Chemotherapy is a cornerstone of cancer treatment, but its adverse effects, particularly those related to the cardiovascular and skeletal systems, are drawing more attention. According to studies, the PI3K/AKT signaling pathway may be involved in myelosuppression and cardiotoxicity, two types of multi-organ damage caused by chemotherapy. Despite the absence of thorough research, deer hide gelatin (DHG), a traditional Chinese medicine high in collagen, has shown promise in the prevention and treatment of skeletal and hematological disorders. This study aimed to evaluate the protective effects of DHG on chemotherapy-induced osteoporosis (OP) in rat bone tissue, as well as the material basis and mechanisms of its anti-OP activity. The results showed that DHG reversed the decrease in bone mineral density induced by chemotherapy, improved bone biomechanical properties, and ameliorated bone microstructure. DHG promoted the expression of the osteoblast-related indicators BALP and P1NP while suppressing the expression of the osteoclast-related marker TRACP-5b. Protein mass spectrometry screening was used to find putative anti-OP bioactive peptides. According to network pharmacology predictions, the PI3K signaling pathway may be the mechanism by which the active peptides in DHG produce their anti-OP actions. Additionally, immunofluorescence investigation demonstrated that DHG inhibited MMP9 expression while increasing RUNX2 expression. In vitro experiments also confirmed that DHG active peptides promoted bone formation by activating the PI3K/AKT/ERK signaling pathway, upregulating RUNX2 protein expression, and promoting osteoblast differentiation and mineralization. In conclusion, DHG has protective benefits against OP caused by chemotherapy. This also raises the possibility that DHG could be useful in the broader management of chemotherapy side effects (e.g., potentially related to cardio-oncology, considering the pathway’s important role in organs like the heart), warranting further investigation.

Aconiti Lateralis Radix Praeparata (Fuzi) represents a significant traditional Chinese medicine (TCM) that exhibits both notable pharmacological effects and toxicity. Various processing methods are implemented to reduce the toxicity of raw Fuzi by modifying its toxic and effective components, primarily diterpenoid alkaloids. To comprehensively analyze the chemical variations between different Fuzi products, ultra-high performance liquid chromatography-linear ion trap quadrupole Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS) was employed to systematically characterize Shengfuzi, Heishunpian and Baifupian. A total of 249 diterpenoid alkaloids present in Shengfuzi were identified, while only 111 and 61 in Heishunpian and Baifupian were detected respectively, indicating substantial differences among these products. An untargeted metabolomics approach combined with multivariate statistical analysis revealed 42 potential chemical markers. Through subsequent validation using 52 batches of commercial Heishunpian and Baifupian samples, 8 robust markers distinguishing these products were identified, including AC1-propanoic acid-3OH, HE-glucoside, HE-hydroxyvaleric acid-2OH, dihydrosphingosine, N-dodecoxycarbonylvaline and three unknown compounds. Additionally, the MS imaging (MSI) technique was utilized to visualize the spatial distribution of chemical constituents in raw Fuzi, revealing how different processing procedures affect the chemical variations between Heishunpian and Baifupian. The distribution patterns of different diterpenoid alkaloid subtypes partially explained the chemical differences among products. This research provides valuable insights into the material basis for future investigations of different Fuzi products.

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 & mu;M.

The composition and quantity of amino acids (AAs) in seeds are complicated due to the various origins and modifications of different species. In this study, a novel automatic neutral loss filtering (ANLF) strategy based on accurate mass searching by Python was developed to analyze the free and hydrolyzed AA-phenyl isothiocyanate (PITC) derivatives from seeds of Gymnosperm and Angiosperm phyla. Compared with traditional strategies, ANLF showed much higher accuracy in screening AA derivatives by filtering nitrogen-containing non-AA compounds and efficiency in processing large datasets. Meanwhile, the content phenotype of 20 proteinogenic AAs from seeds of these two families was characterized by a 35-min HPLC method combined with an automated peak-matching strategy. AA profiles of 232 batches of seeds from 67 species, consisting of 19 proteinogenic AAs, 21 modified AAs, and 77 unknown AAs, would be a good reference for their application in food and medicine.

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.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

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.

