Antioxidant property of an active component purified from the leaves of paraquat-tolerant Rehmannia glutinosa

Abstract

Acteoside extracted from the leaves of Rehmannia glutinosa was examined to determine the mechanism(s) of its antioxidant properties. The deoxyribose assay system showed that acteoside has a high redox potential as electron donor, which generates hydroxyl radicals in an Fe³⁺-dependent manner similar to ascorbic acid. However, the antioxidant properties of acteoside differ from those of ascorbic acid in that the superoxide anion-mediated reduction of nitroblue tetrazolium was actively inhibited by acteoside but not by ascorbic acid. Acteoside protected cells against glucose oxidase-mediated cytotoxicity and apoptosis in a dose-dependent manner. In addition, acteoside had immune stimulating effects, as shown by the acteoside-mediated increase in the level of DNA synthesis, viability, and cytokine secretion in mouse splenocytes. Moreover, acteoside inhibited the gelatinolytic activity of MMP proteins in a dose-dependent manner. Considering these results and the fact that acteoside is a water-soluble natural product, acteoside might have potential as a preventative treatment for oxidative stress-mediated diseases and have possibilities in the cosmetic industry.     

Phytochemical Profiles and Antioxidant Activity of Rehmannia glutinosa from Different Production Locations

The chemical components and antioxidant activity of 16 Rehmannia glutinosa samples were investigated to reveal the high‐quality raw resource for pharmaceutical products. 22 main chemical components were detected with significant content differences (P<0.05). The contents of 14 substances reached the maximum in S1 sample such as catalpol (6.74 mg g^?1), rehmaionoside A (1.93 mg g^?1) and rehmannioside D (5.13 mg g^?1). However, the content distribution of the other eight substances had no obvious change regulation. Three antioxidant evaluation methods commonly showed that S1 sample had strong antioxidant activity with a low IC₅₀ value of 0.022 mg mL^?1, a high ABTS value of 524.196 μmol equiv. Trolox g^?1, and a high FRAP value of 200.517 μmol equiv. Trolox g^?1. Considered the medicinal value, S1 had high quality based on the present phytochemical profiles and antioxidant activity. These results also indicated that the root extracts of R. glutinosa could become useful supplement for pharmaceutical products as new antioxidant agents.     

The activities of antioxidant enzymes in response to oxidative stresses and hormones in paraquat-tolerant Rehmannia glutinosa plants

All members of R. glutinosa show the unique characteristic of intrinsic tolerance to paraquat (PQ). Antioxidant enzymes have been proposed to be the primary mechanism of PQ resistance in several plant species. Therefore, the antioxidant enzyme systems of R. glutinosa were evaluated by comparatively analyzing cellular antioxidant enzyme levels, and their responses of oxidative stresses and hormones. The levels of ascorbate peroxidase (APX), glutathione reductase (GR), non-specific peroxidase (POX), and superoxide dismutase (SOD) were 7.3-, 4.9-, 2.7- and 1.6-fold higher in PQ-tolerant R. glutinosa than in PQ-susceptible soybeans. However, the activity of catalase (CAT) was about 12-fold higher in the soybeans. The activities of antioxidant enzymes reduced after PQ treatment in the two species, with the exception of POX and SOD in R. glutinosa, which increased by about 40 %. Interestingly, the activities of APX, SOD and POX in R. glutinosa, relative to those in soybeans, were further increased by 49, 67 and 93 % after PQ treatment. The considerably higher intrinsic levels, and increases in the relative activities of antioxidant enzymes in R. glutinosa under oxidative stress support the possible role of these enzymes in the PQ tolerance of R. glutinosa. However, the relatively lower levels of SOD versus PQ tolerance, and the mixed responses of antioxidant enzymes to stresses and hormones, suggest a possible alternative mechanism(s) for PQ tolerance in R. glutinosa.     

