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.

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.

Fingerprinting Evaluation and Gut Microbiota Regulation of Polysaccharides from Jujube (Ziziphus jujuba Mill.) Fruit.

Jujube fruit was well-loved and praised by the broad masses due to its delicious taste, abundant nutritional value, and medicinal properties. Few studies reported the quality evaluation and gut microbiota regulation effect of polysaccharides of jujube fruits from different producing areas. In the present study, multi-level fingerprint profiling, including polysaccharides, oligosaccharides, and monosaccharides, was established for the quality evaluation of polysaccharides from jujube fruits. For polysaccharides, the total content in jujube fruits ranged from 1.31% to 2.22%, and the molecular weight distribution (MWD) ranged from 1.14 × 105 to 1.73 × 106 Da. The MWD fingerprint profiling of polysaccharides from eight producing areas was similar, but the profile of infrared spectroscopy (IR) showed differentiation. The characteristic signals were screened and used to establish a discrimination model for the identification of jujube fruits from different areas, and the accuracy of identification reached 100.00%. For oligosaccharides, the main components were galacturonic acid polymers (DP, 2-4), and the profile of oligosaccharides exhibited high similarity. The monosaccharides, GalA, Glc, and Ara, were the primary monosaccharides. Although the fingerprint of monosaccharides was semblable, the composing proportion of monosaccharides revealed significant differences. In addition, the polysaccharides of jujube fruits could regulate the gut microbiota composition and possess potential therapeutic effects on dysentery and nervous system diseases.

Systematical characterization and comparison of saponins in Achyranthes bidentata Blume and its three analogous species.

INTRODUCTION: Achyranthes bidentata Blume (AB) has been used for a long time and is recorded in the Chinese Pharmacopoeia 2020 edition. It is commonly confused with Achyranthes aspera Linn (AA), Cyathula officinalis Kuan (CO) and Cyathula capitata (Wall.) Moq. (CC), belonging to the Achyranthes and Cyathula genera of the Amaranthaceae family. It is of great significance to recognize and distinguish chemical components of AB, AA, CO and CC. OBJECTIVE: The purpose of this study was to develop an analytical method for in-depth characterization and comparison of saponins in AB, AA, CO and CC. METHODS: The extracts of AB, AA, CO and CC were analyzed by an RP × RP (C18 × Phenyl-Hexyl) 2D LC system, eluted by acidic × ion pair mobile phases and detected by high resolution mass spectrometry. Fragmentation patterns of saponins were elucidated and proposed according to reference compounds or literature reports. RESULTS: As a result, 839 saponins consisting of 81, 415, 99 and 392 components corresponding to AB, AA, CO and CC, respectively, were characterized, including 594 potentially new saponins. Meanwhile, 29 kinds of aglycones were elucidated, among which 25 were new ones. Besides, 14, 91, 37 and 174 characteristic potential quality markers with MS intensities exceeding 10,000 were found in AB, AA, CO and CC, respectively. CONCLUSION: This comprehensive study not only expands our knowledge of the types of saponins in Achyranthes and Cyathula, but also reveals the differences among four kinds of analogous herbs (AB, AA, CO and CC), which facilitates the quality control of these herbal medicines in the future.

Authentication of three main commercial Pheretima based on amino acids analysis.

Pheretima has been used as an animal-derived traditional Chinese medicine for thousands of years in Asian countries due to its multi-activities. However, more than half of the commercial Pheretima are adulterants according to the previous research. Besides, the standards of Pheretima are still inadequate in the identification of Pheretima species. In this study, an amino acids (AAs) analytical method established based on the ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-QqQ-MS) in multiple reaction monitoring (MRM) mode through derivatization with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) was used for qualitative and quantitative analysis of the total AAs of three main commercial Pheretima (two major Pheretima species, Amynthas aspergillum, Metaphire vulgaris, and one main counterfeit, M. magna). As a result, 16 AAs were detected and quantitated in their hydrolyzed samples. Then, multivariate statistical analysis was applied to distinguish the three commercial Pheretima based on their AAs level. Finally, four AAs (Thr, Glu, Asp, and Arg) were screened as species-differential AAs, which may be used as chemical markers to distinguish the three commercial Pheretima. This study deeply described the outline of AAs in Pheretima and offered a good reference for its species authentication.