Natural peptides (NPs) are ubiquitous throughout the organism with diversified structures and wide dynamic concentration range, which leads to serious challenge for their comprehensive analysis. Herein, a pseudotargeted peptidomics strategy based on dynamic multiple reaction monitoring (DMRM) with high-throughput data acquisition and accurate quantification was established. Specifically, an in-house software Pep-MRMer was developed for extraction of ions information of peptides, followed by de-redundancy and fusion to construct a transition list. Additionally, retention time (RT) calibration method was optimized by using high content components for effective transference of ion pairs between different detection conditions (e.g., instruments, chromatographic gradients). This strategy was applied for profiling NPs and screening signature peptides for species authentication of Pheretima, a multi-source animal-derived drug widely used in Asian countries. In all, a total of 3307 peptides had excellent quantitative detection after transference. Subsequently, nine signature peptides with extraordinary specificity were screened, identified, and confirmed by comparing with the synthetic standards. Ultimately, a 10 min specific chromatogram based on signature peptides was developed, which realized rapid and accurate identification of Pheretima. Taken together, this research provides a robust strategy for species discrimination of animal-derived drugs without adequate quality control measures.

Despite Eucommia ulmoides to be served as a crucial source for food additives and healthcare products, unclarity of the metabolomic differences among four different plant parts (bark, leaf, flower, seed) seriously restricts the comprehensive quality control of the herb and its related commercial products. In this study, a strategy integrating in-depth profiling, nontargeted metabolomics, and selected detection was established and exemplarily applied to authenticate E. ulmoides related products. UPLC/Q-TOF Fast DDA approach was utilized for in-depth profiling among four parts to characterize 222 compounds. In addition, 38 batches of different parts of E. ulmoides were compared by metabolomics method, and multi-step chemometrics analysis to unveil 25 chemical markers. Finally, an UPLC/QDa-SIM method was established based 8 most important diagnosis components to identify 12 commercial products. This study provides a novel and comprehensive strategy for the quality assessment of foods and dietary supplements.

The hyphenated technique of offline two-dimensional (2D) chromatography with high resolution mass spectrometry (MS) was an efficient tool for separation and characterization of components in complex systems such as herbal medicines, especially those co-eluting components or isomers. In this study, we constructed the ultra-performance convergence chromatography (UPC2) × reversed phase (RP) chromatographic separation system and developed a mass defect filtering (MDF)-based precursor ion list (PIL) acquisition method to improve the selectivity and sensitivity of this technique, and the systematic characterization of diterpenoid alkaloids in the lateral roots of Aconitum carmichaelii (namely "Fuzi" in Chinese) was used as an example. The constructed offline 2D separation system showed a good orthogonality of 0.77. Besides, the in-house databases for known and predicted C19- and C20-diterpenoid alkaloids were established by molecular design in Compound Discoverer software for MS data matching and filtering, and two MDF windows were further constructed to screen out more potential diterpenoid alkaloids with novel structures and to obtain the PIL (mass range: even values between 298 and 1020 Da, parent mass width: ±100 mDa) for data acquisition by calculating the m/z values of potential ions using mass range and corresponding mass defect in the MDF windows. In addition, an integrative structure interpretation strategy was developed by integrating elemental composition analysis, ring double bond analysis, neutral loss filtering, diagnostic ion filtering and database matching, etc. As a result, a total of 659 components in the lateral roots of A. carmichaelii were exposed and characterized, including 526 potential new compounds. This strategy showed significant advantages in improving the coverage and selectivity of screening, and could also be applied in systematic characterization of components in other herbal medicines.

Lilium lancifolium and Lilium brownii viridulum were two common cultivars of Lilium in China, which have been used as a source of food in ancient China, and as a traditional herbal medicine in most northern hemispheres countries continues today. However, only a few secondary metabolites in Lilium closely related to human health have been reported. In this research, an offline two-dimensional (HILIC and RP C18) separation system combined with multimode high-resolution mass spectrometry data acquisition was established for in-depth exploration and comparison of the chemical components in Lilium. In total, 331 components were identified, among which phenylpropanoid derivatives and steroidal saponins were the most abundant components. Furthermore, sulfur derivatives and steroidal alkaloids were systematically characterized in Lilium for the first time. These results provided valuable information for in-depth differentiating types of components characterization, which may be applied to assess and improve the edible and medicinal values of Lilium.

The complexity of metabolites in traditional Chinese medicine (TCM) hinders the comprehensive profiling and accurate identification of metabolites. In this study, an approach that integrates enhanced column separation, mass spectrometry post-processing and result verification was proposed and applied in the identification of flavonoids in Dalbergia odorifera. Firstly, column chromatography fractionation, followed by liquid chromatography–tandem mass spectrometry was used for systematic separation and detection. Secondly, a three-level data post-processing method was applied to the identification of flavonoids. Finally, fragmentation rules were used to verify the flavonoid compounds. As a result, a total of 197 flavonoids were characterized in D. odorifera, among which seven compounds were unambiguously identified in level 1, 80 compounds were tentatively identified by MS-DIAL and Compound Discoverer in level 2a, 95 compounds were annotated by Compound discoverer and Peogenesis QI in level 2b, and 15 compounds were exclusively annotated by using SIRIUS software in level 3. This study provides an approach for the rapid and efficient identification of the majority of components in herbal medicines.

