Determination of isoflavones in red clover and related species by high-performance liquid chromatography combined with ultraviolet and mass spectrometric detection

High-performance liquid chromatography-UV-electrospray ionization-mass spectrometric detector (HPLC-UV-ESI-MSD) method for determination of isoflavones in red clover (Trifolium pratense L.) and related species has been developed. The separated isoflavones including aglycones, glycosides and glycoside malonates, were individually analyzed and identified by their molecular ions and characteristic fragment ion peaks using LC-MSD under MS and MS-MS mode, and in comparison with the standard isoflavones. A total of 31 isoflavones were detected in red clover. Several isoflavones were also identified for the first time in related species, T. repense L. (white clover), T. hybridum L. (alsike clover) and T. campestre Schreber (hop trefoil). Based on reversed phase HPLC, all 10 isoflavone aglycones, daidzein, formononetin, genistein, pseudobaptigenin, glycitein, calycosin, prunetin, biochanin A, irilone and pratensein in acidic hydrolyzed extracts were successfully separated within 40 min and quantified individually by UV and MS detectors. For the 10 target compounds, the investigated concentrations ranged from approximately 24 to approximately 12500 ng/ml for UV detection and approximately 6 to approximately 3125 ng/ml for MS detection, and good linearities (r2 > 0.999 for UV and r2 > 0.99 for MS) for standard curves were achieved for each isoflavone. The accuracy and repeatability (n = 10) were within 15% for these 10 compounds. This is the first method reported that enables the simultaneous quantitation of all 10 isoflavone aglycones in red clover and related species.     

Effects of aqueous extracts from the stems and leaves of Trifolium pratense cv. Minshan on germination and seedling growth of three crops

以岷山红三叶（Trifolium pratense cv.Minshan）的茎、叶为供体植物,用蒸馏水浸泡48h制备水浸液。设0.05、0.10、0.15和0.20g·mL^-1 4个浓度梯度,以蒸馏水为对照,对燕麦（Avena sativa）、小白菜（Brassicachinensis）、披碱草（Elymus dahuricus）3种受体植物种子进行发芽试验和幼苗生长的生物测定。结果表明,不同浓度岷山红三叶茎叶水浸液对不同受体植物种子萌发和幼苗的生长发育有不同的化感效应。各浓度水浸液对种子萌发均没有显著影响（P〉0.05）,但对小白菜幼苗的生长均有显著的抑制作用（P〈0.05）;0.20g.mL-1浓度的水浸液对燕麦、披碱草和小白菜根生长抑制率分别为23.76%、35.73%和62.50%,且抑制作用显著;0.05g.mL-1浓度水浸液对披碱草幼芽的生长促进作用显著;在3种受体植物中,小白菜受到抑制作用较强,燕麦和披碱草则较弱。     

Characteristics of a 4-fold segmented clover detectore

Four high-purity germanium 4-fold segmented Clover detectors have been     

Rapid-resolution HPLC with spectrometric detection for the determination and identification of isoflavones in soy preparations and plant extracts

A rapid-resolution HPLC/UV-VIS DAD separation method (which takes <1 min) for the determination and identification of genistin, genistein, daidzein, daidzin, glycitin, glycitein, ononin, formononetin, sissotrin and biochanin A in fmol quantities in submicroliter sample volumes was optimized. A linear gradient elution (0 min 22% B, 1.0 min 80% B, 1.4 min 100% B, 1.8 min 22% B) using a mobile phase containing 0.2 % (v/v) acetic acid (solvent A) and methanol (solvent B) was applied on a Zorbax SB C₁₈ column (1.8 μm particle size) at 80 °C. The method was verified using samples of bits of soy and methanolic extracts from Trifolium pratense, Iresine herbstii and Ononis spinosa plants. Pseudobaptigenin glucoside, irilone, prunetin, texasin, tlatlancuayin and other isoflavones, in addition to aglycones of isoflavones and their β-glucosides and malonyl and acetyl derivatives, were identified by UV-VIS DAD and electrospray mass spectrometric (ESI-MS) detection in the extracts. Figure Rapid resolution HPLC for determination and identification of isoflavones in soy preparations and plant extracts     

Mass Spectrometric Identification of Antimicrobial Peptides from Medicinal Seeds

Traditional medicinal plants contain a variety of bioactive natural products including cysteine-rich (Cys-rich) antimicrobial peptides (AMPs). Cys-rich AMPs are often crosslinked by multiple disulfide bonds which increase their resistance to chemical and enzymatic degradation. However, this class of molecules is relatively underexplored. Herein, in silico analysis predicted 80–100 Cys-rich AMPs per species from three edible traditional medicinal plants: Linum usitatissimum (flax), Trifolium pratense (red clover), and Sesamum indicum (sesame). Bottom-up proteomic analysis of seed peptide extracts revealed direct evidence for the translation of 3–10 Cys-rich AMPs per species, including lipid transfer proteins, defensins, α-hairpinins, and snakins. Negative activity revealed by antibacterial screening highlights the importance of employing a multi-pronged approach for AMP discovery. Further, this study demonstrates that flax, red clover, and sesame are promising sources for further AMP discovery and characterization.     

