Genome-wide identification and functional characterization of borneol dehydrogenases in Wurfbainia villosa.

MAIN CONCLUSION: Genome-wide screening of short-chain dehydrogenases/reductases (SDR) family reveals functional diversification of borneol dehydrogenase (BDH) in Wurfbainia villosa. Wurfbainia villosa is an important medicinal plant, the fruits of which accumulate abundant terpenoids, especially bornane-type including borneol and camphor. The borneol dehydrogenase (BDH) responsible for the conversion of borneol to camphor in W. villosa remains unknown. BDH is one member of short-chain dehydrogenases/reductases (SDR) family. Here, a total of 115 classical WvSDR genes were identified through genome-wide screening. These WvSDRs were unevenly distributed on different chromosomes. Seven candidate WvBDHs based on phylogenetic analysis and expression levels were selected for cloning. Of them, four BDHs can catalyze different configurations of borneol and other monoterpene alcohol substrates to generate the corresponding oxidized products. WvBDH1 and WvBDH2, preferred (+)-borneol to (-)-borneol, producing the predominant ( +)-camphor. WvBDH3 yielded approximate equivalent amount of (+)-camphor and (-)-camphor, in contrast, WvBDH4 generated exclusively (+)-camphor. The metabolic profiles of the seeds showed that the borneol and camphor present were in the dextrorotatory configuration. Enzyme kinetics and expression pattern in different tissues suggested WvBDH2 might be involved in the biosynthesis of camphor in W. villosa. All results will increase the understanding of functional diversity of BDHs.

Terpenoid VOC profiles and functional characterization of terpene synthases in diploid and tetraploid cytotypes of Chrysanthemum indicum L.

Chrysanthemum indicum L. is a valuable medicinal plant with diploid and tetraploid forms that are widely distributed in central and southern China, and it contains abundant volatile organic compounds (VOCs). Despite the discovery of some terpene synthase (TPS) in C. indicum (i.e., CiTPS) in previous studies, many TPSs and their corresponding terpene biosynthesis pathways have yet to be discovered. In the present study, terpenoid VOCs in different tissues from two cytotypes of C. indicum were analyzed. We identified 52 types of terpenoid VOCs and systematically investigated the content and distribution of these compounds in various tissues. The two cytotypes of C. indicum exhibited different volatile terpenoid profiles. The content of monoterpenes and sesquiterpenes in the two cytotypes showed an opposite trend. In addition, four full-length candidate TPSs (named CiTPS5-8) were cloned from Ci-GD4x, and their homologous TPS genes were screened based on the genome data of Ci-HB2x. These eight TPSs displayed various tissue expression patterns and were discovered to produce 22 terpenoids, 5 of which are monoterpenes and 17 are sesquiterpenes. We further proposed corresponding terpene synthesis pathways, which can enable the establishment of an understanding of the volatile terpenoid profiles of C. indicum with different cytotypes. This knowledge may provide a further understanding of germplasm in C. indicum and may be useful for biotechnology applications of Chrysanthemum plants.

