Intelligent chemical profiling of 73 edible flowers by liquid chromatography-high resolution mass spectrometry combined with HRMS database and their authentication based on large-scale fingerprints.

A commercial high-resolution MS database "TCM-PCDL" was innovatively introduced to automatically identify multi-components in 73 edible flowers rapidly and accurately by liquid chromatography-high resolution mass spectrometry, which can be time-consuming and labor-intensive in traditional manual method. The database encompasses over 2565 natural products with various energy levels. Unknown compounds can be identified through direct matching and scoring MS2 spectra with database. A total of 870 compounds were identified from 73 flowers, with polyphenols constituting up to 75%. Focusing on polyphenols, a high performance liquid chromatography (HPLC) method was developed to generate fingerprints from 510 batches, establishing an "HPLC database" that enabled accurate authentication using similarity scores and rankings. This method demonstrated an accuracy rate of 100% when applied to 30 unknown samples. For flowers prone to confusion, additional statistical analysis methods could be employed as aids in authentication. This study provides valuable insights for large-scale sample chemical profiling and authentication.

Diastereomers of Steroidal Alkaloids with Cytotoxic Activities against Non-Small-Cell Lung Cancer from the Bulbs of Fritillaria sinica.

Eleven new steroidal alkaloids, along with nine known related compounds, were isolated from the bulbs of Fritillaria sinica. Seven pairs of diastereomers were identified, including six and four 20-deoxy cevanine-type steroidal alkaloid diastereomers with molecular weights of 413 and 415, respectively. Structures were elucidated based on spectroscopic data analysis, chemical derivatization, and single-crystal X-ray diffraction analysis. Compounds 5, 9, 11, 12, 16, and 20 exhibited significant in vitro cytotoxic activity against non-small-cell lung cancer with CC50 values from 6.8 ± 3.9 to 12 ± 5 µM.

Undescribed steroidal alkaloids from the bulbs of Fritillaria sinica.

Eight undescribed steroidal alkaloid derivatives, including three cevanine-type isosteroidal alkaloids (two N-oxide glycosides and one D-ring aromatization) (1-3), one verazine-type steroidal alkaloid derivative (4), three solanidine-type steroidal alkaloid glycosides (5-7), and one veratramine-type analogue (8), along with three known compounds (9-11) were isolated from the bulbs of Fritillaria sinica. Their structures were elucidated by comprehensive analysis of spectroscopic data, acidic hydrolysis, and X-ray crystal diffractions. In the in vitro bioassay, the anti-cancer effect, anti-oxidation and anti-inflammatory activities for the isolates were evaluated at a concentration of 10 µM.

Characterization of the natural peptidome of four leeches by integrated proteogenomics and pseudotargeted peptidomics.

Animal-derived drugs are an indispensable part of folk medicine worldwide. However, their chemical constituents are poorly approached, which leads to the low level of the quality standard system of animal-derived drugs and further causes a chaotic market. Natural peptides are ubiquitous throughout the organism, especially in animal-derived drugs. Thus, in this study, we used multi-source leeches, including Hirudo nipponica (HN), Whitmania pigra (WP), Whitmania acranulata (WA), and Poecilobdella manillensis (PM), as a model. A strategy integrating proteogenomics and novel pseudotargeted peptidomics was developed to characterize the natural peptide phenotype and screen for signature peptides of four leech species. First, natural peptides were sequenced against an in-house annotated protein database of closely related species constructed from RNA-seq data from the Sequence Read Archive (SRA) website, which is an open-sourced public archive resource. Second, a novel pseudotargeted peptidomics integrating peptide ion pair extraction and retention time transfer was established to achieve high coverage and quantitative accuracy of the natural peptides and to screen for signature peptides for species authentication. In all, 2323 natural peptides were identified from four leech species whose databases were poorly annotated. The strategy was shown to significantly improve peptide identification. In addition, 36 of 167 differential peptides screened by pseudotargeted proteomics were identified, and about one-third of them came from the leucine-rich repeat domain (LRR) proteins, which are widely distributed in organisms. Furthermore, six signature peptides were screened with good specificity and stability, and four of them were validated by synthetic standards. Finally, a dynamic multiple reaction monitoring (dMRM) method based on these signature peptides was established and revealed that one-half of the commercial samples and all of the Tongxinluo capsules were derived from WP. All in all, the strategy developed in this study was effective for natural peptide characterization and signature peptide screening, which could also be applied to other animal-derived drugs, especially for modelless species that are less studied in protein database annotation.

