Rapid screening of lignans from Phyllanthus myrtifolius and stilbenoids from Syagrus romanzoffiana by HPLC-SPE-NMR

Introduction – Application of on‐line solid‐phase extraction (SPE) as an interface between HPLC and NMR has gained great improvement in solving sensitivity problems and signal interferences by the eluents. Objective – Rapid analysis and characterisation by HPLC‐SPE‐NMR and LC/MS of the arylnaphthalene‐type lignans present in Phyllanthus myrtifolius and the minor stilbenoids present in the polyphenol‐rich fraction from the ethanol extract of the seeds of Syagrus romanzoffiana. Methodology – Pretreatment of fractions by liquid–liquid partitioning, followed by Sephadex LH‐20 fractionation, was found very useful to facilitate the focusing and analysis of the polyphenolic fraction. HPLC‐DAD‐SPE‐NMR (400 MHz and 600 MHz) analysis was carried out using an Agilent 1100 liquid chromatography, followed by a Prospekt 2 automated solid‐phase extraction unit, containing 96 HySphere‐Resin GP cartridges (10 × 2 mm, 10–12 µm), which was connected to a 120 or 60 µL LC probe. Results – Seven arylnaphthalene‐type lignans from the chloroform‐soluble fraction of P. myrtifolius and nine stilbenoids from a polyphenol‐rich butanol‐soluble fraction of the seeds of S. romanzoffiana were characterised. Conclusion – HPLC‐SPE‐NMR associated with HR‐ESI/MS, which consumed only analytical amounts of partially purified mixtures, was demonstrated to be a good tool for rapid screening of both known and new natural products. Copyright © 2011 John Wiley & Sons, Ltd.     

Chemical investigation of Phyllanthus reticulatus by HPLC-SPE-NMR and conventional methods

Eight compounds, including two flavonoid glycosides, were isolated from the butanol-soluble fraction of the methanolic extract of the leaves of Phyllanthus reticulatus by conventional methods. A polyphenol rich fraction, obtained by Sephadex LH-20 fractionation, was also studied using an HPLC-SPE-NMR technique leading to the characterization of six compounds including three additional flavonoid glycosides. The latter approach used only 1 mg of samples, theoretically equivalent to 0.3 g of dry leaves. This study demonstrates that HPLC-SPE-NMR is very useful for thorough chemical investigation and also offers the advantages of saving time, plant materials and consumables over more conventional methods.     

Phenolic antioxidants from the whole plant of Phyllanthus urinaria

The 1,1-diphenyl-2-picrydydrazyl (DPPH) assay on the extract of Phyllanthus urinaria L. (Euphorbiaceae) displayed considerable radical-scavenging activity (SC50 = 14.3 microg/ml). Further bioassay-guided purification of the extract led to the isolation of a series of 15 phenolic compounds, including the ellagitannins 1-7, the flavonoids 8-10, and the simple hydroxylated (or glycosylated) aromatic acids 11-15. Their structures were identified by spectroscopic analyses and comparison with authentic samples or literature data. The structure of repandinin B (1) was for the first time fully assigned by 1D- and 2D-NMR experiments. The phenolic compounds 1, 3, 4, 6, 9, 11, and 15 have not been isolated before from the title plant. The antioxidant activities and mushroom-tyrosinase-inhibitory activities of all compounds were determined by DPPH-radical-scavenging and mushroom-tyrosinase-inhibitory assays (Table 2).     

One step purification of corilagin and ellagic acid from Phyllanthus urinaria using high-speed countercurrent chromatography

High-speed countercurrent chromatography (HSCCC) has been successfully applied to the preparative separation of corilagin and ellagic acid in one step from the Chinese medicinal plant Phyllanthus urinaria L. by use of direct and successive injections of a crude methanolic extract. Some aspects concerning the practical use of this technique in the described application are considered.     

Development of species-specific SCAR markers for identification of three medicinal species of Phyllanthus

Phyllanthus amarus Schum. & Thonn. has been widely used in traditional medicine in Thailand as an antipyretic, a diuretic, to treat liver diseases and viral infections. Two closely related species, P. debilis L. and P. urinaria Klein ex Willd., with different and less effective medicinal properties, are less commonly used. These three species are similar in morphology and often occur in overlapping populations in nature. The latter two species can easily be mistaken for P. amarus and collected for medicinal uses, which can lead to undesirable results. DNA fingerprints of these species were obtained using RAPD-PCR techniques. RAPD markers specific for each species were identified. Primers for highly specific sequence-characterized-amplified-regions (SCAR) were then designed from nucleotide sequences of specific RAPD markers. These primers efficiently amplified SCAR markers of 408, 501 and 319 bp unique to P. amarus, P. debilis and P. urinaria respectively. This method of plant identification was rapid and highly specific when tested against DNA of several closely related species and was able to amplify specific markers from mixed DNA samples.     