Intestines-erythrocytes-mediated bio-disposition deciphers the hypolipidemic effect of berberine from Rhizoma Coptidis: A neglected insight.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhizoma Coptidis (RC), the dried rhizome of Coptis Chinensis Franch., can dispel dampness and heat within the body and has been traditionally used for the treatment of cardiovascular disease (CVD)-associated problems including hyperlipidemia in China. Berberine (BBR) is the main active component of RC, which has been shown to possess significant therapeutic potential. However, only 0.14% of BBR is metabolized in the liver, and the extremely low bioavailability (<1%) and blood concentration of BBR in experimental and clinical settings is insufficient to achieve the effects as observed under in vitro conditions, which imposes challenges to explain its excellent pharmacological actions. Intense efforts are currently being devoted to defining its specific pharmacological molecular targets, while the exploration from the perspective of its pharmacokinetic disposition has rarely been reported to date, which could hardly make a comprehensive understanding of its hypolipidemic enigma. AIM OF THE STUDY: This study made a pioneering endeavor to unveil the hypolipidemic mechanism of BBR from RC focusing on its unique intestines-erythrocytes-mediated bio-disposition. MATERIALS AND METHODS: The fate of BBR in intestines and erythrocytes was probed by a rapid and sensitive LC/MS-IT-TOF method. To analyze the disposition of BBR, a reliable HPLC method was subsequently developed and validated for simultaneous determination of BBR and its key active metabolite oxyberberine (OBB) in whole blood, tissues, and excreta. Meanwhile, the enterohepatic circulation (BDC) of BBR and OBB was verified by bile duct catheterization rats. Finally, lipid overloading models of L02 and HepG2 cells were employed to probe the lipid-lowering activity of BBR and OBB at in vivo concentration. RESULTS: The results showed that BBR underwent biotransformation in both intestines and erythrocytes, and converted into the major metabolite oxyberberine (OBB). The AUC0-t ratio of total BBR to OBB was approximately 2:1 after oral administration. Besides, the AUC0-t ratio of bound BBR to its unbound counterpart was 4.6:1, and this ratio of OBB was 2.5:1, indicative of abundant binding-type form in the blood. Liver dominated over other organs in tissue distribution. BBR was excreted in bile, while the excretion of OBB in feces was significantly higher than that in bile. Furthermore, the bimodal phenomenon of both BBR and OBB disappeared in BDC rats and the AUC0-t was significantly lower than that in the sham-operated control rats. Interestingly, OBB significantly decreased triglycerides and cholesterol levels in lipid overloading models of L02 and HepG2 cells at in vivo-like concentration, which was superior to the prodrug BBR. CONCLUSIONS: Cumulatively, BBR underwent unique extrahepatic metabolism and disposition into OBB by virtue of intestines and erythrocytes. BBR and OBB were mainly presented and transported in the protein-bound form within the circulating erythrocytes, potentially resulting in hepatocyte targeting accompanied by obvious enterohepatic circulation. The unique extrahepatic disposition of BBR via intestines and erythrocytes conceivably contributed enormously to its hypolipidemic effect. OBB was the important material basis for the hypolipidemic effect of BBR and RC.

GC-MS and UHPLC-QTOFMS-assisted identification of the differential metabolites and metabolic pathways in key tissues of Pogostemon cablin.

Pogostemon cablin is an important aromatic medicinal herb widely used in the pharmaceutical and perfume industries. However, our understanding of the phytochemical compounds and metabolites within P. cablin remains limited. To our knowledge, no integrated studies have hitherto been conducted on the metabolites of the aerial parts of P. cablin. In this study, twenty-three volatile compounds from the aerial parts of P. cablin were identified by GC-MS, predominantly sesquiterpenes. Quantitative analysis showed the highest level of patchouli alcohol in leaves (24.89 mg/g), which was 9.12 and 6.69-fold higher than in stems and flowers. UHPLC-QTOFMS was used to analyze the non-volatile compounds of leaf, stem and flower tissues. The differences in metabolites between flower and leaf tissues were the largest. Based on 112, 77 and 83 differential metabolites between flower-leaf, flower-stem and leaf-stem, three tissue-specific biomarkers of metabolites were identified, and the differential metabolites were enriched in several KEGG pathways. Furthermore, labeling differential metabolites in the primary and secondary metabolic pathways showed that flowers accumulated more lipids and amino acids, including proline, lysine and tryptophan; the leaves accumulated higher levels of terpenoids, vitamins and flavonoids, and stems contained higher levels of carbohydrate compounds. Based on the role of acetyl coenzyme A, the distribution and possible exchange mechanism of metabolites in leaves, stems and flowers of P. cablin were mapped for the first time, laying the groundwork for future research on the metabolites in P. cablin and their regulatory role.

The Underling Mechanisms Exploration of Rubia cordifolia L. Extract Against Rheumatoid Arthritis by Integrating Network Pharmacology and Metabolomics.

