckwheat honey was as effective as 1 mM α-tocopherol. Superoxide-scavenging activities of propolis and royal jelly were strongest among the honey species tested. 1,1-Diphenyl-2-picrylhydrazyl radical scavenging ability of sample species were lower than those of 1 mM ascorbic acid and α-tocopherol. Hydroxyl radical scavenging activity was very high in all honeys (over 77% inhibition). From the results of the bacterial test on storage of meat and muscle, each honey exhibited the inhibition of bacterial growth. In particular, propolis and royal jelly exhibited the strongest inhibitory effects against bacterial growth. This suggests that honey species from different floral sources possess strong antioxidative and antibacterial activities and are scavengers of active oxygen species.     

Analysis of chloramphenicol in honey and royal jelly by LC/MS/MS

A sensitive and selective method using liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was developed for the determination of chloramphenicol (CAP) in honey and royal jelly. Mass spectral acquisition was performed in the negative mode by applying multiple reaction monitoring. In LC separation, Mightyl RP-18GP and 10 mmol/L ammonium acetate-acetonitrile were used as the column and mobile phase, respectively. CAP in honey samples was diluted with water, while CAP in royal jelly was extracted with 1% metaphosphoric acid-methanol (4 : 6). The solutions were cleaned up with an Oasis HLB cartridge. The quantification limits of CAP in honey and royal jelly were 0.3 ng/g and 1.5 ng/g, respectively. The recoveries of CAP from both honey and royal jelly at the quantification limits were over 92%. Twenty honey products and seven royal jelly products were analyzed by the developed method. CAP was detected in one honey product at 0.6 ng/g and in six royal jelly products at the level of 1.5-17.8 ng/g. These results show that the developed method has satisfactory sensitivity selectivity and is useful for the determination of CAP residues in honey and royal jelly.     

Tocopherols in espresso coffee: Analytical method development and validation

The present paper reports the development and validation of an analytical micro-method for tocopherols quantification in espresso coffee by normal-phase HPLC with fluorescence detection. The proposed method consists in a liquid–liquid extraction with n-hexane:ethyl acetate, followed by a clean-up with dimethylformamide to eliminate co-eluting interferences. The method showed good intra- and inter-day precisions (coefficient of variation < 6.5%), good accuracies (98 ± 6%), and high correlation coefficients (r > 0.999) for standards subjected to the entire procedure. Only α- and β-tocopherols were identified in the brews. The detection and quantification limits were 0.5 and 1.4 ng/ml, for α-tocopherol, and 0.4 and 1.1 ng/ml, for β-tocopherol, respectively. A mean total tocopherol content (α + β) of 3.5 ± 0.9 μg in commercial espresso coffee blends (30 ml) was detected. The proposed method requires low solvent consumption and proved to be sensitive, precise and accurate.     

The content of astilbin and taxifolin in concentrated extracts of Rhizoma Smilacis Glabrae and turtle jelly vary significantly

Extract of Rhizoma Smilacis Glabrae (RSG) is one of the main ingredients in turtle jelly, which is a traditional functional food in Southern China and Hong Kong. A capillary electrophoresis method was successfully developed for determination of astilbin and taxifolin in turtle jelly and RSG concentrated extract samples. For six determinations of astilbin and taxifolin at concentrations of 20 μg ml⁻¹, the relative standard deviations of migration time were 0.62% and 0.87%, while those of the peak area ratios were 2.17% and 3.62%, respectively. Eighteen turtle jelly samples manufactured in mainland China and Hong Kong were collected for analysis. The results show that astilbin and taxifolin were all from the RSG ingredient. The contents of astilbin and taxifolin in turtle jelly were distinctly different between brands, and some were found not to contain these substances.     

