Liping Kang

Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones

Cytochromes P450 (CYPs) play a key role in generating the structural diversity of terpenoids, the largest group of plant natural products. However, functional characterization of CYPs has been challenging because of the expansive families found in plant genomes, diverse reactivity and inaccessibility of their substrates and products. Here we present the characterization of two CYPs, CYP76AH3 and CYP76AK1, which act sequentially to form a bifurcating pathway for the biosynthesis of tanshinones, the oxygenated diterpenoids from the Chinese medicinal plant Danshen (Salvia miltiorrhiza). These CYPs had similar transcription profiles to that of the known gene responsible for tanshinone production in elicited Danshen hairy roots. Biochemical and RNA interference studies demonstrated that both CYPs are promiscuous. CYP76AH3 oxidizes ferruginol at two different carbon centers, and CYP76AK1 hydroxylates C-20 of two of the resulting intermediates. Together, these convert ferruginol into 11,20-dihydroxy ferruginol and 11,20-dihydroxy sugiol en route to tanshinones. Moreover, we demonstrated the utility of these CYPs by engineering yeast for heterologous production of six oxygenated diterpenoids, which in turn enabled structural characterization of three novel compounds produced by CYP-mediated oxidation. Our results highlight the incorporation of multiple CYPs into diterpenoid metabolic engineering, and a continuing trend of CYP promiscuity generating complex networks in terpenoid biosynthesis.

Global profiling and rapid matching of natural products using diagnostic product ion network and in silico analogue database: Gastrodia elata as a case study.

Rapid discovery of novel compounds of a traditional herbal medicine is of vital significance for pharmaceutical industry and plant metabolic pathway analysis. However, discovery of unknown or trace natural products is an ongoing challenge. This study presents a universal targeted data-independent acquisition and mining strategy to globally profile and effectively match novel natural product analogues from an herbal extract. The famous medical plant Gastrodia elata was selected as an example. This strategy consists of three steps: (i) acquisition of accurate parent and adduct ions (PAIs) and the product ions data of all eluting compounds by untargeted full-scan MS(E) mode; (ii) rapid compound screening using diagnostic product ions (DPIs) network and in silico analogue database with SUMPRODUCT function to find novel candidates; and (iii) identification and isomerism discrimination of multiple types of compounds using ClogP and ions fragment behavior analyses. Using above data mining methods, a total of 152 compounds were characterized, and 70 were discovered for the first time, including series of phospholipids and novel gastroxyl derivatives. Furthermore, a number of gastronucleosides and phase II metabolites of gastrodin and parishins were discovered, including glutathionylated, cysteinylglycinated and cysteinated compounds, and phosphatidylserine analogues. This study extended the application of classical DPIs filter strategy and developed a structure-based screening approach with the potential for significant increase of efficiency for discovery and identification of trace novel natural products.

Structural characterization and discrimination of the Paris polyphylla var. yunnanensis and Paris vietnamensis based on metabolite profiling analysis

Graphical abstract Figure. No caption available. HighlightsAn integrated strategy to characterize and distinguish two Paris herbs is developed.146 metabolites were characterized from two Paris herbs, including 42 new compounds.PCA analysis can clearly separate of metabolic profile data of two Paris herbs.15 biomarkers for discrimination of PPY and PV were screened. Abstract This study aimed to distinguish the rhizomes of Paris polyphylla var. yunnanensis (Franch) Hand Mazz (PPY) and Paris veitnamensis (Takht.) H. Li (PV) using metabolomics‐based ultra high‐performance liquid chromatography coupled with quadrupole time‐of‐fligh mass spectrometry (UHPLC/Q‐TOF MS). First, the UHPLC/Q‐TOF MS approach was optimized for metabolite profiling. Then, the MS data were processed using UNIFI™ combined with an in‐house library to automatically characterize the metabolites. Based on the exact mass information, the fragmentation characteristics, and the retention time of compounds, and the fragmentation mechanism and retention behavior of steroidal glycosides in the references, the structures identified by UNIFI were further verified. Overall, 146 metabolites, including 42 potential new compounds, were identified or tentatively identified. Pattern recognition analysis of the PPY and PV MS data revealed that they were clearly separated, and 15 potential biomarkers for differentiating between them were selected. These biomarkers were subsequently used to successfully predict the genus of PPY and PV samples. These results indicated that metabolite profiling by UHPLC/Q‐TOF MS is an effective, robust approach for determining the characteristic biomarkers that differentiate between TCM species with multiple botanical origins.