叶下珠有效部位对血栓形成的影响及其作用机制初探

采用Born方法和改良的Hamburger方法分别测定叶下珠 (Phyllanthusurinaria)含corilagin的有效部位 (代号PUW )在体内外对血小板聚集功能和对血小板与中性粒细胞之间粘附反应的影响 ;应用Myers方法评价PUW灌胃对小鼠尾静脉注射花生四烯酸 (AA)引起猝死的保护作用 ;运用改良的Charl ton方法及陈长勋等方法分别观察PUW灌胃对电刺激大鼠颈动脉血栓形成和下腔静脉血栓形成的影响 ;采用Tomihisa方法 ,观察PUW对大鼠尾尖出血时间的影响。结果显示 ,PUW在体内外对ADP、AA或血小板活化因子 (PAF)诱导的血小板聚集均无明显抑制作用 ;PUW呈浓度依赖性明显阻抑AA激活的血小板与中性粒细胞之间的粘附反应 ,其半数抑制浓度 (IC50 )为 39 7mg/kg。PUW (10、2 0和 4 0mg/kg)灌胃呈剂量依赖性显著减少AA致小鼠死亡的数量 ,明显延长电刺激大鼠颈动脉血栓形成时间 ,减轻大鼠下腔静脉血栓的干、湿重。 2 0mg/kg的PUW对出血时间无明显影响 ,4 0mg/kg的PUW虽延长出血时间 ,但与阿司匹林 (2 0mg/kg)比较 ,出血时间明显缩短 (P <0 0 5 )。本实验结果提示 ,PUW灌胃在多种体内血栓模型中均具有明显的抗血栓形成作用 ,其机制可能与阻抑血小板和中性粒细胞之间的的粘附作用密切相关。     

Quantitative determination of phyllanthin and hypophyllanthin in Phyllanthus species by high-performance thin layer chromatography

A simple, precise and rapid high-performance thin-layer chromatographic method has been developed for the estimation of phyllanthin (1) and hypophyllanthin (2), the important lignans of Phyllanthus species, especially Phyllanthus amarus. Separation of 1 and 2 was carried out on silica gel 60 F254 layers eluted with hexane:acetone:ethyl acetate (74:12:8), and the analytes were visualised through colour development with vanillin in concentrated sulphuric acid and ethanol. Scanning and quantification of spots was performed at 580 nm. Recoveries of 1 and 2 were 98.7 and 97.3%, respectively. The method was validated and the peak purities and limits of detection and quantification were determined.     

Development of species-specific SCAR markers for identification of three medicinal species of Phyllanthus

Phyllanthus amarus Schum.& Thonn.has been widely used in traditional medicine in Thailand as an antipyretic.a diuretic.to treat liver diseases and viml infections.Two closely related species,P. debills L.and P.urinaria KIein ex Willd.,with different and less effective medicinal properties,are less commonly used.These three species are similar in morphology and often Occur in overlapping populations in nature.The latter two species can easily be mistaken for P.amarus and collected for medicinal uses, which can lead to undesirable results.DNA fingerprints of these species were obtained using RAPD-PCR techniques.RAPD markers specific for each species were identified.Primers for highly specific sequence-characterized-amplified-regions (SCAR) were then designed from nucleotide sequences of specific RAPD markers.These primers efficiently amplified SCAR markers of 408,501 and 319 bp unique to P.amarus,P.debilis and P.urinaria respectively.This method of plant identification Was rapid and highly specific when tested against DNA of several closely related species and was able to amplify specific markers from mixed DNA samples.     

Salicylic acid‐enhanced biosynthesis of pharmacologically important lignans and neo lignans in cell suspension culture of Linum ussitatsimum L

Linum usitatsimum L. (flax) is a perennial herb with magnitude of medicinal and commercial applications. In the present study, we investigated the effects of salicylic acid (SA) on biosynthesis of lignans (secoisolariciresinol diglucoside (SDG) and lariciresinol diglucoside (LDG)) and neolignans (dehydrodiconiferyl alcohol glucoside (DCG) and guaiacylglycerol‐β‐coniferyl alcohol ether glucoside (GGCG)) in cell cultures of flax. Moderate concentration of SA (50 μM) enhanced biomass accumulation (10.98 g/L dry weight (DW)), total phenolic content (37.81 mg/g DW), and antioxidant potential (87.23%) to two‐fold than their respective controls after 72 h of exposure. However, higher levels of total flavonoid content (5.32 mg/g DW) were noted after 48 h of exposure to 50 μM of SA. HPLC analyses revealed that 50 μM SA, significantly enhanced biosynthesis of SDG (7.95 mg/g DW), LDG (7.52 mg/g DW), DCG (54.90 mg/g DW), and GGCG (16.78 mg/g DW), which was almost 2.7, 1.8, 3.88, and 3.98 fold higher than their respective controls after 72 h of exposure time, respectively. These results indicated that moderate concentrations of SA had significant effects on biosynthesis and productivity of lignans and neolignans in cell culture of L. usitatissimum.     

