Three types of enzymes complete the furanocoumarins core skeleton biosynthesis in Angelica sinensis.

Furanocoumarins (FCs) are widely distributed secondary metabolites found in higher plants, including Apiaceae, Rutaceae, Moraceae, and Fabaceae. They play a crucial role in the physiological functions of plants and are well-known for their diverse pharmacological activities. As a representative plant of the Apiaceae family, Angelica sinensis is highly valued for its medicinal properties and FCs are one of the main ingredients of A. sinensis. However, the biosynthetic mechanism of FCs in A. sinensis remains poorly understood. In this study, we successfully cloned and verified three types of enzymes using genome analysis and in vitro functional verification, which complete the biosynthesis of the FCs core skeleton in A. sinensis. It includes a p-coumaroyl CoA 2'-hydroxylase (AsC2'H) responsible for umbelliferone formation, two UbiA prenyltransferases (AsPT1 and AsPT2) that convert umbelliferone to demethylsuberosin (DMS) and osthenol, respectively, and two CYP736 subfamily cyclases (AsDC and AsOD) that catalyze the formation of FCs core skeleton. Interestingly, AsOD was demonstrated to be a bifunctional cyclase and could catalyze both DMS and osthenol, but had a higher affinity to osthenol. The characterization of these enzymes elucidates the molecular mechanism of FCs biosynthesis, providing new insights and technologies for understanding the diverse origins of FCs biosynthesis.

Corydalis tomentella Franch. Exerts anti-inflammatory and analgesic effects by regulating the calcium signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Corydalis tomentella Franch. is a perennial cespitose plant commonly used to treat stomachaches as a folk medicine. The C. tomentella total alkaloids have good protective effects against acute liver injury and potential anti-hepatoma and anti-Alzheimer's disease activities. AIM OF THE STUDY: To establish an effective purification process for total alkaloids from C. tomentella and investigate the mechanism of their anti-inflammatory effects. MATERIALS AND METHODS: Corydalis tomentella were purified using macroporous resin. Then the crude and purified C. tomentella extracts (cCTE and pCTE) were qualitatively analyzed using UPLC-Triple-TOF-MS/MS. The cCTE and pCTE were used to investigate and compare their anti-inflammatory effects on lipopolysaccharide (LPS)-induced RAW264.7 cells. Doses at 100, 200 and 400 mg/kg/d of pCTE were used to study their anti-inflammatory and analgesic activities in mice with xylene-induced ear swelling and acetic acid-induced writhing tests. Content of nitric oxide (NO), interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) were determined both in RAW264.7 cells and mice. Network pharmacology was used to predict the anti-inflammatory mechanism of C. tomentella, and the key enzymes were validated using qPCR and Western Blot analysis. Concentration of intracellular Ca2+ was detected using flow cytometric analysis. RESULTS: The C. tomentella total alkaloid purity increased from 6.29% to 47.34% under optimal purification conditions. A total of 54 alkaloids were identified from CTE. Both cCTE and pCTE could suppress the LPS-induced production of NO, IL-6, IL-1ß, and TNF-α in RAW264.7 cells. The pCTE exhibited a more potent anti-inflammatory effect; it also inhibited pain induced by xylene and acetic acid in mice. The calcium signaling pathway is associated with the anti-inflammatory and analgesic activities of C. tomentella. The mRNA expression of nitric oxide synthase (NOS) 2, NOS3 and calmodulin1 (CALM1) was regulated by C. tomentella through the reduction of inflammation-induced Ca2+ influx, and it also exhibited a more pronounced effect than the positive control (L-NG-nitro arginine methyl ester). CONCLUSIONS: Purified C. tomentella extract shows anti-inflammatory effect both in vitro and in vivo. It exerts anti-inflammatory and analgesic effects through the calcium signaling pathway by down-regulating NOS2 and CALM1 expression and up-regulating NOS3 expression in LPS-induced RAW264.7 cells, and decreasing intracellular Ca2+ concentration.

The chemical profiling of Salvia plebeia during different growth periods and the biosynthesis of its main flavonoids ingredients.