Identification of Five Gelatins Based on Marker Peptides from Type I Collagen by Mass Spectrum in Multiple Reaction Monitoring Mode.

In this study, a novel pseudo-targeted peptidomics strategy, integrating the transition list generated by an in-house software (Pep-MRMer) and the retention time transfer by high-abundance ion-based retention time calibration (HAI-RT-cal), was developed to screen marker peptides of gelatins from five closely related animal species, including porcine, bovine, horse, mule, and donkey. Five marker peptides were screened from the molecular phenotypic differences of type I collagen. Furthermore, a simple and robust 10 min multiple reaction monitoring (MRM) method was established and performed well in distinguishing different gelatins, particularly in discerning horse-hide gelatin (HHG) and mule-hide gelatin (MHG) from donkey-hide gelatin (DHG). The market investigation revealed the serious adulteration of DHG. Meantime, the pseudo-targeted peptidomics could be used to screen marker peptides of other gelatin foods.

Identification of Pinelliae Rhizoma and its counterfeit species based on enzymatic signature peptides from toxic proteins.

BACKGROUND: Pinelliae Rhizoma (PR), a toxic medication, with long history, is commonly used for eliminating phlegm. Due to the shortage of wild resources and the relative lacking of cultivation technology, it is often confused with its counterfeit species in the market, such as Typhonii Rhizoma (TR), Arisaematis Rhizoma (AR) and tubers of Typhonium flagelliforme (TF) and Pinellia pedatisecta (PP). PURPOSE: It was aimed to screen signature enzymatic peptides from toxic proteins to identify PR and its four counterfeit species. STUDY DESIGN: A comparative proteogenomics strategy based on open-source transcriptome data was applied for screening signature peptides from toxic proteins, which were applied for species authentication of PR and its counterfeit species. METHODS: Firstly, the open-source transcriptome data was used for constructing the annotated protein database, which was used for peptides identification. Secondly, the toxicity of different fractions of PR were evaluated by the rat peritoneal inflammation model. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to profile the main proteins bands of five species, whose sequences were identified based on the in-gel digestion experiment by using ultra-high-performance liquid chromatography/quadrupole-Orbitrap mass spectrometry. Finally, the label-free proteomic analysis was performed to character the proteins and screen the signature peptides of five species, which were validated in commercially available products by dynamic multi reaction monitor (DMRM). RESULTS: The results in this study confirmed that protein was the main toxic components of PR. Both Pinellia ternata agglutinin (PTA) and trypsin inhibitor (TI) like proteins are the main proteins, which were characterized by proteomic analysis based on four annotated protein database. Meanwhile, seven signature peptides from toxic proteins were screened and validated with good repeatability and specificity in commercial products. CONCLUSION: Seven signature enzymatic peptides from toxic protein screened by the comparative proteogenomics strategy based on open-source transcriptome data achieved good identification ability of PR and its four counterfeit species.

A rapid and simple signature peptides-based method for species authentication of three main commercial Pheretima.

Pheretima with various activities is a commonly used animal-derived traditional medicine in Asia countries. However, almost half of them are non-pharmacopoeia species in the market due to the similar morphological characteristics between medicinal and non-medicinal species. This study aims to establish an effective method based on signature peptides for species authentication of three main commercial Pheretima, including two major Pheretima species (Amynthas aspergillum, Metaphire vulgaris) and one main adulteration (Metaphire magna). Firstly, the species of 52 batches of commercial Pheretima were authenticated based on DNA barcodes. Secondly, proteomic analysis was performed for protein characterization of three main commercial Pheretima. Furthermore, their signature peptides were screened and validated using ultra-high performance liquid chromatography coupled with mass spectrometry (UPLC-MS/MS) in multiple reaction monitoring (MRM) mode. Moreover, a simplified sample processing method was developed. Finally, large quantities of commercial Pheretima samples were analyzed for further verifying the feasibility of the signature peptides-based method. The result showed that the established method had a great application potential for authenticity identification of commercial Pheretima. SIGNIFICANCE: The authenticity assessment of medicinal materials is a main issue in the quality control process as deceptive practices could imply severe health risks. In this study, a rapid and simple method based on signature peptides was established for species authentication of three main commercial Pheretima, which can be an effective alternative to complex DNA barcoding and difficult morphological identification, and provided a reference for improvement of Pheretima quality standards.