蜂王浆醇溶性成分分析及其抗氧化性研究

蜂王浆经体积分数95%乙醇提取、MCI-Gel、ODS等反相色谱柱分离,得到单磷酸腺苷(AMP)和7种脂肪酸。采用1,1-二苯基苦基苯肼自由基(DPPH.)清除率和总抗氧化能力试剂盒(T-AOC)等方法探索蜂王浆醇溶性物质的抗氧化能力,结果表明:蜂王浆醇提后抗氧化活性提高,分离得到的AMP和3,10-二羟基癸酸(3,10-DDA)的抗氧化性相对较强,且清除DPPH.的IC50值分别为6.274 mg/mL和9.153 mg/mL,是蜂王浆中的抗氧化活性物质。     

Multi-residue method for determination of seven neonicotinoid insecticides in grains using dispersive solid-phase extraction and dispersive liquid–liquid micro-extraction by high performance liquid chromatography

A method using dispersive solid-phase extraction and dispersive liquid–liquid micro-extraction cleanup followed by high performance liquid chromatography (HPLC) has been established for determination of seven neonicotinoid insecticides residues in grains including brown rice, maize, millet and oat. Based on an appraisal of the characteristics of HPLC, validation experiments were conducted for seven neonicotinoid insecticides. In the method, dispersive solid-phase extraction was carried out using PSA and bonded C₁₈ coupled with graphitised carbon black with acetonitrile as the eluted solvent. In the linear range of each pesticide, the correlation coefficient was R² ? 0.99. At the low, medium and high three fortification levels of 0.05–0.8 mg kg⁻¹, recoveries fell within 76–123%. The relative standard deviation was between 0.9% and 12.6% for seven neonicotinoid pesticides. Low limits of detection (0.002–0.005 mg kg⁻¹) and quantification (0.007–0.018 mg kg⁻¹) were readily achieved with this method for all tested pesticides.     

进出口蜂王浆及其制品中10-羟基-α-癸烯酸的分析与评估

目的 了解掌握蜂王浆及其制品中的主要营养成分质量情况,以确保蜂王浆在进出口贸易时得到相应的法律保障。方法 样品经乙醇提取,溶出10-羟基-α-癸烯酸(以下简称10-HDA),并沉淀蛋白质,加内标后用乙醇定容,经离心或放置过夜,上清液过滤,HPLC测定。结果 有代表性的进出口的蜂王浆及蜂王浆胶襄样品均含有一定量10-HDA。结论 蜂王浆含果糖、氨基酸、多种酶、乙酰胆碱及丰富的维生素、芸香甙、促进性腺样物质和抗菌素类物质等,而其中10-HAD由于具有一定稳定性,因此是检验蜂王浆营养品质的主要指标。     

Analysis of tetracyclines in honey and royal jelly by LC/MS/MS

A simple and accurate method using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) was developed for the determination of tetracyclines (TCs), i.e., oxytetracycline (OTC), chlortetracycline (CTC) and tetracycline (TC), in honey and royal jelly. Mass spectral acquisition was performed in the positive mode. In LC separation, L-column ODS and 0.01% formic acid-acetonitrile were used as the column and mobile phase, respectively. TCs in a honey sample were diluted with water, while TCs in royal jelly were extracted with 2% metaphosphoric acid-methanol (6:4). They were cleaned up with Oasis HLB and Sep Pak C18 cartridges, respectively. The quantification limits of TC, OTC, and CTC were 5, 5, and 10 ng/g, respectively, while those in royal jelly were 25, 25, and 50 ng/g, respectively. The recoveries of TCs from both honey and royal jelly were 75-120%.     

Comparison of analytical methods to assay inhibitors of angiotensin I-converting enzyme

The linearity, precision and repeatability of visible spectrophotometric (VSP) and high-performance liquid chromatography (HPLC) methods for analysis of inhibitory activity of angiotensin I-converting enzyme (ACE) were compared by using several inhibitors and Hip-His-Leu (HHL) as substrates. IC₅₀ values (concentration at which ACE activity is inhibited by 50%) of 0.00206 ± 0.00005 μg/mL for captopril, 192 ± 4.53 μg/mL for soybean peptides, and 153 ± 4.29 μg/mL for grass carp peptides determined by the VSP method, and these values were 1.07, 1.07, 1.18 and 1.44-fold, respectively, higher than those from the HPLC method. In addition, the inhibitory constant (K_i value) of captopril was determined to be 7.09 nM and 4.94 nM using VSP and HPLC method, respectively. These results showed that the HPLC method revealed a higher level of sensitivity and precision, suitable for assaying ACE inhibition activity of antihyper-sensitive peptides. In contrast, the VSP method can simultaneously measure several samples with simple operations, suitable for analysis of ACE inhibition activity of food protein enzymatic hydrolysates.     