Salvia plebeia (Lamiaceae) is a valuable medicinal plant widely distributed across Asia and Oceania. However, the composition and accumulation patterns of its active ingredients in different organs during the growth and their biosynthetic mechanism remain unknown. Therefore, we conducted metabolite profiling, transcriptomic analysis, and biological functional verification to explore the distribution, accumulation, and biosynthesis mechanisms of flavonoids in S. plebeia. We identified 70 metabolites including 46 flavonoids, 16 phenolic acids, seven terpenoids, and one organic acid, of which 21 were previously unreported in S. plebeia. Combining metabolomic-transcriptomic analysis and biological functional verification, we identified the key genes involved in biosynthesis of its main active ingredients, hispidulin and homoplantaginin, including SpPAL, SpC4H, Sp4CL2, Sp4CL5, SpCHS1, SpCHI, SpFNS, SpF6H1, SpF6OMT1, SpF6OMT2, SpUGT1, SpUGT2, and SpUGT3. Using the identified genes, we reconstructed the hispidulin and homoplantaginin biosynthesis pathways in Escherichia coli, and obtained a yield of 5.33 and 3.86 mg/L for hispidulin and homoplantaginin, respectively. Our findings provide valuable insights into the changes in chemical components in different organs of S. plebeia during different growth and harvest stages and establishes a foundation for identifying and synthesizing its active components.

Chemometric Discrimination of Cichorium glandulosum Boiss. et Huet and Cichorium intybus L. via Their Metabolic Profiling, Antioxidative, and Hypoglycemic Activities.

Cichorium glandulosum Boiss. et Huet (CG) and Cichorium intybus L. (CI) are widely used as the main raw material of functional food with hepatoprotective and hypoglycemic effects. Due to the lack of comparison on the chemical ingredients and efficacy, they were often used imprecisely and interchangeably. It is necessary to distinguish between them. With the plant metabolomics based on high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS) and multivariate chemometric techniques, the chemical ingredients were characterized and 59 compounds between CG and CI were classified. As for antioxidative and hypoglycemic activities in vitro, CI extraction exhibited better antioxidant activity than CG, while CG extraction showed stronger hypoglycemic activity. Furthermore, a bivariate correlation between the chemical composition and efficacy of the extract was also analyzed, and three differentially strong correlation components between CI and CG were prepared, and the antioxidative and hypoglycemic efficacies were compared in vivo and different active phenotypes were obtained. Finally, we revealed chemical and biological differences between CG and CI, providing a basis for achieving better quality control and developing more effective functional foods.

Strong Reliable Electrostatic Actuation Based on Self-Clearing Using a Thin Conductive Layer.

Electrostatic adhesion, as a promising actuation technique for soft robotics, severely suffers from the failure caused by the unpredictable electrical breakdown. This study proposes a novel self-clearing mechanism for electrostatic actuators, particularly for electrostatic adhesion. By simply employing an enough thin conductive layer (e.g., <7 µm for copper), this method can spontaneously clear the conductor around the breakdown sites effectively once breakdowns onset and survive the actuator shortly after the electrical damage. Compared with previous self-clearing methods, which typically rely on new specific materials, this mechanism is easy to operate and compatible with various materials and fabrication processes. In our tests, it can improve the maximum available voltage by 260% and the maximum electrostatic adhesive force by 276%. In addition, the robustness and repeatability of the self-clearing mechanism are validated by surviving consecutive breakdowns and self-clearing of 173 times during 65 min. This method is also demonstrated to be capable of recovering the electrostatic pad from severe physical damages such as punctures, penetrations, and cuttings successfully and enabling stable and reliable operation of the electrostatic clutch, or gripping, for example, even after the short-circuit takes place for hundreds of times. Therefore, the proposed self-clearing method sheds new light on high performance and more extensive practical applications of electrostatic actuators in the future.

Endophytic Penicillium oxalicum CX-1 prevented Phytophthora cactorum blight on Salvia miltiorrhiza and promoted plant growth.