Secretion of catalpol from Rehmannia glutinosa roots to the rhizosphere

The concentrations of catalpol in the culture solutions, roots, stems and leaves of Rehmannia glutinosa Libosch. were determined by HPLC. The biological activity of catalpol was detected with Arabidopsis thaliana L. seedlings. The results showed that all R. glutinosa Libosch. vegetative organs contained catalpol. Catalpol was also found in culture solutions in which the R. glutinosa Libosch. seedlings were grown. Catalpol inhibited seed germination and root growth in A. thaliana L., respectively, at concentration 80 and 20 μmol/dm³. These results suggest that R. glutinosa Libosch. may produce catalpol and secrete it into the culture solutions. Catalpol acts as an antimicrobial and allelopathic agent; the secretion of catalpol into the R. glutinosa Libosch. rhizosphere may provide a competitive advantage for root establishment through local suppression of soil microorganisms and inhibition of the growth of competing plant species. However, autotoxicity of catalpol in R. glutinosa Libosch. may occur, which may be relevant to the obstacle in its continuous cropping.     

Four caffeoyl glycosides from callus tissue of Rehmannia glutinosa

From the methanolic extract of callus tissue of Rehmannia glutinosa four phenolic glycosides and one aliphatic glycoside were isolated. Two of the phenolic glycosides were identified as acteoside and forsythiaside and the structures of the other two were elucidated as 3,4-dihydroxy-β-phenethyl-0-β-D-glucopyranosyl-(1 → 3) -4-0-calfeoyl-β-D-glucopyranoside and 3,4-dihydroxy-β-phenethyl-0-β-D-glucopyranosyl-(1 → 3) -0-α-L-rhamnopyranosyl-(1 → 6)-4-0-caffeoyl-β-D-glucopyranoside.     

地黄连作的生理生态特性

选择具有典型连作障碍效应的药用植物地黄(Rehmannia glutinosa)为试验材料, 以正茬地黄为对照, 研究连作条件下地黄植株的生理生态特性变化。结果表明, 该试验条件下地黄的连作障碍效应起始于苗期, 在栽种后60天时, 连作引起植株细胞膜质过氧化作用, 使膜的正常结构和功能受到损伤; 重茬地黄的光合速率(P_n)、气孔导度(G_s)和胞间CO₂浓度(C_i)显著低于对照, 而气孔限制值(L_S) 33.97%, 显著高于对照, 同期重茬地黄叶绿素含量(Chl)也开始显著低于对照。相关分析表明, P_n与G_s、Chl含量相关(相关系数分别为0.977和0.814)。对植株叶片叶肉细胞的电镜观察显示, 连作条件下地黄光合细胞结构在其生长中期开始发生变化。因此, 在地黄生长早期由于(连作)环境胁迫植株叶片细胞内活性氧、自由基积累所带来的膜结构损伤, 导致了叶绿素含量的降低, 而气孔关闭以及叶绿素含量的降低共同造成连作地黄早期光合能力降低, 生长受阻, 从而表现出明显的障碍效应。     

Shoot regeneration from mesophyll protoplasts and leaf explants of Rehmannia glutinosa

Mesophyll protoplasts obtained from leaves of shoot cultures of Rehmannia glutinosa were cultured in Murashige and Skoog (1962) liquid or liquid-over-agar medium containing 2.0 mg L^?1 naphthaleneacetic acid and 0.5 mg L^?1 benzylamino purine. An amino acid mixture of glutamine, arginine, glycine, and aspartic acid promoted sustained protoplast division, with an average plating efficiency of 27%. Protoplast-derived colonies formed callus which readily regenerated shoots on fransfer to Murashige and Skoog based agar medium with 2.0 mg L^?1 indoleacetic acid and 1.0 mg L^?1 benzylamino purine. Leaf explants also showed a marked capacity for shoot regeneration in culture.     

地黄的组织培养和快速繁殖

1　植物名称　地黄 (Ｒｅｈｍａｎｎｉａｇｌｕｔｉｎｏｓａ) ,又名生地。2　材料类别　茎段、茎尖。3　培养条件　 ( 1 )无菌苗获得培养基 :ＭＳ ;( 2 )茎尖分化培养基 :ＭＳ ＮＡＡ 0 .0 1ｍｇ·Ｌ- 1 (单位下同 ) 6 ＢＡ 1 ＧＡ30 .2 ;( 3)增殖培养基 :1 /2ＭＳ 6 ＢＡ     

