Comparative transcriptome analysis reveals candidate genes related to the sex differentiation of Schisandra chinensis.

Schisandra chinensis is a monoecious plant with unisex flowers. The fruit of S. chinensis is of high medical with economic value. The yield of S. chinensis fruit is related to the ratio of its female and male flowers. However, there is little research on its floral development and sex differentiation. To elucidate the possible mechanism for the sex differentiation of S. chinensis, we collected 18 samples of female and male flowers from three developmental stages and performed a comparative RNA-seq analysis aimed at identifying differentially expressed genes (DEGs) that may be related to sex differentiation. The results showed 936, 7179, and 6890 differentially expressed genes between female and male flowers at three developmental stages, respectively, and 466 candidate genes may play roles in sex differentiation. KEGG analysis showed genes involved in the flavonoid biosynthesis pathway and DNA replication pathway were essential for the development of female flowers. 51 MADS-box genes and 10 YABBY genes were identified in S. chinensis. The DEGs analysis indicated that MADS-box and YABBY genes were strongly related to the sex determination of S. chinensis. RT-qPCR confirmed the RNA-seq results of 20 differentially expressed genes, including three male-biased genes and 17 female-biased genes. A possible regulatory model of sex differentiation in S. chinensis was proposed according to our results. This study helps reveal the sex-differentiation mechanism of S. chinensis and lays the foundation for regulating the male-female ratio of S. chinensis in the future.

[Anti-inflammatory active component in raw materials of Ligustici Rhizoma et Radix based on spectrum-effect relationship].

The present study investigated the correlation of UPLC fingerprints of raw materials of Ligustici Rhizoma et Radix samples with the anti-inflammatory effect and explored the pharmacodynamic material basis for the anti-inflammatory activity. UPLC fingerprints of 18 batches of raw materials of Ligustici Rhizoma et Radix samples were established for the determination of the content of eight components. The toe swelling rate and the content of IL-1ß, IL-6, and PGE2 in rats with toe inflammation induced by carrageenin were measured. Canonical correlation analysis was used to study the spectrum-effect relationship. Cluster analysis indicated that chemical components of Ligusticum sinense and L. jeholense were similar. Methanol extracts of L. sinense, L. jeholense, and Conioselinum vaginatum significantly reduced the toe swelling rate and the content of IL-1ß, IL-6 and PGE2 in swollen tissues. The anti-inflammatory effect of C. vaginatum was weaker than that of L. sinense and L. jeholense. The results of spectrum-effect relationship indicated that there was an obvious correlation between chemical components and pharmacodynamic indexes. In UPLC fingerprints, compounds 1, 3(chlorogenic acid), 4(cryptochlorogenic acid), 5, 6(ferulic acid), 7(isochlorogenic acid B), 9, 11, 13, 15, 16, 17, 18(coniferyl ferulate), 19, 20(N-butylphthalide), 21, 22, and 23 were significantly correlated with anti-inflammation, among which compounds 5, 11, 13, 15, 17, 21, and 23 had negative correlation. This study screened out the effective components with anti-inflammatory activity in raw materials of Ligustici Rhizoma et Radix, which was of great significance to improve the quality evaluation system of raw materials of Ligustici Rhizoma et Radix.

[Research progress in DIR gene of lignan biosynthesis in medicinal plants].

The active components, mainly derived from secondary metabolites of medicinal plants, are the material basis for the efficacy of medicinal plants. Lignans, the secondary metabolites in plants with high bioactivity, are widely distributed in a variety of plant species, and their antiviral, antitumor, antibacterial, and antioxidant activities have been proved in clinical practice. Generally, lignans are diverse in structures with many chiral centers, and most of them are optically active. The biosynthesis of lignans depends on the oxidative coupling reaction through site selection and stereo selection, which impedes synthesized lignans to form racemates, but makes them in a three-dimensional configuration. Dirigent protein(DIR) plays an important role in guiding location selection and stereo selection of lignans in biosynthesis. In vitro studies on lignan biosynthesis have shown that racemic end products are obtained in the absence of DIR proteins, while the products in a three-dimensional configuration can be yielded in the presence of DIR proteins, indicating that DIR proteins play an asymmetric role in the biosynthesis of plant secondary metabolites. The present study reviewed the biolo-gical significance of DIR protein, the cloning of DIR gene, gene structure, catalytic mechanism, and the research progress in Isatis indigotica, Eucommia ulmoides, Forsythia suspensa, Salvia miltiorrhiza, Panax pseudoginseng var. notoginseng, and Schisandra chinensis, which provides a reference for the follow-up research of DIR gene.