AIM: The main purpose of this study was to study the preventive effect of Penicillium sp. CX-1 on Phytophthora cactorum causing Salvia miltiorrhiza blight and its positive effect on plant growth. METHODS AND RESULTS: The endophytic strain CX-1 was isolated from the medicinal plant Corydalis saxicola Bunting and identified as Penicillium oxalicum. The growth inhibitory capacity of CX-1 against Ph. cactorum was 74.4% in the strain co-culture test and 86.2% in filtrate-modified plates. In the pot experiment, the in vivo control of CX-1 against Ph. cactorum in S. miltiorrhiza was 36.0%, which was higher than that of an anti-Phytophthora fungicide (23.4%). In addition, CX-1 had a potent ability to solubilize phosphate and also showed the ability to produce the plant hormone indole-3-acetic acid (IAA) and siderophores, which increase the bioavailability of iron to plants. It was demonstrated through pot experiments that CX-1 could significantly promote plant growth. As determined by real-time quantitative PCR, the expression of some S. miltiorrhiza tanshinone-related biosynthesis genes was significantly upregulated following colonization by CX-1. CONCLUSION: Strain CX-1 could effectively inhibit Ph. cactorum, the causative agent of S. miltiorrhiza blight, and significantly promoted the growth of plants through several different routes.

Influence of different pretreatments and drying methods on the chemical compositions and bioactivities of Smilacis Glabrae Rhizoma.

BACKGROUND: The processing of medicinal plant materials is one of the important factors influencing the components and biological activities of TCMs. Smilax glabra Roxb. is an herbal vine widely distributed in China, and its dried rhizome (Smilacis Glabrae Rhizoma, SGR) is often used in traditional medicines and functional foods. The processing methods of fresh cutting for SGR slices have been included in ancient Chinese herbal works, some local standards of TCMs, and the current Chinese Pharmacopoeia. Nevertheless, to date, the scientific basis for the processing of fresh medicinal materials for SGR slices has not been revealed. METHODS: To optimize the processing method for preparing SGR slices from the fresh rhizomes, the chemical compositions of the un-pretreated and pretreated (boiling, steaming) samples before and after drying (sun-drying, shade-drying, oven-drying), and the contents of astilbin isomers in dried SGR were analyzed by UHPLC-Q-TOF-MS/MS and UHPLC-DAD methods, respectively. Then, the antioxidant, anti-inflammatory, xanthine oxidase and α-glucosidase inhibitory activities of the prepared SGR slices were investigated by biological assays. RESULTS: A total of fifty-two compounds were identified from the un-pretreated and pretreated samples and a total of forty-nine compounds were identified from the subsequently dried samples. After pretreated by boiling and steaming, the contents of neoastilbin, neoisoastilbin, and isoastilbin in the prepared samples all increased. As a quality marker of SGR, the content of astilbin was unchanged or decreased slightly compared with that in the un-pretreated samples. During the drying process, the contents of the four astilbin stereoisomers in the un-pretreated samples increased significantly, while those in the pretreated samples had a slight increase or decrease. The effects of different processing methods were sorted according to the bioactivities of the prepared SGR. As a result, SGR slices prepared with no pretreatment followed by a sun-drying process have a higher astilbin content, better bioactivities and more energy savings, representing the optimum processing method for SGR slices. CONCLUSIONS: This study reveals the scientific basis for the processing of fresh medicinal materials for SGR slices. The results provide scientific information for the quality control of SGR and its rational applications in herbal medicines and functional foods.

An integrated study of Violae Herba (Viola philippica) and five adulterants by morphology, chemical compositions and chloroplast genomes: insights into its certified plant origin.

BACKGROUND: Viola philippica Cav. is the only original plant for Violae Herba, as described in the Chinese Pharmacopoeia. The quality of this crude drug is affected by several adulterants from congeneric Viola species, and the authentic plant origin of Violae Herba is still controversial. Genome-based identification offers abundant genetic information and potential molecular markers that can be used for the authentication of closely related species. This study aims to investigate the certified origin of Violae Herba and to develop more effective markers for these easily confused species at the genetic level. METHODS: We compared the morphology and chemical composition of 18 batches of commercial samples and six widespread medicinal Viola plants used as Violae Herba or its substitutes by TLC and HPLC-Triple-TOF-MS/MS analyses. The complete chloroplast genomes of these species were sequenced and analyzed, including the general features, repeat sequences, mutational hotspots and phylogeny. The complete chloroplast genomes used as superbarcodes and some specific barcodes screened from mutational hotspots were tested for their ability to distinguish Viola species. RESULTS: A comparative study showed that Violae Herba is a multi-origin traditional Chinese medicine. Commercial decoction pieces and the standard reference drug were mainly derived from V. prionantha, clashing with the record in the Chinese Pharmacopoeia. Chloroplast genome analyses of V. philippica and five adulterants indicated that sequence divergence was relatively low within Viola species. By tree-based approaches, the complete chloroplast genomes showed a better discrimination ability and phylogenetic resolution for each Viola species. These results indicate that the whole chloroplast genomes can be used as superbarcodes to differentiate Viola medicinal plants. More specific DNA barcodes could be further developed from the Viola chloroplast genomes for more efficient and rapid identification of commercial Violae Herba and its adulterants. CONCLUSIONS: This study has implications for chloroplast genome-based phylogenetic analysis and the authentication of multiple Viola species used as Violae Herba. The legal origin recorded in the Chinese Pharmacopoeia should be further revised to V. prionantha, in line with the commercial Violae Herba in the TCM markets.