Infrared-assisted extraction of salidroside from the root of Rhodiola crenulata with a novel ionic liquid that dissolves cellulose

Tetrabutylphosphonium hydroxide (TBPH) aqueous solution, a novel ionic liquid that could dissolve cellulose rapidly at ambient temperature (25 °C), was used for the first time to develop an extraction method for salidroside from Rhodiola crenulata, used as the model sample, with infrared-assisted extraction (IRAE) in this paper. IRAE-TBPH procedures were optimized using a series of single-factor experiments and under optimal conditions, the IRAE-TBPH technique not only took a shorter time (from 1.0 h to 8 min) but also afforded a higher extraction rate of salidroside from the herbs (increased by 15.41–38.65%) compared with other extraction techniques, such as TBPH-based heat reflux extraction (HRE-TBPH), ultrasound-assisted extraction (UAE-TBPH) and conventional solvent (methanol, ethanol and pure water) based IRAE. The results indicated IRAE-TBPH to be a fast and efficient extraction technique. Furthermore, the mechanism of IRAE-TBPH was preliminarily studied by means of the surface structures and chemical compositions of the samples before and after different extraction techniques. On the basis of the destruction of herb surface microstructures, the cellulose dissolving property of TBPH and high efficiency heating of infrared irradiation in the IRAE-TBPH process, the IRAE-TBPH technique eventually achieved the maximum yield value. Therefore, TBPH solution as a novel, effective and alternative solvent with higher extraction efficiency in the IRAE of active compounds from medicinal plants showed a great promising prospect.

A practical protocol for comprehensive evaluation of sulfur-fumigation of Gastrodia Rhizoma using metabolome and health risk assessment analysis

Gastrodia Rhizoma is one of the most heavily sulfur-fumigated edible and medical herbs in the marketplace. We developed a practical protocol using an ultra-performance liquid chromatography coupled with quadrupole time-of-flight-MSE (UPLC/QTOF-MSE)-based metabolome and health risk assessment model to identify characteristic sulfur-fumigated markers, dissect chemical transformation mechanisms, and control the quality of sulfur-fumigated Gastrodia Rhizoma. Two sulfur-containing p-hydroxybenzyl products, one sulfur-containing disaccharide, one glycolipid, and two phospholipids were selected and identified as markers based on multivariate statistical analysis. In particular, the sulfur-containing markers p-hydroxybenzyl hydrogen sulfite and trace p-mercaptobenzyl hydrogen sulfate were positively correlated with the active major phenolics. Moreover, a practical index the time of the minimum content was useful for evaluating the extent of the sulfur-fumigation under different weight ratios of the sulfur to herbal materials (1:20, 1:40, and 1:80). Ultimately, the 1:40 ratio within 1h of sulfur-fumigation was considered as safe and efficient for herb quality preservation under the maximum residue limit of 750mg/kg. This study shows that the practical protocol-based discriminated markers and practical limits can be applied to quality assurance of sulfur-fumigation and non-fumigation Gastrodia Rhizoma and other edible or medical materials.

Untargeted metabolite analysis-based UHPLC-Q-TOF-MS reveals significant enrichment of p-hydroxybenzyl dimers of citric acids in fresh beige-scape Gastrodia elata (Wutianma)

&NA; In order to comprehensively elucidate the chemical biosynthesis process of the beige‐scape Gastrodia elata Blume (Wutianma) as a traditional herbal medicines, the untargeted analysis–based UHPLC‐PDA‐ESI‐Q‐TOF‐MS reveals the metabolites ranging from the skeletons to novel dimers of citric acids in fresh and dried immature/mature stem tubers. Interestingly, two novel types of dimers for citric acids with the anhydride groups at sn‐1 and/or sn‐5 were discovered in fresh samples. Moreover, the classical mono‐ versus novel di‐mers, and the aglycons versus the glycosides could be easily discriminated by signature fragmentation patterns and some novel adduct ions. The heat map of contents demonstrated more p‐hydroxybenzyl metabolites than gastroxyl ones were determined in fresh Wutianma revealing a significant specificity with the lack of the sufficient gastrodin and gastroxyl products in biosynthetic pathway. Graphical abstract Figure. No caption available. HighlightsA total of 51 critic acids comprising 19 novel dimers were identified.Novel adduct ions for discrimination of chemical type and regioisomers.Significant enrichment of p‐hydroxybenzyl dimers in fresh Wutianma.Metabolome analysis for discoursing biosynthesis process of Wutianma.

Chemical profiling in Moutan Cortex after sulfuring and desulfuring processes reveals further insights into the quality control of TCMs by nontargeted metabolomic analysis

HighlightsThe mechanisms of compound changes were illustrated in sulfuring and desulfuring processes.One hundred nineteen metabolites were characterized from two Paris herbs, including seven potential new compounds.PCA analysis can clearly separate the metabolic profile data of normal, sulfured and desulfured Moutan Cortex.Twenty‐eight biomarkers for discrimination of normal, sulfured and desulfured Moutan Cortex were screened. ABSTRACT As a traditional processing method, sulfuring has been used in the processing of many traditional Chinese medicines (TCMs). Desulfuring, which has emerged in recent years, is a new method applied to sulfured herbs so they can comply with regulations regarding residual SO2. Due to the chemical transformations and the residual SO2 in the herbs, both sulfuring and desulfuring have negative effects on the safety and therapeutic effects of TCMs, and Moutan Cortex is one of the TCMs most susceptible to these effects. Here, a new strategy was developed to differentiate normal, sulfured and desulfured Moutan Cortex, and the transformations of compounds in sulfuring and desulfuring processes were analyzed using ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry (UPLC/Q‐TOF‐MSE) method based on metabolomic analysis. Our findings were as follows: (1) a total of 119 compounds were identified or tentatively identified, including 9 compounds that are being reported for the first time as natural products; (2) 15 sulfocompounds were generated during the sulfuring process; (3) these sulfocompounds could not be converted back into their corresponding glycosides by the desulfuring process, and the desulfuring decreased the residual SO2,while also removing some soluble compounds in the sulfured Moutan Cortex; and (4) 28 compounds were screened and tentatively identified as markers for distinguishing normal, sulfured and desulfured Moutan Cortex. Our findings provide a new practical strategy for evaluating how sulfuring and desulfuring affect the quality of TCMs.