[Identification and characterization of DIR gene family in Schisandra chinensis].

Dirigent(DIR) proteins are involved in the biosynthesis of lignin, lignans, and gossypol in plants and respond to biotic and abiotic stresses. Based on the full-length transcriptome of Schisandra chinensis, bioinformatics methods were used to preliminarily identify the DIR gene family and analyze the physico-chemical properties, subcellular localization, conserved motifs, phylogeny, and expression patterns of the proteins. The results showed that a total of 34 DIR genes were screened and the encoded proteins were 156-387 aa. The physico-chemical properties of the proteins were different and the secondary structure was mainly random coil. Half of the DIR proteins were located in chloroplast, while the others in extracellular region, endoplasmic reticulum, cytoplasm, etc. Phylogenetic analysis of DIR proteins from S. chinensis and the other 8 species such as Arabidopsis thaliana, Oryza sativa, and Glycine max demonstrated that all DIR proteins were clustered into 5 subfamilies and that DIR proteins from S. chinensis were in 4 subfamilies. DIR-a subfamily has the unique structure of 8 ß-sheets, as verified by multiple sequence alignment. Finally, through the analysis of the transcriptome of S. chinensis fruit at different development stages, the expression pattern of DIR was clarified. Combined with the accumulation of lignans in fruits at different stages, DIR might be related to the synthesis of lignans in S. chinensis. This study lays a theoretical basis for exploring the biological functions of DIR genes and elucidating the biosynthesis pathway of lignans in S. chinensis.

[Molecular identification and efficacy analysis of herbs at Orussey Herbal Market, Phnom Penh, Cambodia].

Cambodia is rich in medicinal plant resources. One hundred and thirty-three medicinal material samples, including the hole herb, root, stem/branch, leaf, flower, fruit, seed, and resin, were collected from the Orussey Herbal Market in Phnom Penh, Cambodia, and then authenticated by ITS and psbA-trnH. A total of 46 samples were identified based on ITS sequences, belonging to 24 families, 40 genera, and 42 species. A total of 100 samples were identified by psbA-trnH sequences to belong to 42 families, 77 genera, and 84 species. A total of 103 samples were identified by two DNA barcodes. According to the morphological characteristics of the medicinal materials, 120 samples classified into 50 species, 86 genera, and 86 families were identified, and the majority of them were from Zingiberaceae, Fabaceae, and Acanthaceae. Such samples have been commonly used in traditional Cambodian medicine, Ayurvedic medicine, Unani medicine, traditional Chinese medicine, and ethnomedicine, but different medical systems focus on different functional aspects of the same medicinal material. The results of this study have demonstrated that DNA barcoding has a significant advantage in identifying herbal products, and this study has provided basic data for understanding the traditional medicinal materials used in Cambodia.

[Simultaneous determination of six major isosteroidal alkaloids in Beimu by UPLC-ELSD].