Novel amorphous solid dispersion based on natural deep eutectic solvent for enhancing delivery of anti-tumor RA-XII by oral administration in rats.

At present, oral chemotherapy showing the advantages of non-invasiveness, convenience, and high patient compliance, is gradually replacing traditional intravenous chemotherapy to treat patients with cancer. RA-XII, a unique natural cyclopeptide, exhibits various biological activities, such as anti-tumor, anti-angiogenic, and anti-metastatic activities. Designing an orally available formulation of RA-XII is of great importance in the development of clinically useful anticancer agents. However, RA-XII shows low oral bioavailability in rats due to its poor solubility and low permeability. To overcome these limitations, in this work, a natural deep eutectic solvent (NADES) was designed to efficiently deliver RA-XII by oral administration. A novel NADES composed of betaine and mandelic acid in the molar ratio of 1:1 (Bet-Man NADES) was successfully prepared based on a binary phase diagram of Bet and Man. Acute toxicity studies indicated that Bet-Man NADES was well tolerated with acceptable toxicity. In Bet-Man NADES solutions, the solubility of RA-XII was increased by up to 17.54-fold, and the diffusion and permeability of RA-XII carried out in a Franz cell was also significantly improved 10.35 times. In terms of biopharmaceutical classification this is translated into a change for RA-XII from class IV to class II systems. More importantly, Bet-Man NADES was transferred into the solid formulation by the inclusion of a polymer, and amorphous solid dispersions based on Bet-Man NADES (PVP K30/NADES/RA-XII, ASDs) were successfully prepared to improve uniformity, apparent solubility, dissolution, and cytotoxicity in vitro. Consequently, the oral bioavailability of RA-XII in NADES solutions and ASDs was enhanced by approximately 11.58 and 7.56 times compared with that of pure RA-XII in 0.5% CMCNa. Thus, it can be seen that a natural deep eutectic solvent and its modified amorphous solid dispersions are appropriate novel strategies for improving dissolution rate and bioavailability of poor soluble natural products such as RA-XII.

Structurally diverse diterpenoid alkaloids from the lateral roots of Aconitum carmichaelii Debx. and their anti-tumor activities based on in vitro systematic evaluation and network pharmacology analysis.

Thirty-seven diterpenoid alkaloids (DAs) with diverse structures were isolated and identified from the lateral roots of Aconitum carmichaelii Debx., comprising eight C20-DAs and twenty-nine C19-DAs. Besides the 31 known DAs identified by comparing the 1H NMR and 13C NMR data with those reported in the literature, the structures of four new compounds (1, 14, 17, and 25), and two other compounds (26 and 37) which were reported to be synthesized previously, were also elucidated based on the comprehensive analysis of their HR-ESI-MS, 1D and 2D NMR spectra, including 1H-1H COSY, HSQC and HMBC and NOESY/ROESY. Among them, compound 1 represents the first example of a C20-DA glucoside. Besides, the anti-tumor activities of all the isolated compounds against human non-small-cell lung cancer A549 and H460 cells were systematically evaluated by MTT methods. The results revealed that all of the C19-DAs possessed moderate activities against both of the two cell lines with IC50 values ranging from 7.97 to 28.42 µM, and their structure-activity relationships indicated the active sites of C-8, C-10, and C-14 positions and the nitrogen atom in the C19-DA skeleton. In addition, all of the isolated DAs, with chemical structures confirmed, were further applied for network pharmacology analysis, in order to give an insight into the possible mechanisms of their anti-tumor activities. As a result, 173 potential targets and three most important pathways related to non-small-cell lung carcinoma were finally unearthed.

Colletopeptides A-D, Anti-inflammatory Cyclic Tridepsipeptides from the Plant Endophytic Fungus Colletotrichum sp. S8.

Four new hybrid peptide-polyketide cyclic tridepsipeptides, colletopeptides A-D (1-4), were isolated and characterized from the endophytic fungus Colletotrichum sp. S8 derived from the stems of Rubia podantha with the guidance of LC-UV-MS detection. Their structures were elucidated by extensive spectroscopic analysis and X-ray crystallography. Compounds 1-4 are rare natural 12-membered cyclic tridepsipeptides containing a 3,5,11-trihydroxy-2-methyl dodecanoic acid unit in their structures. 1-4 inhibited lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophages with the IC50 values of 8.3, 38.7, 13.5, and 22.2 µM, respectively. 1 also inhibited the production of inflammatory factors IL-6 and TNF-α, and decreased the phosphorylation of NF-κB-associated proteins IκBα and p65.

