地黄炮制过程氨基酸组分分析

利用氨基酸自动分析仪分别测定加工前后地黄中的总氨基酸和游离氨基酸质量分数。分析、比较炮制前后地黄的氨基酸成分变化。炮制前后地黄的氨基酸种类及含量有明显变化,多数氨基酸的含量在炮制后明显下降甚至消失。结论:炮制前后的地黄中氨基酸质量分数差别明显,其中碱性氨基酸质量分数的差别尤其显著,该差异可能是由于炮制过程中发生美拉德反应造成的。     

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.     

地黄源库关系的变化及其与连作障碍的关系

通过正茬剪叶模拟连作地黄光合源不足条件下,测定分析植株源与库的关系,应用同位素示踪技术分析连作地黄光合产物的分配及内源激素的变化,以探讨地黄的连作障碍机制。结果表明:剪叶处理对植株的影响不能达到连作障碍程度,其库容及其活性大于连作地黄;连作地黄出现光合产物滞留于地上部,库的活性影响了植株光合产物向库的运输,在生长前期植株内源激素发生了变化;因此断定,连作地黄库容及其活性过低是限制地黄块根膨大的重要因素,库通过不同激素对源的反馈调节,抑制了地上源的光合能力及光合产物向库的分配,导致连作障碍。     

Alleviatory effect of spent Pleurotus eryngii Quel substrate on replant problem of Rehmannia glutinosa Libosch

Rehmannia glutinosa Libosch. is a medicinal plant cultivated at a commercial scale in China. However, replanting problems result in a severe decline in both the biomass and quality of its roots, which are of greatest medicinal value. This study attempted to remediate the replant soil using spent Pleurotus eryngii Quel substrate for alleviating this issue, and to investigate the underlying mechanisms. Results showed that R. glutinosa grew successfully in fresh soil and remedial replant soil, while no roots were harvested in the unremedied replant soil. Overall, the nutritional status in the remedial soil was higher than that of the unremedied and fresh soil, while the concentration of allelopathic phenolic acids was lower. When planted in unremedied soil, the growth of five plant pathogens was induced and one beneficial fungus was suppressed. When planted in remedied soil, four out of the five pathogens were suppressed, while two beneficial fungi were identified in the remedial soil. This study suggests that the spent P. eryngii substrate significantly alleviates the replant problem of R. glutinosa, and that the alleviatory function reflects a synergetic effect, including the supplementation of soil nutrition, the degradation of allelochemicals, and the remediation of unbalanced microbial community.     

Total phenolics, flavonoids and antioxidant activity of Saptarangi (Salacia chinensis L.) fruit pulp

Salacia chinensis L. has various beneficial properties including antidiabetic and antioxidant activity. The S. chinensis fruit pulp (SCFP) was extracted with four different solvents (Methanol, ethanol, acetone and water) and was screened for total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activity (AOA). The AOA was assessed by evaluating the 1, 1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP) and metal chelating assay. Methanolic SCFP extract exhibited the highest phenolic content (3.20?±?0.12 mg GAE/g FW) whereas, ethanolic extract showed highest flavonoid content (0.31?±?0.68 mg RE/g FW). The methanolic extract possesses highest antioxidant activity towards DPPH (92.44 %), FRAP (1.939 O.D) and metal chelating activity (74.16 %). AOA (DPPH and FRAP) was significantly correlated with TPC. The results indicated that SCFP is a good natural source of antioxidant compounds for use in food and pharmaceutical industry.     

Hairy root cultures of Rehmannia glutinosa and production of iridoid and phenylethanoid glycosides

Hairy root lines through the infection of Agrobacterium rhizogenes strain (A4) were established from shoot tips and leaves of Rehmannia glutinosa Libosch. Ten lines of hairy roots were selected on the basis of biomass increase in half-strength Gamborg medium (¹/₂ B5). Transgenic status of the roots was confirmed by polymerase chain reaction using rolB and rolC specific primers. Iridoid glycosides (catalposide, loganin, aucubin and catalpol) and phenylethanoid glycosides (verbascoside and isoverbascoside) identified using HPLC?CESI?CMS, and their contents were compared with untransformed root culture and roots of 1-year-old field-grown plants of R. glutinosa by RP-HPLC. The growth and production of secondary metabolites in ten hairy root lines varied considerably as to the media. Woody plant (WP) medium displayed higher growth in terms of fresh (FW) and dry weights (DW) compared to ¹/₂ B5 medium. High-yielding hairy root lines produced higher amounts of loganin, catalposide, verbascoside and isoverbascoside in comparison to the untransformed root culture and roots of 1-year-old field-grown plants. The highest amounts of catalposide and loganin in transformed roots were 4.45?mg?g^?1 DW (RS-2 hairy root line) and 4.66?mg?g^?1 DW (RS-1 hairy root line), respectively. Aucubin and catalpol were detected in some lines in trace amounts. The highest amounts of verbascoside (16.9?mg?g^?1 DW) and isoverbascoside (3.46?mg?g^?1 DW) were achieved in RS-2 root line. The contents of catalposide, verbascoside and isoverbascoside in high-producing lines were several times higher than in untransformed root culture and roots of R. glutinosa plants grown in soil. Loganin and aucubin could not be detected in roots of field-grown plants. However, the levels of catalpol were much lower in the in vitro roots.     