An UPLC method was established for the direct determination of six major bioactive isosteroidal alkaloids, namely peimisine, imperialine, sipeimine-3-D-glucoside, verticinone, verticine and hupehenine from the bulbus of Fritillaria(Beimu), a commonly used antitussive traditional Chinese medicinal(TCM) herb. An Acquity UPLC~(TM) CSH C_(18) column(2.1 mm×100 mm, 1.7 µm) was used for all analysis. The investigated six compounds were all separated with gradient mobile phase consisting of 0.02% diethylamine-water-methanol at a flow rate of 0.3 mL·min~(-1). The temperature of sample manager was set at 20 ℃. Drift tube temperature was 45 ℃, and spray parameter was 40% with injection volume of 1 µL. Then, the further quality assessment of Beimu was carried out by cluster analysis(CA) and principal component analysis(PCA). The investigated all had good linearity(r≥0.998 9) over the tested ranges. The method is simple, accurate and reproducible, and can be used for determining the content of six major bioactive isosteroidal alkaloids.

[Decipherment of herb medicine Nanwuweizi and Wuweizi in Chinese ancient literature].

Nanwuweizi( Schisandrae Sphenantherae Fructus) and Wuweizi( Schisandrae Chinensis Fructus) have long-term history of use as common traditional Chinese medicines since the Eastern Han Dynasty( AD.25-220 year).However their information are always confused in ancient literature because they were both used as " Wuweizi". Nanwuweizi and Wuweizi are faced with problems such as confused distribution of producing areas,unclear source plants and efficacy characteristics,which limit modern resource development and application. Based on ancient literatures of materia medica,this study conducted a systematic review from several aspects,i.e. the name,distribution of producing areas,source plants,efficacy characteristics and processing of the two medicines in ancient time. This study clarified five main aspects,as following,ancient production areas and corresponding modern distribution areas; source plants used for medicinal purposes in ancient time; application period and application scope; efficacy characteristics in clinical application;processing method. This study provides a reference for evaluating the quality and for their clinical application and reasonable development of Nanwuweizi and Wuweizi.

[Research methods and applications progress on spectrum-effect relationships in study of traditional Chinese medicine].

The application of chemical fingerprint to evaluate the quality of traditional Chinese medicine has been widely accepted and used in many countries. However,only by analyzing the type and content of its chemical components to evaluate the quality of traditional Chinese medicines,the gold standard of quality evaluation by evaluating pharmacodynamic effects is ignored. The study of Chinese medicinal spectrum-effect relationships combining the chemical composition with the pharmacodynamic activity of traditional Chinese medicine,which can evaluate the quality of traditional Chinese medicine from more comprehensive and different angles,has been applied in many fields of traditional Chinese medicine research. This paper mainly summarizes the research methods of the Chinese medicinal spectrum-effect relationships and its application in the field of traditional Chinese medicine study,and provides reference for the research,development and application of the Chinese medicinal spectrum-effect relationships.

Four New Lignans from Kadsura Interior and Their Bioactivity.

A phytochemical investigation of the stems of Kadsura interior has led to an isolation of four new lignans, named kadsutherin E⁻H (1⁻4), together with two known lignans (5⁻6). The structures of the four new compounds were established on the basis of comprehensive spectroscopic analyses. Compounds 1⁻6 exhibited inhibition against adenosine diphosphate (ADP) induced platelet aggregation. Among the isolated compounds, kadsutherin F (2) showed the strongest anti-platelet aggregation activity with an inhibition of 49.47%.

[Influence of different cultivation measures on the content of three free carbohydrates of Codonopsis Radix].

To provide a scientific evidence for the quality control of Codonopsis Radix, a method was established for determining the content of three free carbohydrates of Codonopsis Radix. The developed method showed good linearity. The calibration curves were linear within the range of 2.312 5-18.500 0 µg for sucrose, 1.500 0-12.000 0 µg for glucose, and 2.000 0-16.000 0 µg for fructose, resgectwely. The recoveries varied between 96.31%-101.8%. The method is simple, accurate and reproducible, and can be used for determining the content of sucrose, glucose and fructose of Codonopsis Radix. The results showed that different cultivation measures had an effect on the content of three free carbohydrates of Codonopsis Radix. According to the content of sucrose, using Zhuanggenling>not using Zhuanggenling. While, not pinching, shelving>not pinching, not shelving>pinching, not shelving>pinching, shelving. According to the content of glucose and fructose, not using Zhuanggenling>using Zhuanggenling. While, pinching, shelving>not pinching, not shelving>not pinching, shelving>pinching, not shelving. In consideration of the differences of sweetness and content of the three free carbohydrates in Codonopsis Radix, we recommend that the content of free carbohydrates could be considered as the marker to evaluate the quality of Codonopsis Radix.