Production of cordycepin by a repeated batch culture of a Cordyceps militaris mutant obtained by proton beam irradiation

Cordycepin (3′-deoxyadenosine) is one of the most versatile metabolites of Cordyceps militaris due to its broad spectrum of biological activity. In our previous study, the C. militaris mutant G81-3, which produces higher levels of cordycepin, was obtained by high-energy proton beam irradiation. In this study, the effects of adenosine on cordycepin production in a surface liquid culture of the mutant and the wild type strains were investigated. For the mutant strain, the optimum dose of adenosine yielded a 30% increase in cordycepin production; the maximum levels of production with adenosine and without adenosine were 8.6 g/l and 6.7 g/l, respectively. In contrast, the increase due to adenosine supplementation for the wild type strain was only 15% (3.1 g/l with adenosine and 2.7 g/l without adenosine). Furthermore, a repeated batch culture, an efficient production method, was carried out to eliminate the relatively long lag phase of the mutant culture. Over four cycles, both the mutant and the wild type strain maintained a production level of more than 85% of that of the initial cycle. As a result, the disadvantage of the mutant was successfully overcome, resulting in a productivity (0.48 g/(l d)) higher than that of the batch culture (0.29 g/(l d)). The productivity for cordycepin obtained in this study is the highest reported value to date, and this method could be applied to large-scale production of cordycepin at industrial levels.     

不同花期蜂王浆主要成分和抗氧化活性分析

为探究不同花期蜂王浆品质和抗氧化活性的差异,以同产地同蜂种同饲养条件下所生产的蜂王浆作为研究对象,分析不同花期蜂王浆的10-羟基-2-癸烯酸（10-HDA）、总蛋白、水分、总酚酸的含量变化,同时采用DPPH法和FRAP法分析比较其体外抗氧化能力。结果表明,不同花期之间蜂王浆的10-HDA含量存在显著差异（P<0.05）,葡萄蜂王浆10-HDA含量最高达到了2.20%。总蛋白含量在13.58%~15.26%之间。蜂王浆的水分含量均≤67.5%,达到国标（GB 9697-2008）关于优等品蜂王浆水分含量的要求。与其他花期蜂王浆相比,荆条花期的总酚酸含量、DPPH自由基清除能力和总抗氧化能力均为最优,且不同花期蜂王浆之间的DPPH自由基清除率存在显著差异（P<0.05）。相关性分析表明,DPPH自由基清除能力和总酚酸以及总抗氧化能力之间均存在极显著正相关性（P<0.01）。     

Liquid chromatography–tandem mass spectrometry for the determination of chrysoidine in yellow-fin tuna

A high performance liquid chromatography (HPLC) coupled to electrospray ionisation tandem mass spectrometry (MS/MS) method was described for the residue detection of chrysoidine in yellow-fin tuna in the present study. Samples were cleaned up with solid phase extraction (SPE) cartridge, and then injected into HPLC for separation. Multiple-reaction monitoring (MRM) was applied for quantitative determination. Results showed that the low limit of detection (LOD) of the method was 1.25 × 10⁻¹² g, and the low limit of quantification (LOQ) was 0.42 μg/L. The standard calibration curve was y = 2333.9x −845 (r² > 0.99) with the linear range of 0.63–100 μg/L. The average recoveries of chrysoidine ranged from 86.0% to 108.0% when the spiked concentration was from 0.5 μg/kg to 20 μg/kg. And the developed method also showed the good test precisions (RSD%: 4.38–14.27%).     

