Sarcandra glabra is a widely distributed and valuable plant in food and daily chemical industries, and is also a common-used medicinal plant for treating inflammatory diseases and tumors. Rosmarinic acid (RA) with significant pharmacological activity is an abundant and important constituent in S. glabra, however, little information about key enzymes involving the biosynthesis of RA in S. glabra is available and the underlying biosynthesis mechanisms of RA in S. glabra remain undeciphered. Therefore, in this study, by full-length transcriptome sequencing analyses of S. glabra, we screened the RA biosynthesis candidate genes based on sequence similarity and conducted enzymatic function characterization in vitro and in vivo. As a result, a complete set of 7 kinds of enzymes (SgPALs, SgC4H, Sg4CL, SgTATs, SgHPPRs, SgRAS and SgC3H) involving the biosynthesis route of RA from phenylalanine and tyrosine, were identified and fully characterized. This research systematically revealed the complete biosynthesis route of RA in S. glabra, which helps us better understand the process of RA synthesis and accumulation, especially the substrate promiscuities of SgRAS and SgC3H provide the molecular biological basis for the efficient biosynthesis of specific and abundant RA in S. glabra. The 7 kinds of key enzymes revealed in this study can be utilized as tool enzymes for production of RA by synthetic biology methods.

Download This Paper Open PDF in Browser Add Paper to My Library Share: Permalink Using these links will ensure access to this page indefinitely Copy URL The Total Biosynthesis Route of Rosmarinic Acid in Sarcandra Glabra 22 Pages Posted: 27 Feb 2024 See all articles by Qianqian LiQianqian LiChina Pharmaceutical UniversityShuai ZhangChina Pharmaceutical UniversityYingying WangChina Pharmaceutical UniversityZhirong CuiChina Pharmaceutical UniversityHansheng LvChina Pharmaceutical UniversityNan WangChina Pharmaceutical UniversityLingyi KongChina Pharmaceutical UniversityJun LuoChina Pharmaceutical University Abstract Sarcandra glabra is a widely distributed and valuable plant in food and daily chemical industry, and is also a common-used medicinal plant for treating inflammatory disease and tumor. Rosmarinic acid (RA) with significant pharmacological activity is its abundant constituent, however, the underlying biosynthesis mechanisms of RA in S. glabra remain undeciphered. Here, a complete set of 7 kinds of enzymes involving the biosynthesis route from phenylalanine and tyrosine, SgPALs, SgC4H, Sg4CL, SgTATs, SgHPPRs, SgRAS and SgC3H, were identified and fully characterized by combining transcriptome data and enzyme function characterization in vivo and in vitro. The substrate promiscuities of SgRAS and SgC3H provide the molecular biological basis for the efficient biosynthesis of specific and abundant RA in S. glabra. This research systematically revealed the complete biosynthesis route of RA in S. glabra, which also provided tool enzymes for production of RA by synthetic biology methods. Keywords: Biosynthesis, Rosmarinic acid (RA), Rosmarinic acid synthase (RAS), Phenylpropanoid pathway, Sarcandra glabra Suggested Citation: Suggested Citation Li, Qianqian and Zhang, Shuai and Wang, Yingying and Cui, Zhirong and Lv, Hansheng and Wang, Nan and Kong, Lingyi and Luo, Jun, The Total Biosynthesis Route of Rosmarinic Acid in Sarcandra Glabra. Available at SSRN: https://ssrn.com/abstract=4733722 Qianqian Li China Pharmaceutical University ( email ) Nanjing Medical UniversityJiangningNanjing, 211166China Shuai Zhang China Pharmaceutical University ( email ) Nanjing Medical UniversityJiangningNanjing, 211166China Yingying Wang China Pharmaceutical University ( email ) Nanjing Medical UniversityJiangningNanjing, 211166China Zhirong Cui China Pharmaceutical University ( email ) Hansheng Lv China Pharmaceutical University ( email ) Nanjing Medical UniversityJiangningNanjing, 211166China Nan Wang China Pharmaceutical University ( email ) Lingyi Kong China Pharmaceutical University ( email ) Jun Luo (Contact Author) China Pharmaceutical University ( email ) Nanjing Medical UniversityJiangningNanjing, 211166China Download This Paper Open PDF in Browser Do you have negative results from your research you’d like to share? Submit Negative Results Paper statistics Downloads 2 Abstract Views 3 41 References PlumX Metrics Feedback Feedback to SSRN Feedback (required) Email (required) Submit If you need immediate assistance, call 877-SSRNHelp (877 777 6435) in the United States, or +1 212 448 2500 outside of the United States, 8:30AM to 6:00PM U.S. Eastern, Monday - Friday.