[Influence of different cultivation measures on chemical quality of Codonopsis Radix].

To observe the influence of different cultivation measures on the chemical constituents of Codonopsis Radix and provide reference for its reasonable cultivation, Codonopsis Radix samples cultivated by different cultivation measures were collected from the planting base in Min county,and their quality were evaluated by establishing HPLC fingerprint and determining the content of lobetyolin and Codonopsis Radix polysaccharide. The results show that different cultivation measures have an effect on the quality of Codonopsis Radix and the contents of lobetyolin and Codonopsis Radix polysaccharide are obviously different. According to the content of lobetyolin, not using Zhuanggenling>using Zhuanggenling. While, not pinching, shelving>not pinching, not shelving>pinching, shelving>pinching, not shelving. According to the content of Codonopsis Radix polysaccharide, not using Zhuanggenling>using Zhuanggenling. While, not pinching, shelving>not pinching, not shelving>pinching, not shelving>pinching, shelving. Based on the chemical quality evaluation results, the appropriate cultivation measure of Codonopsis Radix is not using Zhuanggenling, not pinching and shelving.

[Evaluation for heavy metal pollution of soil and herb from main producing area of Phellodendron amurense in China].

In order to evaluate the heavy metal potential pollution of soil and medicinal materials in main producing area of Phellodendron amurense, we collected 32 soil samples and 32 herb samples from northeast and north of China covering four provinces. In this study, the detection of heavy metal contents was conducted by ICP emission spectroscopy and atomic fluorescence spectrometry. The results showed that the soil from all areas of Ph amurense generally reached the national standard. As, Hg, Cr, Cd, Pb and Cu content of herb samples met the requirtment of the national standard except Hg content exceeding standard slight in a few samples. The reason of excessive Hg was the ability of Hg accumulation in Ph. amurense and atmospheric environment was polluted. So, national standard and Good Agricultural Practice (GAP) must be carried out severely in Ph. amurense resources production.

[Establishment of traceability system of Chinese medicinal materials' quality].

The quality of Chinese medicinal materials relates greatly to the clinical curative effect and security. In order to ensure the quality and safety of Chinese medicinal materials, a systematic and operable traceability system needs to be established. It can realize the whole process of quality and safety management of Chinese medicinal materials "from production to consumption" through recording and inquiring information and recalling defective products, which is an important direction for the future development of traditional Chinese medicine. But it is still at the exploration and trial stage. In this paper, a framework of Chinese medicinal materials' quality and safety traceability system was established on the basis of the domestic and international experience about the construction of food and agricultural products traceability systems. The relationship between traceability system of Chinese medicinal materials' quality and GAP, GMP, GSP was analyzed, and the possible problems and the corresponding solutions were discussed.

[Chemical constituents of Kadsura oblongifolia and evaluation of their toxicity].

To study the chemical constituents of K. oblongifolia, silica gel column chromatography, MCI and Sephadex LH-20 were used to separate the 70% acetone extract of the stems of K. oblongifolia. The structures of the isolated compounds have been established on the basis of physicochemical and NMR spectroscopic evidence as well as ESI-MS in some cases. Twenty compounds were obtained and identified as heteroclitalignan A (1), kadsulignan F (2), kadoblongifolin C (3), schizanrin F (4), heteroclitalignan C (5), kadsurarin (6), kadsulignan O (7), eburicol (8), meso-dihydroguaiaretic acid (9), kadsufolin A (10), tiegusanin M (11), heteroclitin B (12), (7'S)-parabenzlactone (13), angeloylbinankadsurin B (14), propinquain H (15), quercetin (16), kadsulignan P (17), schizanrin G (18), micrandilactone C (19) and (-)-shikimic acid (20). Compouds 1, 5, 8, 11-15, 18 and 20 were isolated from this plant for the first time. Toxicity of compounds 1-10 were evaluated with zebrafish model to observe the effect on its embryonic development and heart function. The results showed that compounds 7, 9 and 10 caused edema of zebrafish embryo and decreased the heart rate of zebrafish, which exhibited interference effect on heart development of zebrafish.