The inherent complexity of traditional Chinese medicines necessitates the application of multi-dimensional information to accomplish comprehensive profiling and confirmative identification of their chemical components. In this study, we display an enhanced strategy by integrating offline superimposed two-dimensional separation (S-2D-LC) with mass defect filter and diagnostic ion filter to comprehensively characterize the alkaloid composition of Fritillariae Pallidiflorae Bulbus (FPB). The superimposed HILIC × RP and UPCC × RP offline two-dimensional liquid chromatography system was constructed with superior orthogonality (R2=0.004 and R2=0.001) for chromatographic separation. In total, 70 fractions were collected after the first-dimensional chromatographic separation (HILIC and UPCC) and then analyzed by the second-dimensional reversed phase (RP) liquid chromatography coupled with Q-TOF/MS/MS in FAST DDA acquisition mode. A four-step interpretation strategy combining mass defect filter with diagnostic ion filter was developed to rapidly characterize alkaloids in Fritillaria species. Ultimately, a sum of 529 Fritillaria alkaloids were characterized from two botanical origins of FPB. The integrated strategy is practical to efficiently expose and comprehensively characterize more trace and isomeric components in complex herbal medicines.

A key segment in medicinal plant authentication is the establishment of quality markers that embody the intrinsic metabolites difference independent of instruments and experiment conditions. A strategy integrating nontargeted metabolomics and multi-criteria decision making model for robust quality markers discovery is presented and applied to authenticate Ophiopogon japonicus (L. f.) Ker-Gawl. First, an ultra-performance liquid chromatography/quadrupole time-of-flight MSE approach was established for global metabolites profiling and identification. Second, multivariate statistical analysis was performed to explore potential quality markers of different origins of ophiopogonis radix. Third, potential quality markers were ordered and filtered by multi-criteria decision making model to infer robust quality markers and further validated in different instruments and experiment conditions by validation model. Forth, The validation model using the robust quality markers managed to discriminate the origins of ophiopogonis radix samples procured from the herbal markets. Consequently, 2 robust quality markers, cixi-ophiopogon B and ophiopogonin D were discovered and further validated on different instruments and experiment conditions. This integrated strategy provided a practical solution for reliable and convenient authentication of geo-authentic herb. This article is protected by copyright. All rights reserved.

Nano-LC MS/MS was used to analyze trypsin digested deer-hide gelatin(DHG) samples, hydroxylation and O-glycosylation on lysine sites of DHG were comprehensive identified by using PEAKS Studio software. The sites, sorts and amounts of hydroxylation and O-glycosylation on Type Ⅰ collagen α1 chain(COL1 A1) and α2 chain(COL1 A2) of DHG were revealed. As a result, 5 284 peptides were identified from DHG samples, which were mainly from COL1 A1 and COL1 A2. Among these peptides, there were 449 peptides with hydroxylysine, 442 with galactosyl-hydroxylysine, 449 with glucosyl-galactosyl-hydroxylysine. The major modified sites of hydroxylation and O-glycosylation in DHG were shown as follow: α1-9 N and α2-5 N in N-telopeptides, α1-87, α1-174, α1-930, α2-87, α2-174, α2-933 in triple helix domain, and α1-16 C in C-telopeptides. These hydroxylation and O-glycosylation were correlated with the formation and stability of collagen molecules and collagen fibrils. It is feasible for the collagens and peptides dissolving from deer skin collagen fibrils under high temperature and pressure decocting, high temperature and pressure also might destroy inter-molecular covalent cross-linking and help those glycol-peptides formations. The present study provided ideas and strategies for the in-depth investigation on DHG chemical constituents, and showed good theoretical significance and application value.

Objective: To develop a reversed-phase high-performance liquid chromatography method for the quantification of major ginsenosides in red ginseng (RG, the steamed and dried root of the cultivar of Panax ginseng C. A. Mey). Methods: A feasible method was developed in strict accordance with chromatographic properties of eight ginsenosides. Their contents could be unveiled with conventional external standard method, or as an alternative, using ginsenoside Rg1 as the single reference standard by means of seven conversion factors. Those parameters had been validated on different chromatographic columns and instruments. Results: Twenty-one batches of RG samples were determined. In addition, the chromatograms of RG and confusing species, including Panax ginseng, Panax quinquefolium, and Panax notoginseng, were apparently different. Conclusions: The method was proved to be efficient for the quality control of RG.