地黄组织培养及植株再生的研究

以地黄根茎所获无菌苗为材料,对其愈伤组织诱导、分化和再生植株的获取进行了初步研究。结果表明：取叶片、茎段、叶柄进行愈伤组织诱导,筛选出最适培养基为MS附加2,4-D0．5mg／L、BA1．0mg／L,愈伤组织诱导率可达100％。将叶片接种在分化培养基中,诱导不定芽,其最适分化培养基为MS附加BA 3mg／L、NAA 0．1mg／L,分化率为77．5％。试管苗在改良的MS(大量与微量元素、铁盐和有机物质各1／2)附加NAA 0．05mg／L的培养基上,经过15～20d培养,生根率可达100％。     

怀地黄ISSR扩增条件优化的研究

用CTAB法提取怀地黄嫩叶DNA,进行简单重复间序列标记(ISSR)分析.通过单因子实验分别研究了退火温度、Taq酶单位、Mg2+浓度、dNTP浓度、引物浓度和模板DNA浓度对ISSR-PCR反应的影响,找出各自的合适条件,而且每一个合适条件确定以后都被作为后续研究的一个条件.通过各个因子的组合研究建立了适宜于怀地黄ISSR分析的扩增体系25 μL PCR反应体积,1×Taq DNA酶缓冲液(10 mmol/L Tris-HCl,50 mmol/L KCl,0.1% Trion X-100,pH9.0 ),2.5 mmol/L MgCl2,1.5～1.0 U Taq酶,60 ng模板DNA,0.4 μmmol/L引物,各0.4 mmol/L的dATP、dGTP、dCTP和dTTP.合适的退火温度为53～55℃.为用ISSR技术分析鉴定怀地黄种质资源奠定了良好的基础.     

Identification of autotoxic compounds in fibrous roots of Rehmannia (Rehmannia glutinosa Libosch.)

Rehmannia is a medicinal plant in China. Autotoxicity has been reported to be one of the major problems hindering the consecutive monoculture of Rehmannia. However, potential autotoxins produced by the fibrous roots are less known. In this study, the autotoxicity of these fibrous roots was investigated. Four groups of autotoxic compounds from the aqueous extracts of the fibrous roots were isolated and characterized. The ethyl acetate extracts of these water-soluble compounds were further analyzed and separated into five fractions. Among them, the most autotoxic fraction (Fr 3) was subjected to GC/MS analysis, resulting in 32 identified compounds. Based on literature, nine compounds were selected for testing their autotoxic effects on radicle growth. Seven out of the nine compounds were phenolic, which significantly reduced radicle growth in a concentration-dependent manner. The other two were aliphatic compounds that showed a moderate inhibition effect at three concentrations. Concentration of these compounds in soil samples was determined by HPLC. Furthermore, the autotoxic compounds were also found in the top soil of the commercially cultivated Rehmannia fields. It appears that a close link exists between the autotoxic effects on the seedlings and the compounds extracted from fibrous roots of Rehmannia.     

怀地黄叶片中总环烯醚萜苷及总苯乙醇苷含量快速分析模型的建立

本文旨在建立地黄叶片中总环烯醚萜苷及苯乙醇苷定量分析模型。利用紫外-可见分光光度法测定不同种质怀地黄生育期内的128份地黄叶片中总环烯醚萜苷及总苯乙醇苷的含量,并将其作为基础值,结合地黄叶片的近红外光谱图,利用TQ8.0分析软件结合偏最小二乘法(PLS),分别建立地黄叶片中总环烯醚萜苷及总苯乙醇苷的定量分析模型。地黄叶片中总苯乙醇苷定量校正模型决定系数(R2)为0.998 2,校正均方根偏差(RMSEC)为0.089 9,预测均方决定差(RMSEP)为0.142,交叉验证均方根偏差(RMSECV)为0.707 2;总环烯醚萜苷定量校正模型的内部交叉验证决定系数(R2)为0.972 1,校正均方差(RMSEC)为0.259,预测均方决定差(RMSEP)为0.095 4,交叉验证均方根偏差(RMSECV)为0.869 4。预测值与实测值差异无统计学意义。该定量模型可用于怀地黄叶片中总环烯醚萜苷及总苯乙醇苷含量的快速测定。     

不同品种的地黄低聚糖含量的比较研究

采用HPLc-ELSD法,对6个不同品种(包括野生和栽培)地黄的低聚糖进行含量测定.测定结果表明:不同的地黄品种低聚糖含量差异比较显著;栽培品种‘85 -5’、‘9302’和‘北京1号’低聚糖含量比较接近;野生品种‘BA3’的水苏糖含量普遍较低,而蔗糖含量比较高;‘北京1号’水苏糖含量较高,总低聚糖含量比较均衡,占干重...     