[Phylogenetic analysis for Fritillaria hupehensis: evidence from ITS, rpl16 and matK sequences].

The systematic position of Fritillaria hupehensis has been in dispute. Phylogentic analyses were conducted on sequences of ITS, rpl16, matK sequences for species of F. hupehensis and allies. Lilium davidii was designed as outgroup. The analyses were performed using MP and ML methods. Conclusions could be achieved as follow. The topologies of MP and ML are consistent. The samples of F. hepehensis from different places form a supported clade with a strong bootstrap. And then form a strongly supported clade with F. anhuiensis, F. monantha. The results suggests that although F. hupehensis has a closet relation with the two ones, it exists some difference.

[Chemical constituents of Conioselinum vaginatum].

Sixteen compounds were isolated from Conioselinum vaginatum by silica column chromatography over silica gel and Sephadex LH-20, as well as recrystallization. On the basis of their physical and chemical properties and spectral data, their structures were identified as ligustilide (1), 1,3-dilinolein (2), coniferaldehyde (3), myristicin (4), stigmasterol (5), beta-sitosterol (6), vanillin (7), pregnenolone (8), bergapten (9), xanthotoxin (10), methyl indole-3-carboxylate (11), ferulic acid (12), (E)-3-methoxy-4,5-methylenedioxy-cinnamic alcohol (13), p-hydroxybenzaldehyde (14), 3-methoxy-4,5-methylenedioxy-acetophenone (15), and alpha-(ethoxymethyl)-4-hydroxy- benzenemethanol (16). Among them, compound 15 was a new natural product, and compounds 2, 3, 10, 11, 14, and 16 were obtained from this genus for the first time.

A novel computed tomography angiography technique: guided preoperative localization and design of anterolateral thigh perforator flap.

BACKGROUND: Anterolateral thigh perforator (ALTP) flap is considered a versatile flap for soft tissue reconstruction. Computed tomography angiography (CTA) is used for mapping perforator in abdominal-based reconstruction; however, it is less commonly used in ALTP due to its poor imaging efficacy. In this study, we introduced a novel CTA technique for preoperative localization and design of ALTP flap and evaluated its value in directing surgical reconstruction. RESULTS: Thirty-five patients with soft tissue defects were consecutively enrolled. Modified CTA procedures, such as sharp convolution kernel, ADMIRE iterative reconstruction, 80 kV tube voltage, high flow contrast agent and cinematic rendering image reconstruction, were used to map ALTPs. A total of 287 perforators (including 884 sub-branches) were determined, with a mean of 5 perforators per thigh (range 2-11). The ALTPs were mainly concentrated in the "hot zone" (42%, 121/287) or the distal zone (41%, 118/287). Most perforators originated from the descending branch of the lateral circumflex femoral artery (76%, 219/287). Three perforator types, namely musculocutaneous (62%, 177/287), septocutaneous (33%, 96/287), and mixed pattern (5%, 14/287), were identified. The median pedicle length measured by two methods was 4.1 cm (range 0.7-20.3 cm) and 17.0 cm (range 4.7-33.9 cm), respectively, and the median diameter of the skin flap nourished by one perforator was 3.4 cm (IQR 2.1-5.7 cm). Twenty-eight ALTP flaps were obtained with the guidance of CTA, and 26 flaps survived after follow-up. CONCLUSIONS: The proposed CTA mapping technique is a useful tool for preoperative localization and design of ALTP flap.

Molecular Structure and Phylogenetic Analyses of the Complete Chloroplast Genomes of Three Medicinal Plants Conioselinum vaginatum, Ligusticum sinense, and Ligusticum jeholense.