Purpose: Rubia cordifolia L. (RC) is a classic herbal medicine for the treatment of rheumatoid arthritis (RA) and has been used since ancient times. The ethanol extract of Rubia cordifolia L. (RCE) showed obvious anti-RA effects in our previous study. However, further potential mechanisms require more exploration. We aimed to investigate the mechanism of RCE for the treatment of RA by integrating metabolomics and network pharmacology in this study. Methods: An adjuvant-induced arthritis (AIA) rat model was established, and we evaluated the therapeutic effects of RCE. Metabolomics of serum and urine was used to identify the differential metabolites. Network pharmacology was applied to determine the key metabolites and potential targets. Finally, the potential targets and compounds of RCE were verified by molecular docking. Results: The results indicated that RCE suppressed foot swelling and alleviated joint damage and also had anti-inflammatory properties by inhibiting the expressions of tumor necrosis factor (TNF)-α, Interleukin (IL)-1ß, prostaglandin E2 (PGE2), and P65. Ten and seven differential metabolites were found in the serum and urine, respectively, of rats. Six key targets, ie, phospholipase A2 group IIA (PLA2G2A), phospholipase A2 group X (PLA2G10), cytidine deaminase (CDA), uridine-cytidine kinase 2 (UCK2), charcot-leyden crystal galectin (CLC), and 5',3'-nucleotidase, mitochondrial (NT5M), were discovered by network pharmacology and metabolite analysis and were found to be related to glycerophospholipid metabolism and pyrimidine metabolism. Molecular docking confirmed that the favorable compounds showed affinities with the key targets, including alizarin, 6-hydroxyrubiadin, ruberythric acid, and munjistin. Conclusion: This study revealed the underlying mechanisms of RCE and provided evidence that will allow researchers to further investigate the functions and components of RCE against RA.

Comparative Analysis of the Metabolites and Biological Activity of Cultivated and Wild Lignosus rhinocerotis.

In this paper, Lignosus rhinocerotis (Cooke) Ryvarden (L. rhinocerotis) cultivated in rice medium (LRR) and in sawdust medium (LRS) was harvested. Then, in terms of the LRR, LRS, and wild L. rhinocerotis (LRW), the total flavonoid contents, total polyphenol contents, total polysaccharide contents, and metabolites were detected; antioxidants of their aqueous extracts and anti-inflammatory of their polysaccharides were performed. In addition, the possible mechanism of the polysaccharides of L. rhinocerotis inhibiting lung damage was elucidated. The results showed that 32 compounds were characterized in L. rhinocerotis, including flavonoids, terpenoids, lignans, and steroids and there were 20 compounds in cultivated and wild L. rhinocerotis; LRR has the highest total polyphenol and flavonoid contents, as well as ABTS and DPPH scavenging capacity. The total polysaccharide contents and the FRAP scavenging capacity of wild L. rhinocerotis were higher than those of cultivated L. rhinocerotis. The inhibition of polysaccharides of LRW (PLRW) on LPS-induced MRC-5 damage was stronger than that of the polysaccharides from cultivated L. rhinocerotis. The PLRW may alleviate lung damage by inhibiting the NLRP3 pathway and thereby suppressing the inflammatory response. In summary, both cultivated and wild L. rhinocerotis are abundant in bioactive components and have antioxidant and anti-inflammatory activities.

Chromosome-level genome assembly and functional characterization of terpene synthases provide insights into the volatile terpenoid biosynthesis of Wurfbainia villosa.

Wurfbainia villosa is a well-known medicinal and edible plant that is widely cultivated in the Lingnan region of China. Its dried fruits (called Fructus Amomi) are broadly used in traditional Chinese medicine for curing gastrointestinal diseases and are rich in volatile terpenoids. Here, we report a high-quality chromosome-level genome assembly of W. villosa with a total size of approximately 2.80 Gb, 42 588 protein-coding genes, and a very high percentage of repetitive sequences (87.23%). Genome analysis showed that W. villosa likely experienced a recent whole-genome duplication event prior to the W. villosa-Zingiber officinale divergence (approximately 11 million years ago), and a recent burst of long terminal repeat insertions afterward. The W. villosa genome enabled the identification of 17 genes involved in the terpenoid skeleton biosynthesis pathway and 66 terpene synthase (TPS) genes. We found that tandem duplication events have an important contribution to the expansion of WvTPSs, which likely drove the production of volatile terpenoids. In addition, functional characterization of 18 WvTPSs, focusing on the TPS-a and TPS-b subfamilies, showed that most of these WvTPSs are multi-product TPS and are predominantly expressed in seeds. The present study provides insights into the genome evolution and the molecular basis of the volatile terpenoids diversity in W. villosa. The genome sequence also represents valuable resources for the functional gene research and molecular breeding of W. villosa.

[Main components in different varieties of Xihuangcao by UPLC-Q-TOF-MS and UPLC-DAD].