Abstract Hyperoside is a bioactive flavonoid galactoside in both medicinal and edible plants. It plays an important physiological role in the growth of flower buds. However, the hyperoside biosynthesis pathway has not been systematically elucidated in plants, including its original source, Hypericaceae. Our group found abundant hyperoside in the flower buds of Hypericum monogynum, and we sequenced its transcriptome to study the biosynthetic mechanism of hyperoside. After gene screening and functional verification, four kinds of key enzymes were identified. Specifically, HmF3Hs (flavanone 3-hydroxylases) and HmFLSs (flavonol synthases) could catalyze flavanones into dihydroflavonols, as well as catalyzing dihydroflavonols into flavonols. HmFLSs could also convert flavanones into flavonols and flavones with varying efficiencies. HmF3′H (flavonoid 3′-hydroxylase) was found to act broadly on 4′-hydroxyl flavonoids to produce 3′,4′-diydroxylated flavanones, dihydroflavonols, flavonols, and flavones. HmGAT (flavonoid 3-O-galactosyltransferase) would transform flavonols into the corresponding 3-O-galactosides, including hyperoside. The parallel hyperoside biosynthesis routes were thus depicted, one of which was successfully reconstructed in Escherichia coli BL21(DE3) by feeding naringenin, resulting in a hyperoside yield of 25 mg/l. Overall, this research not only helped us understand the interior catalytic mechanism of hyperoside in H. monogynum concerning flower development and bioactivity, but also provided valuable insights into these enzyme families.

Rhamnosyltransferase (RT) and rhamnose synthase (Rhs) are the key enzymes that are responsible for the biosynthesis of rhamnosides and UDP-L-rhamnose (UDP-Rha) in plants, respectively. How to discover such enzymes efficiently for use is still a problem to be solved. Here, we identified HmF3RT, HmRhs1, and HmRhs2 from Hypericum monogynum, which is abundant in flavonol rhamnosides, with the help of a full-length and high throughput transcriptome sequencing platform. HmF3RT could regiospecifically transfer the rhamnose moiety of UDP-Rha onto the 3-OH position of flavonols and has weakly catalytic for UDP-xylose (UDP-Xyl) and UDPglucose (UDP-Glc). HmF3RT showed well quercetin substrate affinity and high catalytic efficiency with Km of 5.14 mu M and kcat/Km of 2.21 x 105 S-1 M-1, respectively. Docking, dynamic simulation, and mutagenesis studies revealed that V129, D372, and N373 are critical residues for the activity and sugar donor recognition of HmF3RT, mutant V129A, and V129T greatly enhance the conversion rate of catalytic flavonol glucosides. HmRhs1 and HmRhs2 convert UDP-Glc to UDP-Rha, which could be further used by HmF3RT. The HmF3RT and HmRhs1 co-expressed strain RTS1 could produce quercetin 3-O-rhamnoside (quercitrin), kaempferol 3-Orhamnoside (afzelin), and myricetin 3-O-rhamnoside (myricitrin) at yields of 85.1, 110.7, and 77.6 mg L-1, respectively. It would provide a valuable reference for establishing a better and more efficient biocatalyst for preparing bioactive flavonol rhamnosides by identifying HmF3RT and HmRhs.

Guided by MS/MS molecular networks strategy, chlospicenes A and B (1 and 2), the first example of cyclopropane moiety cracked lindenane sesquiterpene Michael addition dimers, along with their biogenetic analogues (3 and 4), were targetedly discovered from the roots of Chloranthus henryi. Their structures including absolute configurations were characterized by NMR, ECD and X-ray diffraction analysis. The plausible biogenic pathway speculation indicated that cyclopropylcarbinyl rearrangement may dominate the key crack of cyclopropane moiety. In addition, compounds 1 and 2 showed significant anti-nonalcoholic steatohepatitis (NASH) activity in free fatty acid (FFA)-induced HepG2 cells by decreasing intracellular lipid accumulation.

Sarglaoxolane A (1), the first lindenane-normonoterpene heterodimer fused by tetrahydrofuran, was discovered in Sarcandra glabra guided by the first proposed single-node-based molecular networking approach. Moreover, two pseudonatural derivatives (2 and 3) with an oxa-difuranofurone moiety were transformed from 1 and confirmed by X-ray diffraction, and also proven to exist in the plant extract. A combination of molecular networking and biomimetic transformation can significantly promote the discovery and structural elucidation of novel natural products.