Most plants of Ligusticum have an important medicinal and economic value with a long history, Ligusticum sinense and L. jeholense ("Gaoben") has long been used in traditional Chinese medicine for the treatment of carminative, dispelling cold, dehumidification, and analgesia. While in the market Conioselinum vaginatum (Xinjiang Gaoben) is substitution for Gaoben, and occupies a higher market share. These three Gaoben-related medicinal materials are similar in morphology, and are difficult to distinguish from each other by the commonly used DNA barcodes. The chloroplast genome has been widely used for molecular markers, evolutionary biology, and barcoding identification. In this study, the complete chloroplast genome sequences of C. vaginatum, L. sinense, and L. jeholense were reported. The results showed that the complete chloroplast genomes of these three species have typical quadripartite structures, which were comprised of 148,664, 148,539, and 148,497 bp. A total of 114 genes were identified, including 81 protein-coding genes (PCGs), 29 tRNA genes, and four rRNA genes. Our study indicated that highly variable region ycf2-trnL and accD-ycf4 that can be used as specific DNA barcodes to distinguish and identify C. vaginatum, L. sinense, and L. jeholense. In addition, phylogenetic study showed that C. vaginatum nested in Ligusticum and as a sister group of L. sinense and L. jeholense, which suggested these two genera are both in need of revision. This study offer valuable information for future research in the identification of Gaoben-related medicinal materials and will benefit for further phylogenetic study of Apiaceae.

Genome sizes of four important medicinal species in Kadsura by flow cytometry.

Objective: Dianjixueteng is a geoherb in Yunnan Province, the source plant of which is Kadsura interior. However, the formation of this geoherb is not clear in genetic mechanism, in which genome size is the first step that should be known on the genomic level. In this study we aimed to estimate the genome sizes of source plants of K. interior and three related herbs K. heteroclita, K. longipedunculata, and K. coccinea by flow cytometry (FCM) and make a comparison. Methods: The genome sizes of K. interior, K. heteroclita, K. longipedunculata and K. coccinea, i.e., the source plants of Dianjixueteng and its relative medicinal materials, were estimated by FCM. The nuclei of K. interior were isolated using modified LB01 buffer, for the rest species, by the Galbraith's buffer. Results: The genome sizes of K. interior, K. heteroclita, K. longipedunculata, and K. coccinea were 7.36, 7.12, 7.01, and 5.15 pg/1C, respectively. Genome size of K. interior had no significant variation with those of K. heteroclita and K. longipedunculata (P = 0.296), which was significantly larger than that of K. coccinea. Conclusion: Genome size can not distinguish K. interior from K. heteroclita and K. longipedunculata, but could distinguish them from K. coccinea, which lays the foundation for future studies on genetic mechanism of the geoherb formation.

Comparison of anti-inflammatory effects of Lonicerae Japonicae Flos and Lonicerae Flos based on network pharmacology.

Objective: In Chinese herbal medicine (CHM) history, Lonicerae Japonicae Flos and Lonicerae Flos were used clinically as one drug, but now they are admitted as two herbal medicines in Chinese Pharmacopoeia (2010 edition). This study used network pharmacology to investigate whether the two can be used interchangeably for the treatment of inflammatory diseases in TCM clinical practice. Methods: Lonicerae Japonicae Flos and Lonicerae Flos were compared in the inflammation mechanism including core targets, Gene Ontology (GO), pathway and principle chemical components by the method of network pharmacology. Results: Lonicerae Japonicae Flos and Lonicerae Flos shared in six targets accounting for 66.7% of the entire core targets and more than half of the GO terms and pathways are similar. Organic acids are dominent compounds responsible for anti-inflammatory effects. Three of the compounds that bind to core targets including luteolin, quercetin and kaempferol, are shared in both herbs. Conclusion: Due to high similarity between Lonicerae Japonicae Flos and Lonicerae Flos, we believe that they can be used interchangeably for the inflammation in clinical treatment.