A monoclonal antibody-based enzyme-linked immunosorbent assay for the determination of chlorogenic acid in honeysuckle

Graphical abstract Figure. No Caption available. HighlightsA highly specific monoclonal antibody against Chlorogenic acid (CGA) was produced.An indirect competitive enzyme‐linked immunosorbent assay (icELISA) was developed based on the monoclonal antibody.The proposed icELISA is a reliable, simple and cost effective system for the rapid detection of CGA in honeysuckle. ABSTRACT The edible flower buds of honeysuckle are traditionally used as herbal medicine or food additives in China. Chlorogenic acid (CGA) serves as the quality control marker of honeysuckle for its high content and antipyretic property. In this paper, a specific monoclonal antibody (mAb2E2) against CGA was produced. After optimization, mAb2E2‐based indirect competitive enzyme‐linked immunosorbent assay (icELISA) was developed. The concentrations of CGA producing 50% inhibition and the calibration range of the icELISA were 0.39 and 0.10–1.51 ng/mL, respectively. The icELISA had cross‐reactivity with 3,5‐Dicaffeoylquinic acid (17.53%), and the cross‐reactivity levels with other analogs were all below 5%. The average recoveries obtained by standard CGA addition to honeysuckle samples were from 88.4 to 104.8%. The icELISA was applied to CGA detection in different honeysuckle samples and the results were confirmed by high‐performance liquid chromatography analysis. The correlation coefficient between the two assays was 0.97. The proposed icELISA provides a feasible analytical method for highly sensitive and specific, simple, fast, and high‐throughput determination of CGA in honeysuckle samples.

Production of 3-geranyl-4-hydroxybenzoate acid in yeast, an important intermediate of shikonin biosynthesis pathway

Abstract Shikonin and its derivatives are the main active components in the medicinal plant Arnebia euchroma and possess extensive pharmaceutical properties. In this study, we developed an optimized yeast system to obtain high‐level production of 3‐geranyl‐4‐hydroxybenzoate acid (GBA), an important intermediate involved in shikonin biosynthesis pathway. For host selection, recombinant expression of p‐hydroxybenzoate:geranyltransferase (PGT) derived from A. euchroma was performed in Saccharomyces cerevisiae WAT11U strain and high yield monoterpene strain. In shake flask culture with 1 mM p‐hydroxybenzoate acid (PHBA), they could yield GBA at 0.1567 and 20.8624 mg L−1, respectively. Additionally, AePGT6 showed higher enzymatic activity than its homologs. Moreover, by combining improvement in the homologous mevalonate pathway with reconstruction in the heterologous shikimic pathway, a de novo GBA synthesis pathway was constructed in StHP6tHC with co‐overexpressed SctHMG1, optimized EcUbiC and AePGT6. A high titer of 179.29 mg L−1 GBA was achieved in StHP6tHC under shake flask fermentation with 1 mM PHBA. These results suggest that yeast could be engineered systematically to enable an efficient monoterpene‐quinone or naphthoquinone production.

Variation of Sweet Chemicals in Different Ripening Stages of Wolfberry Fruits

Abstract Objective To evaluate the variation of sweet chemicals (sugars and betaine) in fruits of six species in genus Lycium L. i.e. Lycium truncatum, L. cylindricum, L. dasystemum, L. dasystemum var. rubricaulium, L. chinense, and L. barbarum harvested at three different ripening stages. Methods A simple and effective method based on UPLC-ELSD was developed for the simultaneous determination of two monosaccharides (glucose and fructose), two disaccharides (sucrose and maltose), one sugar alcohol (xylitol), and betaine in wolfberry fruits (goji berries) of genus Lycium L. Results The six species of wolfberry fruits harvested at the three different ripening stages were evaluated in sugars and betaine contents. Fructose and glucose were the predominant sugars in mature wolfberry fruits. Fructose, glucose, and betaine, as well as total sugar contents, increased continually over the ripening process. L. truncatum and L. dasystemum had higher contents of sugars and betaine than the other species. Conclusion UPLC-ELSD is a simple, reliable and effective method for analysis of the sweet chemicals in wolfberries. Wolfberry fruits at the different ripening stages were significant different in sweetness. L. truncatum and L. dasystemum could be the potential sources of wolfberry fruit sources.