Systematic characterization of chemical constituents in Mahuang decoction by UHPLC tandem linear ion trap-Orbitrap mass spectrometry coupled with feature-based molecular networking.

Comprehensive characterization of traditional Chinese medicine prescriptions has long been a hurdle due to the chemical complexity and the lack of analytical tools. Mahuang decoction is a well-known traditional Chinese medicine prescription widely used for sweating and relieving the exterior, relieving cough and asthma, but it was insufficiently chemically scrutinized. In this study, the chemical component information of Mahuang decoction was investigated by ultrahigh-performance liquid chromatography tandem linear ion trap-Orbitrap mass spectrometry. A new data processing tool, feature-based molecular networking, was introduced for grouping and elucidating the compounds. In this way, 156 chemical components were identified or tentatively characterized, including alkaloids, triterpenoid saponins, flavanone-O-glycosides, flavone-C-glycosides, and procyanidins. Thus, this research provides a solid foundation for further development of Mahuang decoction, and the adopted method is expected to be applied to other traditional Chinese medicine prescriptions.

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.

Characterization and discrimination of steroidal saponins in Tribulus terrestris L. and its three different aerial parts by chemical profiling with chemometrics analysis.

Tribulus terrestris L. is a well-known medicinal plant from subtropical areas. In China Pharmacopoeia, only the fruit of T. terrestris is recorded to be the medicinal part. But some pharmaceutical preparations and food supplements made of total saponins of aerial part of T. terrestris including fruits, stems, and leaves are commercially available. However, steroidal saponins in different parts have not been studied extensively. So differentiating three parts and ensuring reasonable application of T. terrestris has been an important issue. Herein, we developed an integrated platform based on components profiling and chemometrics analysis to comparatively characterize and investigate steroidal saponins in three parts. As a result, a total of 84 steroidal saponins were characterized or tentatively identified, including 20 compounds reported for the first time. A clear separation of the three parts was achieved by partial least squares discriminant analysis based on the identified saponins, and 17 saponins were screened as biomarkers. Support vector machines model established based on 17 markers showed excellent predication accuracy of 100%. Finally, different distribution of steroidal saponins in three parts was shown obviously by heatmap visualization. These results provide promising perspectives for quality control of Chinese medicine, especially those with different medical parts.

[Mass spectrometry guided strategy based on feature fragment ions for guided-separation on quinoline alkaloids from root barks of Dictamnus dasycarpus].

The root bark of Dictamnus dasycarpus is one of common traditional Chinese medicines (TCMs). Quinoline alkaloids are one of the main active substances in this TCM and possess many biological activities including anti-titumor, anti-inflammation, anti-bacteria, anti-oxidation, and anti-platelet aggregation activities. In this study, eight quinoline alkaloids 1-8 were firstly separated from the root barks of D. dasycarpus. It was difficult to isolate more quinoline alkaloids from the remaining fraction 8 in D. dasycarpus by this conventional chemical separation, so the target analysis method combined LC-MS guided-separation of quinoline alkaloids from fraction 8 was established. MS/MS fragmentation patterns of eight quinoline alkaloids reference standard compounds 1-8 were studied by ultra-performance liquid chromatography-electrospary ionization-mass spectrometry (UPLC-ESI-MS/MS). Based on the feature fragment ion m/z 200, the parent ion scan mode was established for the target analysis of quinoline alkaloids in fraction 8. Finally, 8-methoxyflindersine (9) and N-metilatanina (10) were discovered and isolated quickly from fraction 8 guided by LC-MS, and their structures were identified by NMR and MS. Among them, compound 10 was isolated from the genus Dictamnus for the first time. These results indicated that this method is not only quick and sensitive for analyzing the quinoline alkaloids, but also to effectively guided-separate this kind of alkaloids in the root barks of D. dasycarpus.

[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.