Fifteen diterpene derivatives including seven new ones, sinensisins A-G (1, 2, 4, 7, 10, 14, 15), were obtained from the leaves and twigs of Aphanamixis sinensis. Their structures were elucidated by NMR spectroscopic and ECD data analyses. These diverse carbon skeletons containing meroditerpenoids, acyclic diterpenes, and norditerpenoids biogenetically were derived from chain-like diterpenes. Compounds 3, 5, and 6 showed inhibitory effects of nitric oxide (NO) production in LPS-induced RAW 264.7 cells.

Molecular networking (MN) is an efficient tool for natural product research. However, single MN might lead to false annotation due to the limited information, and the importance of combining MN with chromatogram is always ignored. In this study, we proposed a comprehensive MN strategy combining feature-based molecular networking (FBMN) and dual ionization mode MS/MS to improve the annotation accuracy and to achieve structural feature visualization in a chemotaxonomic chromatogram. Three steps were taken: (1) employing FBMN and dual ionization mode MS/MS to distinguish isomers and improve components' identification accuracy. (2) Using a 3-level initiative supported by in-house database to evaluate the annotation confidence. As a result, 95 compounds were successfully identified from Ginkgo biloba leaf extract (GBE) and Ginkgo biloba leaf (GBL), and 70 compounds mainly consisting of flavonoid glycosides, ginkgolides, and lignan glycosides were assigned as high-confidence molecules. (3) Building color-dependent chemotaxonomic chromatograms, to achieve component visualization by connecting FBMN with chromatogram in which the peaks of the same color indicated the compounds with similar structural features. Our research provided a new and efficient strategy for component identification and visualization of herbal medicine.

Sarcandra glabra, a traditional Chinese medicine and herbal tea, has benefits from treating inflammation and fractures to curing cancer. The anti-inflammatory properties of sesquiterpenoids (chloranthalactone A, CTA; atractylenolide II, AT-II) have made S. glabra to a great potential for research and development. Here this study developed S. glabra callus culture to circumvent particular needs of the plant growing seasons and Hardiness zone, with the goal being able to produce sesquiterpenoids in vitro. The half strength Murashige and Skoog (1/ 2MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 2.0 mg/L) and 6-benzylaminopurine (6-BA, 1.0 mg/L) was effective for callus induction. The 1/2MS medium supplemented with 2,4-D (2.0 mg/L) and 6-BA (0.5 mg/L) was superior for callus proliferation. The callus was friable, yellowish and rapidly grown without tissue differentiation. Moreover, high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) analysis indicated that the contents of CTA and AT-II in multiple subculture S. glabra callus were 3.72 +/- 0.51 mg/g and 0.50 +/- 0.12 mg/g in fresh weight (FW), respectively. Using abiotic stresses and elicitors, production of CTA (by ultraviolet-B radiation for 48 h) and AT-II (by methyl jasmonate, 100 mu mol/L) was enhanced by 2.3-fold. In conclusion, this work not only established the S. glabra callus culture for in vitro production of CTA and AT-II, but also provided the foundation for further development and utilization of S. glabra.

INTRODUCTION Melia toosendan Sieb. et Zucc. has been used as a Chinese folk medicine for roundworm treatment since ancient times. Many diverse limonoids have been isolated from Meliaceae plants, but it remains difficult to isolate and identify other limonoids because of their small natural concentrations. OBJECTIVE This study was performed to overcome the difficulties associated with fast and accurate identification of limonoids and establish a reliable and sensitive method for the analysis of minor limonoids in M. toosendan fruits. METHODS An efficient strategy for enrichment, detection, and identification of minor limonoids from M. toosendan fruits using solid-phase extraction with high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (SPE-HPLC-Q-TOF-MS/MS) was developed herein. RESULTS Characteristic fragmentations and fragmentation ions containing trichilin-, nimbin-, and vilasinin-class limonoid skeletons were initially studied, and characteristic diagnostic ions involved retro Diels-Alder (RDA) reactions or homolytic cleavages, which were used to identify minor limonoids. In total, 13 limonoids, including four new ones, were identified. CONCLUSION This is the first report on the analysis of M. toosendan fruits to identify limonoids. This novel analysis method may stimulate further research regarding the identification of limonoids in other plant species.

The formation of HO-1 based oxygen-bridges and new C–C bonds via a Dieckmann reaction provided diverse ring systems, aphanamolide-type skeletons, and a solution for the structural elucidation of prieurianin limonoids with missing NMR signals.