This study established an ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS) method to analyze the main components in different varieties of Xihuangcao and established a UPLC-DAD method to simultaneously determine the five active components(caffeic acid, rosmarinic acid, schaftoside, isoschaftoside, and oridonin).The chromatographic separation was performed on a Waters ACQUITY UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 µm) with a gradient elution of methanol(B)-water containing 0.1% formic acid(A) at a flow rate of 0.3 mL·min~(-1).The column temperature was 30 ℃.The Q-TOF-MS discriminant analysis was performed under positive electrospray ion mode and the split ratio was 1∶1. Quantitative analysis was carried out by UPLC-DAD.The determination wavelength was set at 245 nm.Thirty-two main components of Xihuangcao were separated and identified by UPLC-Q-TOF-MS, where 19 were identified in Rabdosia serra, nine in R.nervosa, 10 in R.lophanthoides, 15 in R.lophanthoides var.graciliflora, 10 in R.lophanthoides var.gerardianus, and seven in R.stracheyi.The UPLC-DVD method was developed for simultaneously determining five active components in different varieties of Xihuangcao.The standard curves for five compounds showed good linearity with correlation coefficients higher than 0.999 0.The precision, repeatability, and stability were good.The average recoveries(n=6) were between 97.01% and 102.7% with RSD<3.0%.The results of UPLC-Q-TOF-MS analysis provided a scientific basis for the use of R.stracheyi as a medicinal material of Xihuangcao and the equivalent use of R.lophanthoides var.gerardianus with R.lophanthoides var.graciliflora to some extent.The UPLC-DAD method for simultaneously determining five active components is simple, rapid, and accurate.This study can provide the basis for the quality control of different varieties of Xihuangcao.

The Antioxidant and Hypolipidemic Effects of Mesona Chinensis Benth Extracts.

In this study, the antioxidant and hypolipidemic effects of Mesona Chinensis Benth (MCB) extracts were evaluated. Seven fractions (F0, F10, F20, F30, F40, F50 and MTF) were obtained from the MCB ethanol extracts. Compared to the commercial antioxidants (vitamin C), MTF and F30 exhibited higher antioxidant activities in the antiradical activity test and the FRAP assay. The half-inhibition concentration (IC50) for MTF and F30 were 5.323 µg/mL and 5.278 µg/mL, respectively. MTF at 200 µg/mL significantly decreased the accumulation of TG in oleic acid (OA)-induced HepG2 cells and reversed the inhibitory effect of Compound C on AMPK (MTF and F30 significantly increased the glucose utilization of insulin-induced HepG2 cells). In addition, the components of MTF were identified by HPLC-MS, which were caffeic acid, quercetin 3-O-galactoside, isoquercetin, astragalin, rosmarinic acid, aromadendrin-3-O-rutinoside, rosmarinic acid-3-O-glucoside and kaempferol-7-O-glucoside. Through statistical correlations by Simca P software, it was found that the main antioxidant and hypolipidemic components of MCB might be caffeic acid, kaempferol-7-O-glucoside, rosmarinic acid-3-O-glucoside and aromadendrin-3-O-rutinoside, which may play important roles in the AMPK pathway. MTF and F30 in MCB could be potential health products for the treatment of hyperlipidemia.

Cytochrome P450 Monooxygenase/Cytochrome P450 Reductase Bi-Enzymatic System Isolated From Ilex asprella for Regio-Specific Oxidation of Pentacyclic Triterpenoids.

Ilex asprella is a plant from Aquifoliaceae. Its root is commonly used as folk medicinal materials in southern China. The chemical compositions of I. asprella are rich in pentacyclic triterpenoids, which show various biological activities and demonstrate a good prospect for drug development. The elucidation of biosynthesis mechanism of triterpenoids in I. asprella could lay important foundations for the production of these precious plant secondary metabolites by metabolic engineering. Our previous studies have revealed IaAO1 (a CYP716A210 homolog) responsible for the C-28 oxidation of α- and ß-amyrin. Herein, we reported the identification of three more cytochrome P450 monooxygenase genes IaAO2 (a CYP716A212 homolog), IaAO4 (CYP714E88), IaAO5 (CYP93A220), and a cytochrome P450 reductase gene IaCPR by using Saccharomyces cerevisiae eukaryotic expression system and gas chromatography-mass spectrometry. Among them, the protein encoded by IaAO2 can catalyze the C-28 oxidation of α-amyrin and ß-amyrin, IaAO4 can catalyze the C-23 oxidation of ursolic acid and oleanolic acid, while IaAO5 is responsible for the C-24 oxidation of ß-amyrin. By introducing three genes IaAO1, IaAO4 and IaCPR into S. cerevisiae. We constructed an engineered yeast strain that can produce C-23 hydroxyl ursane-type triterpenoid derivatives. This study contributes to a thorough understanding of triterpenoid biosynthesis of medicinal plants and provides important tools for further metabolic engineering.