Lignans and secoiridoids from the root bark of Chionanthus virginicus L.: isolation, identification and HPLC analysis

Five lignans, phillyrin (1), pinoresinol-beta-D-glucoside (2), pinoresinol di-beta-D-glucoside (3), phillyrin-2-O-beta-D-glucoside (6), phillyrin-6-O-beta-D-glucoside (7), and three secoiridoids, oleuropein (4), ligustroside (5) and angustifolioside B (8), have been isolated from the root bark of Chionanthus virginicus L. (Oleaceae), a raw material used in the commercial preparation of homeopathy tinctures. Compound 6 is a new lignan, and compounds 2, 3, 4, 5, 7 and 8 are described for the first time in the title plant. The structures of these compounds were elucidated by spectroscopic methods (NMR and HPLC-MS). An isocratic HPLC method for the rapid characterisation and quantification of the major constituents, 1 and 4, of the root bark is described and validated.     

Phyllanthus urinaria induces the Fas receptor/ligand expression and ceramide-mediated apoptosis in HL-60 cells

Phyllanthus urinaria (P. urinaria), a widely used herb medicine, was tested for the anticancer effect on human myeloid leukemia cells in this study. The water extract of P. urinaria induced the apoptosis of HL-60 cells as demonstrated by morphological change, DNA fragmentation and increased caspase-3 activity. However, normal human peripheral mononuclear cells remained viable under the same treatment. The P. urinaria-induced apoptosis of HL-60 cells was associated with the increased Bax gene expression and decreased Bcl-2 gene expression. In addition, the gene expressions of Fas receptor and Fas ligand, but not p53, were also induced in HL-60 cells dose- and time-dependently. The inhibitor of ceramide synthase, fumonisin B1, completely suppressed the apoptosis induced by P. urinaria and this inhibitory effect of fumonisin B1 could be eliminated by the addition of ceramide. It indicated that the activity of ceramide synthase is critical for the P. urinaria-induced apoptosis in HL-60 cells. The P. urinaria-induced apoptosis in HL-60 cells is mediated through a ceramide-related pathway.     

The quantification of ellagic acid in the crude extract of Phyllanthus amarus Schum. & Thonn. (Euphorbiaceae)

Introduction – Phyllanthus amarus Schum. & Thonn. (Euphorbiaceae), already well known for its antiviral, antihyperglycaemic and antihepatotoxic effects, is also investigated for its antimalarial activity. The major constituent of the crude extract of the whole plant was isolated and identified in this research to be ellagic acid, for which antiplasmodial activity already has been reported. Objective – Because of the potential of the plant and the interesting properties of ellagic acid, an analytical method can be useful for the standardisation of the extracts to allow further biological and pharmacological investigations. In order to obtain an easily performable and inexpensive method, an HPLC analysis was developed and validated. Methodology – The samples were dissolved in DMSO, ultrasonicated for 15 min, and diluted with 50% methanol. Analysis was performed using water and methanol containing 0.06% TFA and the peaks were detected at 254 nm. Results – Ellagic acid showed a linear relationship in the range of 1.74–20.91 µg/mL and a single‐point calibration was allowed. The method was shown to be precise with respect to time (RSD of 1.84%, 3 days, n = 6) and concentration (RSD of 2.54%, 3 levels, n = 6). The overall mean content of ellagic acid was 2.06%. A recovery experiment was performed and it showed an accuracy of 100.4%. Conclusion – Based on the obtained results, it can be concluded that the newly developed method is suitable for its purpose, namely the determination of ellagic acid in the crude extract of P. amarus. Copyright © 2010 John Wiley & Sons, Ltd.     