Main observation and conclusion Sarcaglarols A—D ( 1 — 4 ), two pairs of lindenane−monoterpene heterodimers fused by a 1,2‐dioxane moiety, were discovered and isolated from the leaves of Sarcandra glabra guided by MS/MS molecular networking‐based strategy. Their planar structures, absolute configurations of basic skeleton and flexible polyhydric side chain were established by analysis of HRESIMS, NMR spectroscopic data, ECD spectrum, and the X‐ray diffraction study of isopropylidene derivatives. An intermolecular [2+2+2] cycloaddition may play a key role in the biosynthesis pathway of the 1,2‐dioxane moiety fused lindenane−monoterpene heterodimer skeleton, which can be recognized as the biogenetic precursors of our previous reported lindenane−normonoterpene conjugates. In addition, compounds 1 , 3 and 4 exhibited moderate inhibitory effects of lipid accumulation in free fatty acid‐exposed L02 cells.

Toona ciliata Roem., belonging to the Meliaceae family, is now much cultivated throughout the tropics for its colour wood hearts suitable for architecture and furniture. Large amount of barks of T. ciliata are available as the by-product of wood first-stage processing, which also have been used as a Chinese folk medicine to treat diarrhea, dysentery, and ringworm. In order to further exploit their potential uses, 22 new limonoids, ciliatasecones D-Y (1-22) and eight known ones (23-30) were isolated from the barks of this plant in our current research. The cytotoxicity, anti-inflammatory, and anti-multidrug resistance (MDR) activities of these isolates were also evaluated. Compounds 3, 8, 27, and 29 at the concentration of 50 mu M displayed significant MDR reversal activities in MCF-7/DOX cells to conventional anticancer drug doxorubicin. Compounds 3 and 29 exerted potentiation effects of 107.94 and 351.50-fold better than the positive control verapamil. This study added new data to the investigations of the structural diversity and biological activity of limonoids and provided phytochemical and pharmacological evidences for the further industrial and medicinal utilization of the barks of this common plant resource.

Aphamines A-C (1-3), three pairs of acyclic diterpene dimer enantiomers with an unprecedent ploymerization pattern, were discovered from Aphanamixis polystachya by NMR-guided isolation and chiral resolution. The elucidation of their novel carbon skeletons was achieved based on spectroscopic analysis, exciton chirality, and calculated electronic circular dichroism (ECD). Plausible Claisen rearrangement, 5-exo-trig cyclization, and reduction reactions may play important roles in the polymeric biosynthesis pathway. Compounds 1 and 3 showed inhibitory effects on nitric oxide (NO) production (IC50: 6.71-15.36 mu mol/L) and reduced the expression of iNOS in LPS-induced RAW 264.7 macrophages. (C) 2020 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.

Four new limonoids, toonayunnanaes F − I ( 1 − 4 ), and six known compounds ( 5 − 10 ) were isolated from the barks of Toona ciliata . Their structures were elucidated by thoroughly analyzing of NMR and HRMS data, and single-crystal X-ray diffraction of 1 . The oxetane ring moiety in 1 was rare in limonoids and other natural products. Compound 1 showed nitric oxide (NO) inhibitory effect with an IC 50 38.45 ± 0.41 µM in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Graphic Abstract

Melaleucadines A and B (1 and 2), two rare benzylic phloroglucinol-terpene hybrids (meroterpenoids), have been isolated and identified from the branches and leaves of the Australian "tea tree" Melaleuca leucadendron L. Their structures including absolute configurations were determined by 113/2D NMR, HR-MS, and the CD exciton chirality method. Compound I was a hybrid of a benzylic phloroglucinol and rearranged beta-pinene via a rare 2,3,4,5-tetrahydrooxepine ring, and 2 was the adduct of a benzylic phloroglucinol and a humulene-type sesquiterpene via an oxa-heterocycle. Bioactivity screening revealed that compounds 1 and 2 exhibited neuroprotective effects at a concentration of 50.0 mu M. (C) 2019 Elsevier Ltd. All rights reserved.

Ciliatasecones A-C (1-3), three rearranged limonoids with a novel ring-seco model and an unprecedented cycle system, were isolated from the root bark of Toona ciliata var. yunnanensis. Ciliatasecones A-B (1-2) share a novel cyclopenta[b]furan ring C/D system through C-9/11-seco and C-11/14 ether linkage. Ciliatasecone C (3) was found to possess a rare rearranged six-membered lactone ring B between C-7 and C-9. Plausible biogenetic pathway speculation indicated that C-9/11 cleavage and oxygen bridge formation played the key roles in the framework rearrangement of 1-3.