Iris domestica (iso)flavone 7- and 3'-O-Glycosyltransferases Can Be Induced by CuCl2.

In many plants, isoflavones are the main secondary metabolites that have various pharmacological activities, but the low water solubility of aglycones limits their usage. The O-glycosylation of (iso)flavones is a promising way to overcome this barrier. O-glycosyltransferases (UGTs) are key enzymes in the biosynthesis of (iso)flavonoid O-glycosides in plants. However, limited investigations on isoflavonoid O-UGTs have been reported, and they mainly focused on legumes. Iris domestica (L.) Goldblatt et Mabberley is a non-legume plant rich in various isoflavonoid glycosides. However, there are no reports regarding its glycosylation mechanism, despite the I. domestica transcriptome previously being annotated as having non-active isoflavone 7-O-UGTs. Our previous experiments indicated that isoflavonoid glycosides were induced by CuCl2 in I. domestica calli; therefore, we hypothesized that isoflavone O-UGTs may be induced by Cu2+. Thus, a comparative transcriptome analysis was performed using I. domestica seedlings treated with CuCl2, and eight new active BcUGTs were obtained. Biochemical analyses showed that most of the active BcUGTs had broad substrate spectra; however, substrates lacking 5-OH were rarely catalyzed. Real-time quantitative PCR results further indicated that the transcriptional levels of BcUGTs were remarkably induced by Cu2+. Our study increases the understanding of UGTs and isoflavone biosynthesis in non-legume plants.

Dynamic analysis of secondary metabolites in various parts of Scrophularia ningpoensis by liquid chromatography tandem mass spectrometry.

The roots of Scrophularia ningpoensis are used as traditional medicines for thousands of years in China, nevertheless the stems and leaves were discarded as non-medicinal parts. Modern research have indicated the chemical constituents in the stems and leaves are similar to the identified in the roots, and the therapeutic effects of stems and leaves are superior to roots for some disease. In the study, the chemical constituents in roots, stems and leaves of S. ningpoensis were analyzed qualitatively by HPLC-Q-TOF-MS/MS. 40 compounds including 17 iridoid glycosides, 15 phenylpropanoids and 8 flavonoids were identified. Meantime, the dynamic accumulations of six index constituents in various parts were measured by HPLC-DAD. The results indicated the S. ningpoensis stems contained high content of aucubin (30.09 mg/g) and harpagide (28.4 mg/g) in August, and the leaves contained high content of harpagoside (12.02 mg/g) in July. The study provides the basis for the full development and utilization of the resource of stems and leaves from S. ningpoensis.

Separation of acteoside and linarin from Buddlejae Flos by high-speed countercurrent chromatography and their anti-inflammatory activities.

Buddleja officinalis Maxim., a deciduous, flowering shrub, is used as a traditional Chinese medicine; the bioactivity of B. officinalis is primarily due to flavonoids and phenylethanoid glycosides. In the study, acteoside and linarin were successfully isolated from B. officinalis by high-speed countercurrent chromatography with a two-phase solvent system composed of ethyl acetate: n-butanol: water (5:0.8:5, v/v/v). The purities of acteoside and linarin were determined to be 97.3 and 98.2%, respectively, using one-step high-speed countercurrent chromatography separation. The chemical structures of the two compounds were identified by electrospray ionization-mass spectrometry and nuclear magnetic resonance. After separation, the anti-inflammatory effects of the two compounds were evaluated using lipopolysaccharide-induced human umbilical vein endothelial cells. Acteoside and linarin inhibited the expression of nitric oxide, tumor necrosis factor α and interleukin 1ß, which demonstrated that acteoside and linarin possessed anti-inflammatory activity.