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     

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.     

Total phenolics,flavonoids and antioxidant properties of three Ceropegia species from Western Ghats of India

The aim of present work was to assess the total phenolic content (TPC), total flavonoid content (TFC), phenolic compounds and antioxidant properties of various extracts of three Ceropegia spp.: Ceropegia spiralis, Ceropegia panchganiensis and Ceropegia evansii from Western Ghats of India. TPC of the samples varied from 0.3 ± 0.2 to 28.5 ± 0.3 mg TAE/g FW, whereas, TFC of the samples ranged between 0.1 ± 0.1 and 15.3 ± 0.3 mg RE/g FW. The major phenolic compounds identified were gallic acid, vanillin, cathechol and ferulic acid. All the extracts possess 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging activity, ferric reducing antioxidant power (FRAP) as well as metal chelating ability and this was also supported by significant correlation with TPC and TFC. To the best of our knowledge, this is the first paper presenting comprehensive data on TPC, TFC, phenolic profile and antioxidant properties of the Ceropegia spp.     

The antioxidant activity of phloretin: the disclosure of a new antioxidant pharmacophore in flavonoids

Phloretin is a dihydrochalcone flavonoid that displays a potent antioxidant activity in peroxynitrite scavenging and the inhibition of lipid peroxidation. Comparison with structurally related compounds revealed that the antioxidant pharmacophore of phloretin is 2,6-dihydroxyacetophenone. The potent activity of 2,6-dihydroxyacetophenone is due to stabilisation of its radical via tautomerisation. The antioxidant pharmacophore in the dihydrochalcone phloretin, i.e., the 2,6-dihydroxyacetophenone group, is different from the antioxidant pharmacophores previously reported in flavonoids.     

Identification of Novel and Conserved microRNAs in Rehmannia glutinosa L. by Solexa Sequencing

We report the use of a high throughput (Solexa) sequencing platform to obtain the sequences of a large number of microRNAs (miRNAs) from the Chinese medicinal plant Rehmannia glutinosa L. The analysis of >14,000,000 sequence reads allowed the identification of seven novel miRNAs (comprising seven miRNA families) and conserved 89 miRNAs (comprising 25 miRNA families) in R. glutinosa. The relative abundance of some these miRNAs was checked using quantitive PCR (qRT-PCR) analysis. Some putative target genes of the novel miRNAs were predicted.     

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.     

Oxidation of flavonoids by hypochlorous acid: reaction kinetics and antioxidant activity studies

Flavonoids, plant polyphenols, ubiquitous components of human diet, are excellent antioxidants. Hypochlorous acid (HOCl), produced by activated neutrophils, is highly reactive chlorinating and oxidizing species. It has been reported earlier that flavonoids are chlorinated by HOCl. Here we show that flavonoids from flavonol subclass are also oxidized by HOCl, but only if the latter is in a large molar excess (≥?10). The kinetics of this reaction was studied by stopped-flow spectrophotometry, at different pH. We found that flavonols were oxidized by HOCl with the rate constants of the order of 10⁴–10⁵ M^?1 s^?1 at pH 7.5. Antioxidant activity of HOCl-modified flavonoids was measured by 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) method. Slightly higher antioxidant activity, compared to parent compounds, was observed for flavonols after their reaction with equimolar or moderate excess of HOCl whereas flavonols treated with high molar excess of HOCl exhibited decrease in antioxidant activity. The mechanism of flavonoid reaction with HOCl at physiological pH is proposed, and biological consequences of this reaction are discussed.     

半枝莲黄酮的超声提取及其抗氧化活性

通过单因素和正交试验获得超声波辅助乙醇提取半枝莲黄酮的最佳工艺条件,考察了黄酮提取物对油脂的抗氧化活性及对羟基自由基的清除效果,并与常用的抗氧化剂作比较。结果表明,当乙醇体积分数为50%、料液比1∶30（g/mL）、超声提取时间25 min、浸泡温度65℃时为最佳条件,此时黄酮的得率为3.0%,且该提取物对羟基自由基清除效果随浓度的增大而升高,对油脂氧化有较强的抑制作用。     

Interactions of flavonoids with iron and copper ions: a mechanism for their antioxidant activity