Cytotoxic and neuroprotective activities of constituents from Alternaria alternate, a fungal endophyte of Psidium littorale.

Chemical investigation of the EtOAc extract of the plant-associated fungus Alternaria alternate in rice culture led to the isolation of a novel liphatic polyketone, alternin A (1), a new indole alkaloid (8), and a new sesquiterpene (11), together with 12 known compounds. Their structures were elucidated by the interpretation of extensive spectroscopic data, and the absolute configurations of 1-3 were established using calculations of ECD spectra, NMR data, and optical rotation values. Compound 1 possesses an unprecedented C25 liphatic polyketone skeleton. Compounds 5 and 10 exhibited potential cytotoxic activities against MCF-7 and HepG cells, and compounds 2, 7, and 9 exhibited potential neuroprotective activities in glutamate induced-PC12 injured cells.

Systematic characterization and quantification of Rubiaceae-type cyclopeptides in 20 Rubia species by ultra performance liquid chromatography tandem mass spectrometry combined with chemometrics.

Rubiaceae-type cyclopeptides (RAs) are one of characteristic constituents isolated from Rubia species, which are candidates of innovative anti-tumor drugs due to their significant bioactivity. However, approaches on the systematic characterization and quantification of RAs are still not available because of low contents and complicated purification. In this study, an ultra performance liquid chromatography coupled triple quadrupole tandem mass spectrometry (UPLC-QqQ-MS/MS) method was established and validated for qualitative and quantitative analysis of 14 RAs (1-14) in 20 Rubia plants from China. The separation was achieved on a Waters ACQUITY UPLC® BEH C18 column (50 × 2.1 mm, 1.7 µm) using gradient elution with a mobile phase consisting of 0.1% formic acid in acetonitrile and water. Multiple reaction monitoring (MRM) in positive mode was used to enable the selective detection of RAs from the Rubia root and rhizome extracts within 10 min. This method was proved to be specific, sensitive, precise, and accurate with the limits of detection and quantification at 0.6-11.4 ng/mL and 1.9-34.2 ng/mL, respectively, and the relative standard deviation (RSD) of the overall intra-day and inter-day precision less than 5.24%. Satisfactory recovery was obtained from 83.80% to 111.77%, with the RSD less than 5.32%. Totally, 67 samples were then qualitatively and quantitatively analyzed by this method and 51 of them were proved to contain RAs. Thereinto, R. podantha and R. yunnanensis from Yunnan were the two most abundant species. Additionally, RAs were detected in 8 Rubia species for the first time. Then chemometric approaches were revealed to explain the relationship between samples based on their contents of RAs. This study demonstrated that the method was not only useful for RAs source discovery and chemotaxonomy of Rubia species, but also could be extended to standardization of RAs medical materials and their new drug research and development.

[Chemical constituents from root barks of Dictamnus dasycarpus and their cytotoxic activities].

Nineteen compounds, including kihadanin D (1), obacunone (2), kihadanin A (3), kihadanin B (4), kihadanin C (5), limonin (6), evodol (7), fraxinellone (8), furo[2,3-b]quinolin-4-ol (9), preskimmianine (10), ifflaiamine (11), dictamnol (12), naringenin (13), diosmetin (14), wogonin (15), scopoletin (16), cleomiscosin A (17), apocynin (18), and methyl pyroglutamate (19), were isolated from the methanol extract of the root barks of Dictamnus dasycarpus by using various column chromatographies. Their chemical structures were extensively determined on basis of UV, IR, NMR, MS, and CD spectroscopic data analyses. Among them, 1 is a new limonoid, 9 was isolated from plant kingdom for the first time, 11, 13-14 and 17-19 were obtained from the genus Dictamnnus for the first time. Cytotoxicities of compounds 1-18 were tested, and the results indicated that 1 exhibited cytotoxicities against three human cancer cell lines MDA-MB-231, A549 and HT29 with IC58 values of 16.22, 21.72 and 31.06 µmol·L⁻¹, respectively.

Dimeric BODIPY-loaded liposomes for dual hypoxia marker imaging and activatable photodynamic therapy against tumors.