A mild and efficient extraction method for polysaccharides from Sinonovacula constricta and study of their structural characteristic and antioxidant activities.

The aim of this paper is to develop a mild and efficient extraction method for polysaccharides from Sinonovacula constricta (SCP) using enzyme extraction, and analyze the structural characteristics and antioxidant activities of the two purified polysaccharide fractions (SCP-1 and SCP-2). Firstly, enzyme extraction conditions were optimized, and the conditions were found to be, as follows: enzymolysis time 173.0 min, pH 8.2, enzymolysis temperature 50.0 ℃ and enzyme content 4.0%. Comparison between enzymatic extraction and water extraction was obtained from visual, UV-visible and IR spectrum images. The results clearly indicate that there is no significant difference between them with regard to the composition of the SCP fraction, but the polysaccharide content produced by enzymatic extraction is higher. Then, the physicochemical properties and structural characteristics of SCP-1 and SCP-2 were investigated using FT-TR, UV, GC and HPGPC. The carbohydrate content, sulfuric radicals and uronic acids of the two fractions were detected. Both SCP-1 and SCP-2 were mainly consisted of glucose, but their molecular weights were different. In addition, compared the Fe2+ chelating activity, ABTS+ radical and superoxide radical scavenging activity, and lipid peroxidation inhibition activity of SCP-1 and SCP-2, it turned out that SCP-2 had stronger antioxidant activity than SCP-1.

Chemical profiles and quality evaluation of Buddleja officinalis flowers by HPLC-DAD and HPLC-Q-TOF-MS/MS.

The dried flowers and inflorescences of Buddleja officinalis Maxim are used as traditional medicines in China, and aqueous extracts of the flowers have also been used since ancient times as a yellow rice colorant at local festivals. In this study, HPLC-Q-TOF-MS/MS was used to determine the overall chemical composition of this medicine-food plant. A total of 54 compounds, including 23 flavonoids, 19 phenylethanoid glycosides, 7 alkaloids and 5 other compounds, were detected in the methanol extracts of the herb using this method. Among them, 35 compounds were found firstly in this herb. HPLC fingerprints were also developed, together with a method for the simultaneous quantification of 11 constituents that could be used for quality evaluation of B. officinalis. Fingerprint analysis, using 28 characteristic fingerprint peaks, was used to assess the similarities among 12 samples collected from different geographic areas and showed that the similarity was >0.900. Simultaneous quantification of 11 markers in B. officinalis was then performed to determine consistency of quality. Additionally, the total phenolic content and antioxidant capacity of extracts of the 12 samples of B. officinalis flowers were measured using spectroscopic methods. B. officinalis was found to have good antioxidant capacity and to be a potential natural antioxidant. The highest antioxidant capacity was found in the samples from Guizhou, Sichuan and Guangxi Province. Our results provide valuable information for further understanding and exploiting the herb.

Qualitative and Quantitative Analysis of C-glycosyl-flavones of Iris lactea Leaves by Liquid Chromatography/Tandem Mass Spectrometry.

Iris lactea Pall. var. chinensis (Fisch.) Koidz. is a traditional medicinal plant resource. To make full use of the I. lactea plant resources, constituents of I. lactea leaves were determined by high performance liquid chromatography (HPLC)-quadrupole time-of-flight tandem mass spectrometry and 22 C-glycosylflavones were identified or tentatively identified. Optimal extraction of I. lactea leaves was established via single factor investigations combined with response surface methodology. Then, HPLC coupled with a diode array detector was used to quantitatively analyze the six main components of 14 batches of I. lactea leaves grown in different areas. The results showed the C-glycosylflavones were the main components of I. lactea leaves, and the total contents of detected components were relatively stable for the majority of samples. These results provide a foundation for the development and utilization of I. lactea leaves.

Ameliorative effects of protodioscin on experimental diabetic nephropathy.