The metal chelating properties of flavonoids suggest that they may play a role in metal-overload diseases and in all oxidative stress conditions involving a transition metal ion. A detailed study has been made of the ability of flavonoids to chelate iron (including Fe 3+ ) and copper ions and its dependence of structure and pH. The acid medium may be important in some pathological conditions. In addition, the ability of flavonoids to reduce iron and copper ions and their activity-structure relationships were also investigated. To fulfil these objectives, flavones (apigenin, luteolin, kaempferol, quercetin, myricetin and rutin), isoflavones (daidzein and genistein), flavanones (taxifolin, naringenin and naringin) and a flavanol (catechin) were investigated. All flavonoids studied show higher reducing capacity for copper ions than for iron ions. The flavonoids with better Fe 3+ reducing activity are those with a 2,3-double bond and possessing both the catechol group in the B-ring and the 3-hydroxyl group. The copper reducing activity seems to depend largely on the number of hydroxyl groups. The chelation studies were carried out by means of ultraviolet spectroscopy and electrospray ionisation mass spectrometry. Only flavones and the flavanol catechin interact with metal ions. At pH 7.4 and pH 5.5 all flavones studied appear to chelate Cu 2+ at the same site, probably between the 5-hydroxyl and the 4-oxo groups. Myricetin and quercetin, however, at pH 7.4, appear to chelate Cu 2+ additionally at the ortho -catechol group, the chelating site for catechin with Cu 2+ at pH 7.4. Chelation studies of Fe 3+ to flavonoids were investigated only at pH 5.5. Only myricetin and quercetin interact strongly with Fe 3+ , complexation probably occurring again between the 5-hydroxyl and the 4-oxo groups. Their behaviour can be explained by their ability to reduce Fe 3+ at pH 5.5, suggesting that flavonoids reduce Fe 3+ to Fe 2+ before association.     

Effect of NaCl on fatty acids, phenolics and antioxidant activity of Nigella sativa organs

The objective of this study was to investigate the effect of salinity on growth, fatty acid composition, phenol content and antioxidant activity of Nigella sativa organs. Plants were grown hydroponically under NaCl stress (0, 20 40 and 60 mM). The results indicated that salinity affected N. sativa growth. The fatty acid composition of the leaves and the roots was investigated for the first time and major fatty acids were linolenic acid (58.1%) in the leaves and linoleic (43.9%) and palmitic (33.3%) acids and in the roots. Total fatty acid (TFA) content of the leaves decreased at 60 mM NaCl while root TFA increased at 20 and 40 mM NaCl. Moreover, the fatty acid composition was affected by NaCl; in leaves, the double bond index (DBI) decreased accompanied by a decrease of the level of linolenic acid which reached 14% at 60 mM NaCl. However, root DBI degree increased at 40 at 60 mM NaCl provoked mainly by the increase of the amount of linoleic acid by 15 and 8%, respectively, and the decrease of the amount of palmitic acid by 20 and 14%, respectively. Salt stress increased total polyphenol and individual phenolic acid contents in shoots. Moreover, the antiradical activity of the shoots (DPPH) increased at 60 mM NaCl. However, in roots, the total polyphenol content and the antiradical activity decreased sharply with increasing NaCl doses. Data reported here revealed the variation of fatty acids and phenolic compound contents in different organs of N. sativa, and the possible role of theses changes in the plant salt response were discussed.     

Growth characteristics and catalpol production in chinese foxglove (Rehmannia glutinosa Liboschitz) hairy roots transformed withAgrobacterium rhizogenes ATCC15834

Hairy root clones ofRehmannia glutinosa were established via transformation withAgrobacterium rhizogenes ATCC15834. To optimize the culturing conditions for both root growth and catalpol production, effects of various combinations of seven basal media, pH, and carbon sources were examined under darkness. The fastest root growth was obtained in an SH medium containing 4% sucrose (pH 5.8); the highest catalpol content (0.54% of dry weight) was achieved in a WPM medium supplemented with 4% sucrose (pH 5.8). Effects of plant growth regulators and chitosan were also investigated. IAA at 2 mg L^-1 significantly increased root lengths and the frequency of lateral roots. Chitosan (50 mg L^-1) and CA₃ (0.5 mg L^-1) induced catalpol production, with contents calculated at 0.7% dry weight and 0.65% dry weight, respectively.     

Structure-property studies on the antioxidant activity of flavonoids present in diet

The screening of natural flavonoids for their bioactivity as antioxidants is usually carried out by determinination of their profile as chain-breaking antioxidants, by the evaluation of their direct free radical-scavenging activity as hydrogen- or electron-donating compounds. Since this may not be the only mechanism underlying the antioxidant activity it is important to check the ability of these compounds to act as chelators of transition metal ions. Accordingly, in the present study the acidity constants of catechin and taxifolin, as well as the formation constants of the corresponding copper (II) complexes, were investigated by potentiometry and/or spectrophotometry. Moreover, a detailed quantitative examination of the coordination species formed is presented. In addition, the partition coefficients of both catechin and taxifolin in a biomimetic system (micelles) were determined, since these properties may also contribute to the antioxidant behavior of this type of compound. The log P values determined depend on the electrostatic interactions of the compounds with the differently charged micelles (the highest values were obtained for zwitterionic and cationic micelles). The prooxidant behavior of the compounds was assessed through the oxidation of 2'-deoxyguanosine, induced by a Fenton reaction, catalyzed by copper. The data obtained reveal that the flavonoids under study did not present prooxidant activity, in this particular system. The results obtained are evidence of a clear difference among the pKa, the complexation properties, and the lipophilicity of the flavonoids studied, which can partially explain their distinct antioxidant activity. The most stable geometries of the free compounds were determined by theoretical (ab initio) methods, in order to properly account for the electron correlation effects which occur in these systems, thus allowing a better interpretation of the experimental data.