Pharmacodynamical research of extracts and compounds in traditional Chinese medicines for Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease (AD). Currently, there is no cure for PD, and medications can only control the progression of the disease. Various experimental studies have shown the significant efficacy of TCM in treating PD, and combination with western medicine can enhance the effects and reduce toxicity. Thus, exploring effective anti-PD compounds from TCM has become a popular research fields. This review summarizes commonly used TCM extracts and natural products for the treatment of PD, both domestically and internationally. Furthermore, it delves into various mechanisms of TCM in treating PD, such as anti-oxidative stress, anti-inflammatory, anti-apoptotic, improve mitochondrial dysfunction, inhibits α-synuclein (α-Syn) misfolding and aggregation, regulating neurotransmitters, regulates intestinal flora, enhances immunity, and so on. The results reveal that most TCMs exert their neuroprotective effects through anti-inflammatory and anti-oxidative stress actions, thereby slowing down the progression of the disease. These TCM may hold the key to improving PD therapy and have tremendous potential to be developed as novel anti-PD drugs.

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.

Species discrimination of multiple botanical origins of Fritillaria species based on infrared spectroscopy, thin layer chromatography-image analysis and untargeted metabolomics.

BACKGROUND: Fritillaria Bulbus (FB), a precious medicinal herb renowned for its heat-clearing, lung-moistening, cough-relieving and phlegm-eliminating effects. In pursuit of profits, unscrupulous merchants have engaged in the substitution or adulteration of valuable varieties with cheaper alternatives. It is, therefore, urgent to develop effective technical approaches to identify FBs from adulterants. METHODS: This paper employed infrared spectroscopy (IR), thin layer chromatography-image analysis (TLC-IA), and untargeted metabolomics techniques to discriminate ten species of FBs. RESULTS: Five species of FBs were successfully differentiated using mid-infrared spectroscopy. Furthermore, the power of TLC-IA technology allowed the differentiation of five species of FBs and two origins of FCBs (Fritillariae Cirrhosae Bulbus). Remarkably, through the application of untargeted metabolomics technique, the precise discrimination of five species of FBs, as well as three origins of FCBs were accomplished. Moreover, a comprehensive identification of 101 markers that reliably distinguished diverse FBs was achieved through the employment of untargeted metabolomics technique. CONCLUSION: The investigation presented powerful means of detection for assuring the quality control of Fritillaria herbs.

Chemical profiling and comparative analysis of different parts of Asarum heterotropoides using SPME-GC-QTOF-MS and LC- Orbitrap -MS.

Asari radix et rhizoma is the sole plant from the Aristolochiaceae family officially sanctioned for medicinal in China, primarily employed for treating colds and headaches, and is widely utilized in clinical practice. Initially, the entire plant was specified for medicinal use, but since 2005, the authorized part has been restricted to the roots and rhizomes. The chemical constituents are directly linked to its efficacy and safety, yet a comparative analysis of the chemical profiles between the overground and underground parts has not been reported. This paper represents the first comparative study of the chemical constituents in the two parts, achieved through the synergistic application of solid phase micro extraction coupled with gas chromatography mass spectrometry (SPME-GC-MS) and liquid chromatography Orbitrap MS (LC-Orbitrap-MS). Using SPME-GC-MS, 51 constituents were identified from both parts, with 89 % being shared components, indicating a close similarity in their volatile compositions. Through LC-Orbitrap-MS, 308 constituents were identified, sharing 76 % commonality, revealing a more pronounced disparity in non-volatile components. Plant metabolomics screening pinpointed 8 volatile and 14 non-volatile components capable of distinguishing the two parts, with the latter being more stable and thus better suited as markers for differentiation. This research furnishes a scientific rationale for selecting distinct parts of Asari radix et rhizoma and for implementing monitoring strategies in clinical application.

MATLAB language assisted data acquisition and processing in liquid chromatography Orbitrap mass spectrometry: Application to the identification and differentiation of Radix Bupleuri from its adulterants.