BACKGROUND: Diabetic nephropathy is one of the most common and serious complications of diabetes mellitus. HYPOTHESIS/PURPOSE: The present study aimed to investigate the effects of protodioscin on renal damage in high-fat diet-fed and streptozotocin-induced diabetic rats. METHODS: After 4 weeks of feeding a high-fat diet, male Sprague-Dawley rats were injected 35 mg/kg streptozotocin intraperitoneally. The diabetic rats were divided into 4 groups, and treated orally with carboxymethylcellulose sodium, metformin, or protodioscin (20 or 40 mg/kg). After 12 weeks of treatment, blood, urine, and renal tissue were collected for biochemical and histological examination. RESULTS: Protodioscin significantly reduced the levels of blood glucose, serum creatinine, and blood urea nitrogen, and also the excretion of urinary protein and albumin in diabetic rats. Histological examinations showed that protodioscin ameliorated the diabetes-induced glomerular and tubular pathological changes. Furthermore, protodioscin significantly reduced the renal concentrations of total cholesterol, triglycerides, free fatty acids, phospholipids, and TNF-α. CONCLUSION: These results indicate that protodioscin has ameliorative effects on diabetic nephropathy.

Baicalin regulates SirT1/STAT3 pathway and restrains excessive hepatic glucose production.

Sirtuin 1 (SirT1) and signal transducer and activator of transcription 3 (STAT3) oppositely regulate hepatic gluconeogenic genes and the association remains to be elucidated. Baicalin is a natural flavonoid with beneficial effects on glucose and lipid metabolism. This study aims to investigate the effect of baicalin on hepatic gluconeogenesis with focus on the regulation of fatty acid mobilization and SirT1/STAT3 pathway. In HFD feeding or fasting state, hepatic gluconeogenesis and fatty acid oxidation induced SirT1 expression due to the increased nicotinamide adenine dinucleotide+ (NAD+) contents. Baicalin reduces endogenous glucose production via suppression of hepatic gluconeogenesis and decreased SirT1 induction via reducing NAD+ accumulation in an energy-sensing way. Fasting increased SirT1 protein in STAT3 immunoprecipitation products and less in the liver of baicalin-treated mice, indicating that baicalin blocked the binding of SirT1 to STAT3 and thus preserved STAT3 acetylation. SirT1 knockdown enhanced the protective effect of baicalin on pyruvate-induced STAT3 phosphorylation and acetylation, these results further indicated that the regulation of STAT3 activity by baicalin was dependent on SirT1. Moreover, HFD feeding increased gene expression for PGC-1α in the liver, but the transcriptional regulation was inhibited by baicalin treatment. SirT1 overexpression and STAT3 inhibition enhanced pyruvate-mediated PGC-1α gene expression, suggesting that deacetylation of STAT3 by SirT1 is required for PGC-1α activity on hepatic gluconeogenesis. Taken together, these results showed that baicalin restrained HGP via inhibiting SirT1 activity coupled with STAT3 acetylation and subsequent PGC-1α suppression, suggesting that hepatic SirT1 and STAT3 pathway may provide therapeutic advantages for the control of hyperglycemia.

Global Transcriptome Analyses Reveal Differentially Expressed Genes of Six Organs and Putative Genes Involved in (Iso)flavonoid Biosynthesis in Belamcanda chinensis.

Belamcanda chinensis (L.) DC., a perennial herb of the family Iridaceae, is rich in a variety of (iso)flavonoids with significant organ-specific distribution and has a swollen rhizome that is widely used in East Asia as a traditional medicine. In the present study, comprehensive transcriptomes of six organs (root, rhizome, aerial stem, leaf, flower, and young fruit) of B. chinensis were obtained by high-throughput RNA-sequencing and de novo assembly. A total of 423,661 unigenes (mean length = 618 bp, median length = 391 bp) were assembled and annotated in seven databases: Non-redundant protein sequences, Nucleotide sequences, Swiss-Prot, Protein family database, euKaryotic Ortholog Groups, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO). A total of 4995 transcription factors were identified, including 408 MYB, 182 bHLH, 277 AP2/ERF, and 228 WRKY genes. A total of 129 cytochrome P450 unigenes belonging to 10 divergent clans were identified and grouped into clades in a phylogenetic tree that showed their inferred evolutionary relationship. Differentially expressed unigenes among the six organs were subjected to GO and KEGG enrichment analysis to profile the functions of each organ. Unigenes associated with (iso)flavonoid biosynthesis were then profiled by expression level analysis. Additionally, the complete coding sequences of six predicted enzymes essential to the (iso)flavonoid pathway were obtained, based on the annotated unigenes. This work reveals clear differences in expression patterns of genes among the six organs and will provide a sound platform to understand the (iso)flavonoid pathways in B. chinensis.