Development of enzyme-linked immunosorbent assay (ELISA) for the detection of neomycin residues in pig muscle,chicken muscle,egg, fish,milk and kidney

A colorimetric competitive direct enzyme-linked immunosorbent assay (ELISA) method was developed using polyclonal antibody to determine neomycin residues in food of animal origin. No cross-reactivity of the antibody was observed with other aminoglycosides. The limit of detection of the method was 0.1 μg/kg. A simple and efficient sample extraction method was established with recoveries of neomycin ranged from 75% to 105%. The detection limits were 5 μg/kg(l) in pig muscle, chicken muscle, fish and milk, 10 μg/kg in kidney and 20 μg/kg in egg, respectively. Chemiluminescence assay was developed for detecting neomycin residues in pig muscle and chicken muscle. The limit of detection of the method was 0.015 μg/kg, and the detection limits were 1.5 μg/kg in pig muscle and 6 μg/kg in chicken muscle. The ELISA tests were validated by HPLC, and the results showed a good correlation (r²) which was greater than 0.9.     

Quantitative analysis of quercetin using Raman spectroscopy

Raman spectroscopy was used for the quantitative analysis of quercetin. Raman spectra were measured with an Ar ion laser at 488 nm. A 20× objective lens was used for focusing the laser beam on the sample. The back scattering light was passed into the monochrometer and detected with a CCD detector. The Raman band of a solvent (ethanol and methanol) was used as the internal standard to remove the effect of factors such as laser power and other instrumental effect. The band ratio between the Raman intensity of the sample and that of the solvent had good linearity with the analyte concentration. The equations of the calibration curve were y = 9.005x with an R² of 0.9998 and y = 11.50x with R² of 0.9999 in ethanol and methanol, respectively. The limit of detection (LOD) was 5 × 10⁻⁵ mol/L. The quantity of quercetin extracted from onion peels was determined by Raman spectroscopy. Masses in quercetins of 73 mg/100 g and 70 mg/100 g were extracted from dried onion peels by using hot ethanol and hot methanol for three hours. These values are in good agreement with results obtained by HPLC and UV–vis spectroscopy.     

Preparative isolation of triterpenoids from Ganoderma lucidum by counter-current chromatography combined with pH-zone-refining

Ganoderma lucidum is a famous fungus. The triterpenoids are the main bioactive components and exhibit various pharmacological activities. However, the scarcity of the pure triterpenoid has greatly restrained the modern research of G. lucidum. The present paper first describes a convenient method to separate the main triterpenoids from G. lucidum using counter-current chromatography (CCC) technique. Ganoderic acid C6 (38 mg), E (76 mg), F (32 mg) were successfully separated from the crude extract in 1 day with the HPLC purity of above 90% using stepwise CCC. Ganoderic acid G (36 mg), A (64 mg), B (25 mg), D (28 mg) and ganoderenic acid D (11 mg) were separated in 2 days with the HPLC purity of above 90% using a combination of stepwise CCC and pH-zone-refining CCC. The method presented in the paper is much quicker and easier than the previous methods.     

蜂王浆蛋白肽的制备及其降血糖和抗氧化活性研究

目的:研究蜂王浆蛋白的最佳酶解工艺条件,并分析所制备酶解肽的氨基酸组成、分子量分布、降血糖及抗氧化活性。方法:以蜂王浆为原料,采用碱提酸沉法提取蜂王浆粗蛋白,以α-葡萄糖苷酶抑制率和ABTS自由基清除率为评价指标,筛选出酶解蜂王浆蛋白制备降血糖及抗氧化活性肽的最佳蛋白酶,并研究蜂王浆酶解肽的体外降血糖和抗氧化活性。结果:蜂王浆降血糖及抗氧化活性肽的最佳制备工艺为:以酸性蛋白酶为酶制剂,酶添加量为6000 U/g,酶解温度为43℃,酶解pH为4.0,酶解时间为4 h,料液比为1:10。在上述条件下,制备的蜂王浆蛋白肽的氨基酸总量为82.19%,相对分子质量集中在1000 Da以下。蜂王浆蛋白肽对α-葡萄糖苷酶的半抑制浓度(IC₅₀)为6.94 mg/mL,清除ABTS自由基、羟自由基、1,1-二苯基-2-三硝基苯肼(DPPH)自由基及超氧阴离子自由基的半抑制浓度(IC₅₀)分别为14.18、0.45、11.02和18.38 mg/mL。试验结果表明,蜂王浆蛋白肽具有一定的降血糖和抗氧化活性,可为蜂王浆高值化利用及活性肽产品开发提供理论依据。