Comprehensive and rapid analysis of secondary metabolites like saponins remains challenging. This study aimed to establish a semi-automated workflow for filtration, identification, and characterization of saikosaponins in six Bupleurum species. Radix Bupleuri, a high-sales herbal medicine, is often adulterated, restricting its quality control and applications. Two authentic Radix Bupleuri species and four major adulterants were analyzed through UHPLC-LTQ-Orbitrap-MS for targeted saikosaponin analysis. To reveal trace saikosaponins and obtain quality fragment data, a MATLAB-based process automatically enumerating "sugar chain + aglycone + side chain" combinations and deduplicating generated a predicted saikosaponin database covering all possible saikosaponins as a precursor ion list for comprehensive targeted acquisition. To focus on informative ions and reduce MS analysis workload, we utilized MATLAB to automatically filtrate the false positive ions by MS1 and MS2 spectrometry. The newly established MATLAB-assisted data acquisition approach exhibited 50 % improvement in characterization of targeted saikosaponins. Furthermore, positive and negative ionization workflows were designed for accurate saikosaponins characterization based on fragmentation rules. In total, 707 saikosaponins were characterized, including over 500 potential new compounds and previously unreported C29 aglycones. We identified 25 saikosaponins present in both authentic species but absent in adulterants as potential markers. This unprecedented comprehensive multi-origin species differentiation demonstrates the promise of MATLAB-assisted acquisition and processing to advance saponin identification and standardize the Radix Bupleuri market.

Medcheck: a novel software for automated de-formulation of traditional Chinese medicine (TCM) prescriptions by liquid chromatography-mass spectrometry.

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Comparative identification of phytoecdysteroids in Achyranthes bidentata Blume and its three analogous species and application in differentiation between processing products from different species.

The differentiation of raw herbal products from similar species have been achieved by plant metabolomics. However, the distinguishment on various processed products with improved activities and wide clinical utilization from similar species is still tricky due to obscure composition variations during processing. In this study, a comprehensive analysis of phytoecdysteroids in Achyranthes bidentata Blume (AB) and its three analogous species, which were all called Niuxi in Chinese, was conducted on UPLC-HRMS by integrating dynamic exclusion acquisition with data post-processing of targeted multilateral mass defect filter. Two most frequently used species, AB and Cyathula officinalis Kuan (CO) were systematically compared with plant metabolomics methods. And the differential components from the raw materials were evaluated on the ability of distinguishing processed products. The substitution of hydroxyl groups on C-21, C-20, C-22 and C-25 were determined by characteristic mass differences, leading to systematical characterization of 281 phytoecdysteroids. In plant metabolomics studies of raw AB and CO, 16 potential markers were filtered by VIP value > 1, and displayed satisfactory differentiation on the processed AB and CO. The results facilitated the quality control of the four species, especially the processed products of AB and CO, also provided a reference method for the quality control of other processed products.

Systematical characterization of gypenosides in Gynostemma pentaphyllum and the chemical composition variation of different origins.

Gynostemma pentaphyllum (Thunb.) Makino is an herbaceous plant of Cucurbitaceae family, which has been widely used as an herbal tea and traditional Chinese medicine. Since its saponins are similar to ginsenosides and have a wide range of activities, it has attracted wide interest. However, there are still a large number of unknown saponins that have not been isolated, especially some trace gypenosides. In the present study, a HILIC × RP offline two-dimensional liquid separation combined with a multimode data acquisition was developed for the systematical characterization of gypenosides. On top of the negative mode information, considering that saponins are prone to in-source fragmentations in positive ion mode, a precursor ion list data acquisition method was used for the targeted acquisition of multistage positive data. Reference herbal drug was taken as a golden sample to probe the chemical composition of G. pentaphyllum. The mixed sample of commercially available samples were also analyzed in parallel. Furthermore, the chemical compositions of commercially available samples from different sources were compared. In total, 1108 saponins were characterized, among which 588 were accurately characterized, with 574 identified in the reference herbal drug and 700 in the mixed commercially available samples. The commercially available samples showed great composition variation. These findings clarified the material basis and provided clues for quality control of G. pentaphyllum.

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.