[Cloning of transcription factor PcFBA-1 in Pogostemon cabin and its interaction with FPPS promoter].

Farnesyl diphosphate synthase(FPPS) is a key enzyme at the branch point of the sesquiterpene biosynthetic pathway, but there are no reports on the transcriptional regulation of FPPS promoter in Pogostemon cabin. In the early stage of this study, we obtained the binding protein PcFBA-1 of FPPS gene promoter in P. cabin. In order to explore the possible mechanism of PcFBA-1 involved in the regulation of patchouli alcohol biosynthesis, this study performed PCR-based cloning and sequencing analysis of PcFBA-1, analyzed the expression patterns of PcFBA-1 in different tissues by fluorescence quantitative PCR and its subcellular localization using the protoplast transformation system, detected the binding of PcFBA-1 protein to the FPPS promoter in vitro with the yeast one-hybrid system, and verified its transcriptional regulatory function by dual-luciferase reporter gene assay. The findings demonstrated that the cloned PcFBA-1 had an open reading frame(ORF) of 1 131 bp, encoding a protein of 376 amino acids, containing two conserved domains named F-box-like superfamily and FBA-1 superfamily, and belonging to the F-box family. Moreover, neither signal peptide nor transmembrane domain was contained, implying that it was an unstable hydrophilic protein. In addition, as revealed by fluorescence quantitative PCR results, PcFBA-1 had the highest expression in leaves, and there was no significant difference in expression in roots or stems. PcFBA-1 protein was proved mainly located in the cytoplasm. Furthermore, yeast one-hybrid screening and dual-luciferase reporter gene assay showed that PcFBA-1 was able to bind to FPPS promoter both in vitro and in vivo to enhance the activity of FPPS promoter. In summary, this study identifies a new transcription factor PcFBA-1 in P. cabin, which directly binds to the FPPS gene promoter to enhance the promoter activity. This had laid a foundation for the biosynthesis of patchouli alcohol and other active ingre-dients and provided a basis for metabolic engineering and genetic improvement of P. cabin.

A Rapid Screening Method for Sibutramine Hydrochloride in Natural Herbal Medicines and Dietary Supplements.

Herbal weight loss drugs are becoming more widely used in the fight against obesity, but ineffective regulation of these products have resulted in harmful additives. These products may contain adulterants such as sibutramine hydrochloride that may result in serious adverse health events including death. This work established a color precipitation reaction-based rapid screening method for illegal adulteration of sibutramine hydrochloride in natural herbal medicines (NHM) and dietary supplements (DS). While a variety of chromatography- and electrophoresis-based systems have been reported to measure this analyte, they generally suffer from high costs, complicated sample preparation, and a costly analytical infrastructure. In contrast, we present a simple, handheld kit to assay for sibutramine. The performance metrics of this tool include an average detection time of approximately 3 minutes, which is markedly shorter than conventional methods (HPLC or HPLC-MS, etc.), a detection limit of 0.1 mg per aliquot, and an accuracy of 99.02% (n = 820). More strikingly, the sensitivity is 100% (n = 278), and the specificity is 98.52% (n = 542). The rapid test kit developed from this screening method was evaluated by FDA. In summary, this screening method is a rapid, simple, and low-cost tool for the detection of sibutramine in NHM and DS with superior selectivity and sensitivity. For these reasons, this method is especially suitable for underdeveloped settings because it can be employed onsite without any instrumentation. In addition, this approach could rapidly exclude most of the negative samples to boost efficiency in large-scale samples assay. If necessary, positive samples can undergo further alternate testing methods to confirm the positive results of sibutramine hydrochloride content.

Volatile metabolic profiling and functional characterization of four terpene synthases reveal terpenoid diversity in different tissues of Chrysanthemum indicum L.

