Antioxidative Activities and Active Compounds of Extracts from Catalpa

In order to screen the Catalpa plant with high antioxidant activity and confirm the corresponding active fractions from Catalpa ovata G. Don, C. fargesii Bur., and C. bungei C. A. Mey., total flavonoid contents and antioxidant activities of the extracts/fractions of Catalpa plant leaves were determined. The determined total flavonoid content and antioxidant activity were used as assessment criteria. Those compounds with antioxidant activity were isolated with silica gel column chromatography and ODS column chromatography. Our results showed that the total flavonoid content in C. bungei C. A. Mey. (30.07 mg/g·DW) was the highest, followed by those in C. fargesii Bur. (25.55 mg/g·DW) and C. ovata G. Don (24.96 mg/g·DW). According to the determination results of total flavonoid content and antioxidant activity in 3 clones of leaves of C. bungei C. A. Mey., the total flavonoid content and antioxidant activity in crude extracts from C. bungei C. A. Mey. 6 (CA6) leaves were the highest. Moreover, the results showed that the total flavonoid content and antioxidant activities of ethyl acetate (EA) fraction in ethanol crude extracts in CA6 leaves were the highest, followed by n-butanol, petroleum ether (PE), and water fractions. Two flavonoid compounds with antioxidant activity were firstly isolated based on EA fraction. The two compounds were luteolin (1) and apigenin (2), respectively

Metabolite Profiles, Bioactivity, and HPLC Fingerprint of Different Varieties of Eucommia ulmoides Oliv.: Towards the Utilization of Medicinal and Commercial Chinese Endemic Tree

Eucommia ulmoides Oliv. is widely regarded in China as a precious medicinal and commercial endemic tree. Due to cross-breeding or natural variation of E. ulmoides, the metabolite composition may vary significantly, making control of the medical quality difficult. In order to improve the rational development and utilization, the quality of seven varieties of E. ulmoides were evaluated based on metabolite profiles (total phenolic, total flavonoid, gutta-percha, aucubin, geniposidic acid, chlorogenic acid, geniposide, pinoresinol diglucoside, rutin, hyperoside, and astragalin), bioactivities (in vitro, in vivo antioxidant activities, and antibacterial activities) and HPLC fingerprint combined with chemometrics analysis. On this basis, the differences of medicinal parts (leaf and bark) were further carried out. For the traditional use of bark, Purple-leaf E. ulmoides was the most suitable. For the use of leaf, Qinzhong 1 and Purple-leaf E. ulmoides were appropriate. HPLC fingerprint analysis showed that significant differences in metabolite profiles exist among seven varieties of E. ulmoides. Combined with chemometrics analysis, seven varieties of E. ulmoides were divided into three groups from the use of leaf and bark. The analysis not only evaluated quality of seven varieties of E. ulmoides, but also could distinguish different varieties and different regions of origin. The results can provide theoretical basis for E. ulmoides resources utilization and cultivation of fine varieties

Transcriptome Sequencing in Response to Salicylic Acid in Salvia miltiorrhiza.

Salvia miltiorrhiza is a traditional Chinese herbal medicine, whose quality and yield are often affected by diseases and environmental stresses during its growing season. Salicylic acid (SA) plays a significant role in plants responding to biotic and abiotic stresses, but the involved regulatory factors and their signaling mechanisms are largely unknown. In order to identify the genes involved in SA signaling, the RNA sequencing (RNA-seq) strategy was employed to evaluate the transcriptional profiles in S. miltiorrhiza cell cultures. A total of 50,778 unigenes were assembled, in which 5,316 unigenes were differentially expressed among 0-, 2-, and 8-h SA induction. The up-regulated genes were mainly involved in stimulus response and multi-organism process. A core set of candidate novel genes coding SA signaling component proteins was identified. Many transcription factors (e.g., WRKY, bHLH and GRAS) and genes involved in hormone signal transduction were differentially expressed in response to SA induction. Detailed analysis revealed that genes associated with defense signaling, such as antioxidant system genes, cytochrome P450s and ATP-binding cassette transporters, were significantly overexpressed, which can be used as genetic tools to investigate disease resistance. Our transcriptome analysis will help understand SA signaling and its mechanism of defense systems in S. miltiorrhiza

Hydrogen Peroxide Is Involved in Salicylic Acid-Elicited Rosmarinic Acid Production in Salvia miltiorrhiza Cell Cultures

Salicylic acid (SA) is an elicitor to induce the biosynthesis of secondary metabolites in plant cells. Hydrogen peroxide (H2O2) plays an important role as a key signaling molecule in response to various stimuli and is involved in the accumulation of secondary metabolites. However, the relationship between them is unclear and their synergetic functions on accumulation of secondary metabolites are unknown. In this paper, the roles of SA and H2O2 in rosmarinic acid (RA) production in Salvia miltiorrhiza cell cultures were investigated. The results showed that SA significantly enhanced H2O2 production, phenylalanine ammonia-lyase (PAL) activity, and RA accumulation. Exogenous H2O2 could also promote PAL activity and enhance RA production. If H2O2 production was inhibited by NADPH oxidase inhibitor (IMD) or scavenged by quencher (DMTU), RA accumulation would be blocked. These results indicated that H2O2 is secondary messenger for signal transduction, which can be induced by SA, significantly and promotes RA accumulation