白珠树属5种药用植物木脂素苷含量的比较

Ｔ.Ｚ.Ｈｓｕ＆Ｒ.Ｃ.Ｆａｎｇ〕是治疗风湿病的民族药,目前还没有评价质量的定性和定量方法。一般认为水杨酸甲酯是可能的有效成分,但根据作者的最新研究[1～3],建议暂用滇白珠木脂素苷含量作滇白珠的质量标准,同时建立[4]和改进[5]了分析木脂素苷的高效液相法,并研究了木脂素苷在滇白珠体内的分布特点及季节和地区对含量的影响[6]。根据作者对白珠树属(ＧａｕｌｔｈｅｒｉａＫａｌｍｅｘＬ.)的研究[7],发现具有抗风湿活性是该属许多植物的一个共同特点,其中至少有15种(变种)植物在民间被用于风湿病治疗[8],为了分析和比较这些植物在相关化…     

余甘子总黄酮超声波辅助提取工艺的优化

以余甘子干粉为材料,利用超声波辅助提取余甘子总黄酮。单因素试验分析液料比、乙醇浓度、超声时间、超声功率对余甘子总黄酮提取率的影响,在此基础上,利用响应面分析法对提取工艺进行优化。结果表明,余甘子总黄酮最佳提取条件为：液料比82:1(V/W)、乙醇浓度45%、超声时间41 min、超声功率210 W,在此条件下余甘子总黄酮的实际提取率为6.12%。     

Structural determinants of plant lignans for growth of mammary tumors and hormonal responses in vivo

Low risk of breast cancer (BC) has been proposed to be associated with high intake of lignans. Some plant lignans are converted to mammalian lignans, e.g., enterolactone (ENL), suggested to be the biologically active lignan forms. Until now, little attention has been paid to the possible biological activities of plant lignans, even though some plant lignans are absorbed and present in serum and urine. In this study, we have investigated the antitumorigenic and endocrine-modulatory activities of different plant lignans in order to clarify the structure–activity relationships. 7-Hydroxymatairesinol (HMR) is converted to ENL, and both HMR and ENL inhibit the growth of 7,12-dimethylbenz[a]-anthracene (DMBA)-induced mammary cancer. Nortrachelogenin (NTG) resembles HMR, but has a hydroxyl group at C-8 instead of C-7 and is not converted to ENL. In DMBA-model, NTG showed no inhibition of tumor growth, but increased the uterine weight. Furthermore, life-long exposure to NTG increased uterine weight in immature females and ventral prostate weight in adult males. In contrast, life-long exposure to HMR had no effects on uterine or prostate weights at any age. Our results indicate that a difference in the position of one hydroxyl group results in distinct biological responses in vivo, as well as different lignan metabolite profiles.     

重要食药同源植物余甘子转录组微卫星特征分析

为全面了解余甘子转录组SSR位点的分布特征和变异规律,本研究利用Illumina Hiseq 4000平台对余甘子叶片转录组进行测序,通过MISA软件对获得的Unigenes进行SSR位点搜索和统计分析。结果发现9 538条包含SSR位点的Unigenes,共检测到9 991个SSR位点,平均每5.49 kB出现1个SSR。单碱基和二碱基为余甘子转录组SSR主要重复类型,分别占SSR总数的42.3%和30.79%。位于基因编码区的SSR位点共有1 731个,出现频率为0.039 SSRs/kB,优势重复类型为三碱基重复。余甘子转录组SSR中共有169种重复基元,其中所占比例最高的是A/T（42.10%）,其次是AG/CT（22.91%）和AAG/CTT（5.02%）。SSR各基元的重复次数波动于4~75次,且多数集中于4~20次。重复片段长度≥ 20 bp的SSR占21.20%,且SSR发生频率与片段长度呈显著负相关（P<0.01）,相关系数为-0.561。本研究获得的余甘子转录组SSR位点出现频率较高、分布密度较大、低级重复基元较多,重复次数较高、长片段较多,大多数SSR位点的多态性潜能较高,用于余甘子遗传多样性分析的潜力较大,为下一步余甘子转录组SSR标记的大规模开发和群体遗传学研究提供了重要的数据信息,进而为余甘子野生资源的保护和合理开发利用提供了参考依据。     

Determination of lignans in human plasma by liquid chromatography with coulometric electrode array detection

The presence of mammalian lignans, mainly enterolactone, in human plasma has been related to lower incidence of certain cancers and cardiovascular disease. The plant lignans secoisolariciresinol and matairesinol have been reported to be precursors of mammalian lignans, but recently other plant lignans relatively abundant in the diet have also been identified as precursors. To evaluate the importance and contribution of these new dietary precursors to the mammalian lignan formation in vivo, metabolic studies in human subjects must be carried out. For this purpose a method based on high-performance liquid chromatography using coulometric electrode array detection for the simultaneous determination of nine plant lignans and two mammalian lignans in human plasma was developed, validated, and shown to fulfill the reliability criteria.