Chrysanthemum indicum has long been used in traditional Chinese medicine for its health-promoting benefits. Studies on C. indicum have mainly focused on the flowers. Terpenoid distribution in various parts of the plant and characterization of terpene synthases remain unclear. In this study, volatile metabolic profiling was performed to compare the composition and quantity of terpenoids distributed in the root, stem, leaf, flower bud and flower of C. indicum. The potential for extracting active ingredients from the root, stem, and leaf was also examined. In total, 17 monoterpenoids and 27 sesquiterpenoids were identified. Transcriptome data were used to clone two monoterpene synthases and two sesquiterpene synthases highly expressed in the root. The recombinant proteins of full-length and truncated versions of C. indicum terpene synthase (CiTPS1) produced α-pinene, but the truncated one was catalytically more efficient than the full-length version. No product could be detected when full-length version of CiTPS2 was used for catalyzing GPP, but the truncated one can produce a minor amount of α-pinene. CiTPS3 contributed to the production of three sesquiterpenoids, namely ß-farnesene, petasitene, and α-bisabolene. CiTPS4 acted as a difunctional enzyme, contributing to the production of four monoterpenoids and three sesquiterpenoids, including petasitene. The evidence suggests that petasitene and the genes responsible for its biosynthesis were first found in the genus Chrysanthemum. The present findings provide insights into the composition, formation, and regulation of these bioactive compounds.

Identification of α-Amyrin 28-Carboxylase and Glycosyltransferase From Ilex asprella and Production of Ursolic Acid 28-O-ß-D-Glucopyranoside in Engineered Yeast.

Ilex asprella is a medicinal plant that is used extensively in southern China. The plant contains ursane-type triterpenoids and triterpenoid saponins which are known to be responsible for its pharmacological activities. Previously, a transcriptomic analysis of I. asprella was carried out and the gene IaAS1, which is important in the formation of the core structure α-amyrin, was identified. However, the genes related to the subsequent derivatization of the core structures of the triterpenoid remain largely unknown. Herein, we describe the cloning and functional characterization of an amyrin 28-carboxylase IaAO1 (designated as IaCYP716A210) and a glycosyltransferase IaAU1 (designated as UGT74AG5), based on transcriptomic data. The expression of IaAO1 in an α-amyrin producing yeast strain led to the accumulation of ursolic acid. An enzyme assay using recombinant protein IaAU1 purified from E. coli revealed that IaAU1 can catalyze the conversion of ursolic acid to ursolic acid 28-O-ß-D-glucopyranoside. IaAU1 has regiospecificity for catalyzing the 28-O-glucosylation of ursane-/oleanane-type triterpene acids, as it can also catalyze the conversion of oleanolic acid, hederagenin, and ilexgenin A to their corresponding glycosyl compounds. Moreover, co-expression of IaAO1 and IaAU1 in the α-amyrin-producing yeast strain led to the production of ursolic acid 28-O-ß-D-glucopyranoside, although in relatively low amounts. Our study reveals that IaAO1 and IaAU1 might play a role in the biosynthesis of pentacyclic triterpenoid saponins in I. asprella and provides insights into the potential application of metabolic engineering to produce ursane-type triterpene glycosides.

Global analysis of lysine succinylation in patchouli plant leaves.

Lysine succinylation is a novel, naturally occurring posttranslational modification (PTM) in living organisms. Global lysine succinylation identification has been performed at the proteomic level in various species; however, the study of lysine succinylation in plant species is relatively limited. Patchouli plant (P. cablin (Blanco) Benth., Lamiaceae) is a globally important industrial plant and medicinal herb. In the present study, lysine succinylome analysis was carried out in patchouli plants to determine the potential regulatory role of lysine succinylation in patchouli growth, development, and physiology. The global succinylation sites and proteins in patchouli plants were screened with an immunoprecipitation affinity enrichment technique and advanced mass spectrometry-based proteomics. Several bioinformatic analyses, such as function classification and enrichment, subcellular location predication, metabolic pathway enrichment and protein-protein interaction networking, were conducted to characterize the functions of the identified sites and proteins. In total, 1097 succinylation sites in 493 proteins were detected in patchouli plants, among which 466 succinylation sites in 241 proteins were repeatedly identified within three independent experiments. The functional characterization of these proteins indicated that the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, photosynthesis processes, and amino acid biosynthesis may be regulated by lysine succinylation. In addition, these succinylated proteins showed a wide subcellular location distribution, although the chloroplast and cytoplasm were the top two preferred cellular components. Our study suggested the important role of lysine succinylation in patchouli plant physiology and biology and could serve as a useful reference for succinylation studies in other medicinal plants.