This work reports a dimeric BODIPY (BDP)-loaded liposome with conjugation of anti-HIF antibodies for dual hypoxia marker imaging and nitroreductase (NTR)-activatable photodynamic therapy (PDT) against hypoxic tumors. In this theranostic nanosystem (designated Ab-DiBDP NPs), the newly designed orthogonal BDP dimer has high 1O2 quantum yield, and the substitution of a nitro group at the meso-position leads to the NTR-controllable activation of phototoxicity and fluorescence. Both in vivo and in vitro experiments demonstrate that the NTR-activatable PDT liposome can efficiently destroy cancer cells and prevent damage to normal cells. More significantly, the fascinating advantage of the nanoprobe is the synergy between the Cy 7-marked anti-HIF-1α antibody and the NTR-activatable DiBDP, which significantly improves the accuracy of tumor hypoxia imaging by simultaneous detection of NTR and HIF-1α. Therefore, this work presents a new paradigm for NTR-triggered PDT against cancer cells and provides a new avenue for precise tumor hypoxia diagnosis.

Venenum Bufonis induces rat neuroinflammation by activiating NF-κB pathway and attenuation of BDNF.

ETHNOPHARMACOLOGICAL RELEVANCE: Venenum Bufonis (VB), also called toad venom, has been widely used in clinic as a cardiotonic, anohyne and antineoplastic agents both in China and other Asian countries. However, its neurotoxicity and cardiotoxicity limit its wide clinical application. Compared with extensive attention attracted with cardiotoxicity, the toxic effect of VB on Central Nervous System (CNS) is much less studied. AIM OF THE RESEARCH: This study was performed to examine the neurotoxicity caused by VB on Sprague Dawley (SD) rats, then to clarify the mechanism in vivo by investigating its action on the neuroinflammation which possibly attributed to the activation of nuclear factor κB (NF-κB) pathway and the attenuation of brain-derived neurotrophic factor (BDNF). MATERIALS AND METHODS: Rats administrated with 0.5% carboxymethyl cellulose sodium salt (CMC-Na) aqueous solution and VB (100mg/kg, 200mg/kg and 400mg/kg) were sacrificed at 2h, 4h, 6h, 8h, 24h and 48h. The brain level of neurotransmitters and their corresponding receptors, pro-inflammatory cytokines, BDNF/TrkB and NF-κB pathway-related proteins were examined, respectively. RESULTS: VB administration induced severe neurologic damage and neuroinflammation, as indicated by the disordered 5-hydroxytryptamine (5-HT), dopamine (DA) and their corresponding receptors, together with the over production of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α). VB also notably promoted the expression of p-NF-κBp65, p-IκBα, p-IKKα and p-IKKß and down-regulated the expression of BDNF and TrkB. CONCLUSION: This study demonstrates that VB triggers neurotoxicity which probably is induced by neuroinflammation via activating of NF-κB pathway and attenuating the expression of BDNF.

Cloning, expression and characterization of D-aminoacylase from Achromobacter xylosoxidans subsp. denitrificans ATCC 15173.

D-Aminoacylase catalyzes the conversion of N-acyl-D-amino acids to d-amino acids and fatty acids. The aim of this study was to identify the D-aminoacylase gene from Achromobacter xylosoxidans subsp. denitrificans ATCC 15173 and investigate the biochemical characterization of the enzyme. A previously uncharacterized D-aminoacylase gene (ADdan) from this organism was cloned and sequenced. The open reading frame (ORF) of ADdan was 1467 bp in size encoding a 488-amino acid polypeptide. ADdan, with a high amino acid similarity to N-acyl-D-aspartate amidohydrolase from Alcaligenes A6, showed relatively low sequence similarities to other characterized D-aminoacylases. The recombinant ADdan protein was expressed in Escherichia coli BL21 (DE3) using pET-28a with a T7 promoter. The enzyme was purified in a single chromatographic step using nickel affinity gel column. The molecular mass of the expressed protein, calculated by SDS-PAGE, was about 52 kDa. The purified ADdan showed optimal activity at pH 8.0 and 50°C, and was stable at pH 6.0-8.0 and up to 45°C. Its activity was inhibited by Cu(2+), Fe(2+), Ca(2+), Mn(2+), Ni(2+), Zn(2+) and Hg(2+), whereas Mg(2+) had no significant influence on this recombinant D-aminoacylase. This is the first report on the characterization of D-aminoacylase with activity towards both N-acyl derivatives of neutral D-amino acids and N-acyl-D-aspartate. The characteristics of ADdan could prove to be of interest in industrial production of D-amino acids.