Hydrogen Peroxide Is Involved in Salicylic Acid-Elicited Rosmarinic Acid Production in Salvia miltiorrhiza Cell Cultures

Salicylic acid (SA) is an elicitor to induce the biosynthesis of secondary metabolites in plant cells. Hydrogen peroxide (H2O2) plays an important role as a key signaling molecule in response to various stimuli and is involved in the accumulation of secondary metabolites. However, the relationship between them is unclear and their synergetic functions on accumulation of secondary metabolites are unknown. In this paper, the roles of SA and H2O2 in rosmarinic acid (RA) production in Salvia miltiorrhiza cell cultures were investigated. The results showed that SA significantly enhanced H2O2 production, phenylalanine ammonia-lyase (PAL) activity, and RA accumulation. Exogenous H2O2 could also promote PAL activity and enhance RA production. If H2O2 production was inhibited by NADPH oxidase inhibitor (IMD) or scavenged by quencher (DMTU), RA accumulation would be blocked. These results indicated that H2O2 is secondary messenger for signal transduction, which can be induced by SA, significantly and promotes RA accumulation

Lipopolysaccharide Enhances Tanshinone Biosynthesis via a Ca2+-Dependent Manner in Salvia miltiorrhiza Hairy Roots

Tanshinones, the major bioactive components in Salvia miltiorrhiza Bunge (Danshen), are synthesized via the mevalonic acid (MVA) pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway and the downstream biosynthesis pathway. In this study, the bacterial component lipopolysaccharide (LPS) was utilized as a novel elicitor to induce the wild type hairy roots of S. miltiorrhiza. HPLC analysis revealed that LPS treatment resulted in a significant accumulation of cryptotanshinone (CT) and dihydrotanshinone I (DTI). qRT-PCR analysis confirmed that biosynthesis genes such as SmAACT and SmHMGS from the MVA pathway, SmDXS and SmHDR from the MEP pathway, and SmCPS, SmKSL and SmCYP76AH1 from the downstream pathway were markedly upregulated by LPS in a time-dependent manner. Furthermore, transcription factors SmWRKY1 and SmWRKY2, which can activate the expression of SmDXR, SmDXS and SmCPS, were also increased by LPS. Since Ca2+ signaling is essential for the LPS-triggered immune response, Ca2+ channel blocker LaCl3 and CaM antagonist W-7 were used to investigate the role of Ca2+ signaling in tanshinone biosynthesis. HPLC analysis demonstrated that both LaCl3 and W-7 diminished LPS-induced tanshinone accumulation. The downstream biosynthesis genes including SmCPS and SmCYP76AH1 were especially regulated by Ca2+ signaling. To summarize, LPS enhances tanshinone biosynthesis through SmWRKY1- and SmWRKY2-regulated pathways relying on Ca2+ signaling. Ca2+ signal transduction plays a key role in regulating tanshinone biosynthesis in S. miltiorrhiza

mRNA profiling of SA induced <i>S</i>. <i>miltiorrhiza</i> cell cultures by RNA-seq.

<p>(a) The common and unique expression profiles among sample groups. Numbers represent expressed unigenes in control (0 h) and SA (2 h and 8 h) treated cell cultures. (b) Number of DEGs found among different sample groups, according to a FDR< 0.01 and FC ≥ 2 or ≤ -2. T1, T2 belong to control group, T3, T4 and T5 belong to treatment group of SA induction for 2 h, T6, T7 and T8 belong to treatment group of SA induction for 8 h. (c) Length distribution of the 50 778 assembled unigenes (digital details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147849#pone.0147849.s007" target="_blank">S3 Table</a>).</p

KEGG classifications of the DEGs in <i>S</i>. <i>miltiorrhiza</i> cell cultures under SA induction.

<p>A total of 532 DEGs were assigned to 104 KEGG pathways. The DEGs predominantly belonged to ‘Plant hormone signal transduction’ and ‘Plant-pathogen interaction’. The number of DEGs belonging to each category are provided.</p

The most enriched GO terms (level 2) in unigenes of <i>S</i>. <i>miltiorrhiza</i> cell cultures.

<p>All 17 867 unigenes predominantly belonged to ‘Catalytic activity’ and ‘Binding’ under Molecular function, ‘Cell part’ and ‘Cell’ under Cellular component, and ‘Metabolic process’ and ‘Cellular process’ under Biological process. The number of unigenes belonging to each category are provided.</p

Functional analysis of DEGs in <i>S</i>. <i>miltiorrhiza</i> cell cultures after SA induction.

<p>(a) Hierarchically clustered heat map for the expression profile of DEGs (reflected as log₂ FC when compared to control), which consist of 1584 up-regulated (left), 1492 down-regulated (middle) and 2240 inconsistently regulated DEGs (right) after 8h SA induction. Blue represent repression, whereas red represent induction. (b) Analysis of biological process category of DEGs including up-regulated (red) and down-regulated (green) in <i>S</i>. <i>miltiorrhiza</i> cells after 8h SA induction. Enrichment was measured by comparing the number of DEGs from each category with the total number of genes for that GO term and using Fisher’s exact test. Significance indicated <i>p</i>-values below 0.01 or between 0.01 and 0.05, respectively.</p