Full-length transcriptome sequencing and methyl jasmonate-induced expression profile analysis of genes related to patchoulol biosynthesis and regulation in Pogostemon cablin.

BACKGROUND: Pogostemon cablin (Blanco) Benth. (Patchouli) is an important aromatic and medicinal plant and widely used in traditional Chinese medicine as well as in the perfume industry. Patchoulol is the primary bioactive component in P. cablin, its biosynthesis has attracted widespread interests. Previous studies have surveyed the putative genes involved in patchoulol biosynthesis using next-generation sequencing method; however, technical limitations generated by short-read sequencing restrict the yield of full-length genes. Additionally, little is known about the expression pattern of genes especially patchoulol biosynthesis related genes in response to methyl jasmonate (MeJA). Our understanding of patchoulol biosynthetic pathway still remained largely incomplete to date. RESULTS: In this study, we analyzed the morphological character and volatile chemical compounds of P. cablin cv. 'Zhanxiang', and 39 volatile chemical components were detected in the patchouli leaf using GC-MS, most of which were sesquiterpenes. Furthermore, high-quality RNA isolated from leaves and stems of P. cablin were used to generate the first full-length transcriptome of P. cablin using PacBio isoform sequencing (Iso-Seq). In total, 9.7 Gb clean data and 82,335 full-length UniTransModels were captured. 102 transcripts were annotated as 16 encoding enzymes involved in patchouli alcohol biosynthesis. Accorded with the uptrend of patchoulol content, the vast majority of genes related to the patchoulol biosynthesis were up-regulated after MeJA treatment, indicating that MeJA led to an increasing synthesis of patchoulol through activating the expression level of genes involved in biosynthesis pathway of patchoulol. Moreover, expression pattern analysis also revealed that transcription factors participated in JA regulation of patchoulol biosynthesis were differentially expressed. CONCLUSIONS: The current study comprehensively reported the morphological specificity, volatile chemical compositions and transcriptome characterization of the Chinese-cultivated P. cablin cv. 'Zhanxiang', these results contribute to our better understanding of the physiological and molecular features of patchouli, especially the molecular mechanism of biosynthesis of patchoulol. Our full-length transcriptome data also provides a valuable genetic resource for further studies in patchouli.

Profiling and Pharmacokinetic Studies of Alkaloids in Rats After Oral Administration of Zanthoxylum nitidum Decoction by UPLC-Q-TOF-MS/MS and HPLC-MS/MS.

Zanthoxylum nitidum (Roxb.) DC (Rutaceae), called as "liangmianzhen" in China, is well known for its anti-inflammation and analgesic effect. Alkaloids are its main active constituents. However, little has been known about the absorption of main alkaloids in vivo. In this study, an ultra-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry was employed for identification of absorbed alkaloids in rats after oral administration of Z. nitidum decoction. By analyzing the fragmentation patterns, a total of nineteen alkaloids were exactly or tentatively identified in rat plasma after treatment, of which magnoflorine, α-allocryptopine, and skimmianine are dominant. Moreover, a high performance liquid chromatography coupled mass spectrometry method was developed for simultaneous quantification of magnoflorine, α-allocryptopine, and skimmianine, and successfully applied to pharmacokinetic study in rats after oral administration of Z. nitidum decoction. The research would contribute to comprehensive understanding of the material basis and function mechanism of Z. nitidum decoction.

Optimization of Storage Conditions of the Medicinal Herb Ilex asprella against the Sterigmatocystin Producer Aspergillus versicolor Using Response Surface Methodology.

The root of Ilex asprella is a commonly used herb in Southern China, and also constitutes the main raw material of Canton herbal tea. I. asprella is readily contaminated by mildew because of rich nutrients. Aspergillus versicolor producing sterigmatocystin is one of the most common molds that contaminate foodstuffs and medicinal herbs. Previous study on the evaluation of fungal contamination on samples of I. asprella revealed that A. versicolor was the dominant contaminant. In this study, experiments based on response surface methodology combined with central composite design were carried out to determine the optimal storage conditions of I. asprella to minimize the contamination of sterigmatocystin. The herb, manually innoculated with A. versicolor, was stored under different temperatures (20⁻40 °C) and humidity (80⁻95%) for seven days. The effects of temperature and humidity were evaluated using total saponin, polysaccharide and the sterigmatocystin levels as quality indexes. The results showed that A. versicolor grew quickly and produced large amounts of sterigmatocystin on I. asprella, at humidity ranging from 85% to 90% and temperatures above 26 °C. Meanwhile, total saponin and polysaccharide amounts were reduced significantly. These findings suggested that I. asprella samples should be stored in an environment with humidity and temperature below 85% and 26 °C, respectively, to reduce A. versicolor growth and sterigmatocystin production.