In this study, a high performance thin-layer chromatography/single quadrupole mass spectrometry QDa (HPTLC-QDa) method for robust authentication of Ganoderma lucidum, a popular and valuable herbal medicine, has been developed. This method is simple and practical, which allows direct generation of characteristic mass spectra from the HPTLC plates automatically with the application of in situ solvent desorption interface. The HPTLC silica gel plates were developed with toluene-ethyl formate-formic acid (5 : 5 : 0.2, V/V) and all bands were transferred to QDa system directly in situ using 80% methanol with 0.1% formic acid as desorption solvent. The acquired HPTLC-QDa spectra showed that luminous yellow band b3, containing ganoderic acid B/G/H and ganodeneric acid B, the major active components of Ganoderma, could be found only in G. lucidum and G. lucidum (Antler-shaped), but not in G. sinense and G. applanatum. Moreover, bands b13 and b14 with m/z 475/477 and m/z 475/491/495, respectively, could be detected in G. lucidum (Antler-shaped), but not in G. lucidum, thus allowing simple and robust authentication of G. lucidum with confused species. This method is proved to be simple, practical and reproducible, which can be extended to analyze other herbal medicines.

In vivo metabolite profiling of herbal medicines remains a challenge due to the complex chemical composition and drastic interference from biological matrix. In this study, a systematic strategy was established for comprehensive metabolite profiling of Danqi Tongmai (DQTM) tablet, a combination of salvianolic acids and notoginsenosides, in rats after oral administration. This strategy was composed of six steps. Firstly, the rat plasma and tissue samples were collected at multiple time points to increase the representativeness of samples. Secondly, different sample preparation methods were systematically investigated including protein precipitation, liquid-liquid extraction and solid-phase extraction to obtain superior extraction efficiency for both salvianolic acids and notoginsenosides. Thirdly, the MS acquisition method was optimized by splitting the full scan range into two separate segments to improve the detection capability for minor components. Fourthly, an extended polygonal mass defect filter (EP-MDF) model was constructed to filter potential metabolites of salvianolic acids and notoginsenosides, and remove large amounts of interference ions. Fifthly, ion intensity-based time point-staggered precursor ion list (IITPS-PIL) was generated to trigger more targeted MS/MS acquisition for potential metabolites at the highest concentration. Finally, the absorbed prototypes and metabolites were comprehensively characterized by reference standards and MS/MS fragmentation. The proposed strategy significantly improved the detection ability for trace prototypes and metabolites in vivo. A total of 370 components, including 94 prototypes (38 confirmed with reference standards) and 276 metabolites, were tentatively characterized in rat plasma and tissue samples after oral administration of DQTM. Collectively, this paper provided an applicable reference for comprehensive metabolite profiling of herbal medicines in complex biological samples.

Comprehensive analysis and identification of chemical components are of great significance for evaluating the efficacy and safety of herbal medicines, as well as for drug exploitation and development. Here we developed a “force iteration molecular designing” strategy, by combing a database-based in-house software for a precursor ion list (PIL) and PIL-triggered collision-induced dissociation-MS2 and high-energy C-trap dissociation-MS2 (PIL-CID/MS2-HCD/MS2) on an LTQ-Orbitrap mass spectrometer, aiming for the systematic characterization and discovery of new protostane triterpenoids (PTs) from Alisma Rhizoma (AR). AR was a well-known herbal remedy widely used for diarrhea, but its systematic characterization and comparison between two botanical origins have not been reported. Firstly, in-house software was developed based on force iteration, to generate a PIL that contains 483 accurate precursor ions. Secondly, to facilitate the acquisition of rich fragments and diagnostic ions sufficient for the structural elucidation of different types of PTs, a hybrid data acquisition method, namely PIL-CID/MS2-HCD/MS2, was generated. Thirdly, a total of 473 PTs were rapidly characterized from two botanical origins of AR according to an established four-step interpretation method, and the common constituents were 277 with ratio 70% (277/395) and 78% (277/355) in the rhizome of Alisma plantago-aquatica and A. orientale, respectively. Finally, two new PTs were isolated and unambiguously identified by NMR verifying the feasibility of this combined data acquisition strategy. This integrated strategy could improve the efficiency in the detection of new compounds in a single run and is practical to comprehensively characterize the complex components in herbal medicines.

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.