Organ-Specific Metabolic Shifts of Flavonoids in Scutellaria baicalensis at Different Growth and Development Stages.

Scutellaria baicalensis Georgi is a traditional Chinese herbal medicine mainly containing flavonoids that contribute to its bioactivities. In this study, the distributions and dynamic changes of flavonoid levels in various organs of S. baicalensis at different development stages were investigated by UHPLC-QTOF-MS/MS and HPLC-DAD methods. The results indicated that the metabolic profiles of S. baicalensis changed with growth and development. During the initial germination stage, the seeds mainly contained flavonols. With growth, the main kinds of flavonoids in S. baicalensis changed from flavonols to flavanones and flavones. The results also revealed that the accumulation of flavonoids in S. baicalensis is organ-specific. The flavones without 4'-OH groups mainly accumulate in the root and the flavanones mainly accumulate in aerial organs. Dynamic accumulation analysis showed that the main flavonoids in the root of S. baicalensis accumulated rapidly before the full-bloom stage, then changed to a small extent. The results suggested the proper harvest time for the aerial parts was at the initial stage of reproductive growth and the flower buds should be collected before flowering. This study deepening the knowledge of S. baicalensis should provide valuable information for guiding the scientific cultivation of this plant and the development and utilization of S. baicalensis.

Molecular diversity analysis of Tetradium ruticarpum (WuZhuYu) in China based on inter-primer binding site (iPBS) markers and inter-simple sequence repeat (ISSR) markers.

"Wu zhu yu", which is obtained from the dried unripe fruits of Tetradium ruticarpum (A. Jussieu) T. G. Hartley, has been used as a traditional Chinese medicine for treatment of headaches, abdominal colic, and hypertension for thousands of years. The present study was designed to assess the molecular genetic diversity among 25 collected accessions of T. ruticarpum (Wu zhu yu in Chinese) from different areas of China, based on inter-primer binding site (iPBS) markers and inter-simple sequence repeat (ISSR) markers. Thirteen ISSR primers generated 151 amplification bands, of which 130 were polymorphic. Out of 165 bands that were amplified using 10 iPBS primers, 152 were polymorphic. The iPBS markers displayed a higher proportion of polymorphic loci (PPL = 92.5%) than the ISSR markers (PPL = 84.9%). The results showed that T. ruticarpum possessed high loci polymorphism and genetic differentiation occurred in this plant. The combined data of iPBS and ISSR markers scored on 25 accessions produced five clusters that approximately matched the geographic distribution of the species. The results indicated that both iPBS and ISSR markers were reliable and effective tools for analyzing the genetic diversity in T. ruticarpum.

Optimization of the Extraction Conditions for Buddleja officinalis Maxim. Using Response Surface Methodology and Exploration of the Optimum Harvest Time.

The Box-Behnken design was used to evaluate the effects of the methanol concentration (60-100%), liquid to solid ratio (20:1 to 40:1 mL/g) and extraction time (20-40 min) on the yield of 11 constituents from Buddleja officinalis Maxim using ultrasound-assisted extraction. The Derringer's desirability function approach showed that the modified optimum extraction conditions were: 76% methanol concentration, 33 min extraction time and a 34:1 mL/g solvent to solid ratio. Under these conditions, the experimentally measured yields of the compounds were in good agreement with the predicted values. An accurate and sensitive method was also established using high-performance liquid chromatography with diode-array detection for the simultaneous determination of the 11 compounds in Buddleja officinalis. The newly developed method was used to determine the amounts of bioactive components in Buddleja officinalis during four different growth stages. According to these results, we recommend that the full blossom stage is the best time for harvesting this plant to obtain the highest yield of crude materials.