Fungicidal property of active component derived from Acorus gramineus rhizome against phytopathogenic fungi

The fungicidal property of Acorus gramineus rhizome-derived materials against Botrytis cineria, Erysiphe graminis, Phytophthora infestans, Puccinia recondita, Pyricularia grisea, and Rhizoctonia solani was tested using a whole plant method in vivo, and was compared with a synthetic fungicide and seven commercially available A. gramineus rhizome-derived components. The responses varied according to the plant pathogen tested. At 2000 mg/l, the hexane fraction of A. gramineus rhizomes showed strong fungicidal activities against R. solani and P. infestans. The results indicated at least one of the fungicidal actions of alpha-asarone and asaronaldehyde derived from A. gramineus rhizomes.     

怀地黄SRAP扩增体系的建立与引物的筛选

为建立适合怀地黄SRAP-PCR分子标记技术体系,通过单因子实验分别研究了DNA模板浓度、TaqDNA聚合酶浓度、Mg2+浓度、引物浓度以及dNTP浓度对怀地黄SRAP扩增反应的影响,确立了适合怀地黄SRAP最佳反应体系为:在25μL的反应体系中,模板DNA量20ng/25μL、2.5mmol/LMg2+、0.32μmol/L的上下游引物、0.30μmol/L的dNTP以及2.5UTaq酶,并利用确定的体系从88个引物组合中筛选出12对适合怀地黄SRAP-PCR反应的引物。     

Isolation of paraquat-tolerant mutants from tomato cell cultures

Summary Tomato callus clones selected for the ability to grow at paraquat concentrations lethal to wild-type cells were found at an approximate frequency of 5×10^–8 per viable cell. Diploid plants were regenerated from nine of the nineteen paraquat-tolerant callus clones isolated. Although some of these plants appeared normal, others had altered morphology and reduced vigor and fertility. New callus cultures initiated from these regenerated plants typically had at least a 30-fold increase over the wild type in tolerance to paraquat. Tests on callus from sexual progeny showed that the paraquat-tolerant phenotypes of clones PQT⁴, PQT⁶, and probably also PQT¹³ resulted from dominant nuclear mutations, but the number of loci involved is not yet known. Paraquat spray experiments indicated that slight paraquat-tolerance was expressed at the plant level in PQT¹³, but not in any of the other clones tested.     

怀地黄块根的形态发生和结构发育

观察了怀地黄(Rehmannia glutinosa cv．Hueichingensis Hsiao．)块根的形态发生和生长发育过程中的形态结构变化。采用怀地黄的传统栽培方法,即用上一年的块根作母根进行繁殖,分别从母根和不定芽的茎基部发生不定根。怀地黄不定根的初生结构和维管形成层的发生与一般双子叶植物相同,但其次生生长却有两种方式,即正常次生生长和异常次生生长。一类不定根的形成层产生的次生结构与一般双子叶植物相同,即次生木质部中主要是导管,而薄壁细胞较少。这类不定根其次生生长为正常次生生长(normal secondary growth),是担负吸收和固着作用的正常根。另一种类型的不定根,其形成层产生的次生木质部含有大量的薄壁细胞,少量的导管分散在薄壁细胞之间。这种次生生长为异常次生生长(anomalous secondary growth),从而使不定根膨大,形成块根。因此,怀地黄的药用部分在起源和结构上都属于根的性质,其药用部分应称为块根。     

Antioxidant phenylpropanoid glycosides from the leaves of Wasabia japonica

From the MeOH extract of the leaves of W. japonica, seven phenylpropanoid gentiobiosides (1-7) were isolated along with eight known phenylpropanoids (8-15). Structures of 1-7 were determined based on spectroscopic data and chemical evidence. The activity of compounds 1-15 to scavenge superoxide anion radicals was investigated using an electron spin resonance (ESR) method.