[SSR loci information analysis in transcriptome of Andrographis paniculata].

To study the SSR loci information and develop molecular markers, a total of 43 683 Unigenes in transcriptome of Andrographis paniculata were used to explore SSR. The distribution frequency of SSR and the basic characteristics of repeat motifs were analyzed using MicroSAtellite software, SSR primers were designed by Primer 3.0 software and then validated by PCR. Moreover, the gene function analysis of SSR Unigene was obtained by Blast. The results showed that 14 135 SSR loci were found in the transcriptome of A. paniculata, which distributed in 9 973 Unigenes with a distribution frequency of 32.36%. Di-nucleotide and Tri-nucleotide repeat were the main types, accounted for 75.54% of all SSRs. The repeat motifs of AT/AT and CCG/CGG were the predominant repeat types of Di-nucleotide and Tri-nucleotide, respectively. A total of 4 740 pairs of SSR primers with the potential to produce polymorphism were designed for maker development. Ten pairs of primers in 20 pairs of randomly picked primers produced fragments with expected molecular size. The gene function of Unigenes containing SSR were mostly related to the basic metabolism function of A. paniculata. The SSR markers in transcriptome of A. paniculata show rich type, strong specificity and high potential of polymorphism, which will benefit the candidate gene mining and marker-assisted breeding.

Clinical Efficacy of Jinshuibao Capsules Combined with Angiotensin Receptor Blockers in Patients with Early Diabetic Nephropathy: A Meta-Analysis of Randomized Controlled Trials.

BACKGROUND: Jinshuibao capsules (JSB) have been widely used to treat early diabetic nephropathy (DN), but the specific effects are still inconsistent. A meta-analysis of randomized controlled trials (RCTs) was conducted to evaluate the clinical efficacy of JSB for early DN. METHODS: Four international databases and four Chinese databases were searched from publication dates to March 1, 2018. The RCTs reporting the results of JSB's specific effects were included, and comparisons were between JSB combined with Angiotensin Receptor Blockers (ARBs) as experimental intervention and ARBs as the control. Included studies' quality was evaluated and the extracted data were analyzed with RevMan 5.3 software. RESULTS: Twenty-six RCTs including 2198 early DN participants were adopted in the meta-analysis. The results showed that, compared with the ARBs alone, JSB could remarkably improve the ORR (OR = 3.84; 95% CI: 2.37~6.24; P < 0.00001) and decrease 24 h UTP (MD = -93.32; 95% CI: -128.60 ~-58.04; P < 0.00001), UAER (MD = -24.02; 95% CI: -30.93 ~-17.11; P < 0.00001), BUN (MD = -0.26; 95%: -0.44 ~-0.08; P = 0.005), Scr (MD = -9.07; 95% CI: -14.26 ~-3.88; P = 0.0006), ACR (MD = -17.55; 95% CI: -22.81 ~-12.29; P < 0.00001), Cys-C (MD = -0.60; 95% CI: -0.88 ~-0.32; P < 0.00001), SBP (MD = -3.08; 95% CI: -4.65 ~-1.52; P = 0.0001), DBP (MD = -2.09; 95% CI: -4.00 ~-0.19; P = 0.03), and TG (MD = -0.36; 95% CI: -0.50 ~-0.21; P < 0.00001). However, it showed no significant differences in TC (MD = -0.32; 95% CI: -0.69~0.04; P = 0.08), FBG (MD = 0.04; 95% CI: -0.39~0.47; P = 0.87), HbA1c (MD = -0.26; 95% CI: -0.59~0.06; P = 0.11), and ß2-MG (MD = -15.61; 95% CI: -32.95~1.73; P = 0.08). CONCLUSIONS: This study indicates that JSB is an effective accessory therapeutic medicine for patients with early DN. It contributes to decreasing blood pressure and the content of triglyceride and improving the renal function of early DN patients. However, there is still a need to further verify the auxiliary therapeutic effect of JSB with more strictly designed RCTs with large sample and multiple centers in the future.