Use of some Inorganic and Organic Compounds as Decontaminants for Cobalt-60 and Caesium-134 by Clover Plant Grown on Inshas Sandy Soil

Outdoor lysimeter experiments were carried out to elucidate the effect of 4 inorganic and 3 organic salts on ⁶⁰Co and ¹³⁴Cs uptake and dry matter yield of three cuts of clover in the soils of Inshas. A new concept was proposed for assessing the difference in capacity of soil to supply radionuclides to plants as affected by applied inorganic or organic compounds. A relation of plant tissue radionuclide content with time can be expressed in an exponential equation with corresponding regression coefficients. When the same plant species is grown on the same soil differing only in the applied compounds (e.g. Fe-EDDHA, Fe-DTPA, Fe-OAC, Fe(COO)₂, Al(OH)₃, Ca(H₂PO₄)₂, Fe₂O₃) the derived equation will change. Differences of either Co or Cs accumulation with time between control and any other treatment were evaluated. The data also show that Fe-DTPA is preferred for ⁶⁰Co as enhancing compound for plant uptake (for decontamination use), compared with other applied salts, on the other hand, any tested salts did not significantly effect ¹³⁴Cs taken by clover. It was also found that more than 70% of the total cobalt uptake was accumulated in the roots which indicate that Co is less mobile in plants than Cs.     

Biosynthesis of Fe3O4-magnetic nanoparticles using clover leaf aqueous extract: Green synthesis of 1,3-benzoxazole derivatives

In this research, magnetic Fe₃O₄-NP nanoparticles were synthesized employing a green biosynthetic procedure by reduction of ferric chloride solution with clover leaf water extract. The nanoparticles prepared via this biosynthesis method can potentially be valuable for different purposes such as organic synthesis. In this research, 1,3-benzoxazole derivatives were generated via a multicomponent reaction of α-bromo ketones, isothiocyanate, and propiolate in the presence of a catalytic amount of bio-Fe₃O₄ MNPs and sodium hydride in water at 50°C in good yields. The catalyst was reused five times with a minor decrease in its catalytic activity.     

LC Determination of Four Isoflavone Aglycones in Red Clover (Trifolium pratense L.)

Red clover (Trifolium pratense L.) is an important forage plant that contains the isoflavones daidzein, genistein, formononetin, and biochanin A. These compounds have been studied lately due to their human health benefits. The aim of this study was to develop and validate an HPLC method with simplified sample preparation to quantify daidzein, genistein, formononetin and biochanin A simultaneously in red clover leaves. The validation showed that the method is specific, accurate, precise and robust, not to mention that the sample preparation is easier and faster than those described earlier. The response was linear over a range of 0.01–0.2 μg mL⁻¹ for daidzein, 0.05–0.5 μg mL⁻¹ for genistein, 4–40 μg mL⁻¹ for formononetin and 2–20 μg mL⁻¹ for biochanin A. The range of recoveries was 85.6–101.0%. The RSD for intra- and inter-day precision were <2.54 and <7.22%, respectively. Five populations of red clover, from the National Plant Germplasm System-USDA were analyzed and the content of daidzein, genistein, formononetin and biochanin A ranged from 7.87–91.31, 51.60–131.30, 6568.33–23461.82, to 2499.55–10337.33 μg g⁻¹ of dried material, respectively.     

小口径轴对称收缩喷嘴射流冲击大平板噪声指向特性研究

实验研究了3毫米口径轴对称收缩喷嘴在各种压比下射流垂直冲击和倾斜冲击坚固大平板产生的噪声的指向特性。发现噪声在过平板法线和喷嘴轴线的平面内呈近似四瓣分布，当喷嘴与平板距离减小时，指向壁射流下游的瓣得到增强，反之，指向喷嘴上游的瓣得到增强。喷嘴压比增加时，指向壁射流下游的瓣得到增强，反之，指向喷嘴上游的瓣得到增强。根据自由射流噪声的基本指向特性、射流冲击噪声基本指向特性、声波在平板处发生镜面反射和声波能量叠加的设定，建立了一个冲击射流总体噪声指向特性的模型，成功解释了实验结果，并揭示了形成冲击射流总体噪声指向特性的内在机理。