Three undescribed dihydrostilbene glycosides from leaves of Camellia oleifera Abel. And their anti-inflammatory activity.

Three previously unidentified dihydrostilbene glycosides, named oleiferaside A (1), oleiferaside B (2), and oleiferaside C (3), were discovered through a phytochemical exploration on Camellia oleifera Abel. leaves. Additionally, nine known secondary metabolites (4-12) were also identified. The undescribed secondary metabolites 1-3 were elucidated as 3,5-dimethoxydihydrostilbene 4'-O-α-l-arabinofuranosyl-(1 â†’ 6)-ß-d- glucopyranoside, 3,5-dimethoxydihydrostilbene 4'-O-α-l-arabinopyranosyl-(1 â†’ 6)-ß-d- glucopyranoside and 3,5-dimethoxydihydrostilbene 4'-O-ß-d-apiofuranosyl-(1 â†’ 6)-ß-d- glucopyranoside, respectively. HR-MS and NMR spectroscopy were utilized for determining the structures of the isolates. The natural products were assessed for their anti-inflammatory effect using RAW264.7 macrophage stimulated by LPS. The findings demonstrated that compounds 1-4 exhibited inhibitory activities on NO and PGE2 production without causing cytotoxicity. These observations suggest that these compounds may have potential anti-inflammatory properties.

Compounds isolation from Syzygium cumini leaf extract against the Vibrio species in shrimp through bioassay-guided fractionation.

This study was conducted to isolate and identify the bioactive compounds from the ethanolic extract of Syzygium cumini leaf against Vibrio species through a bioassay-guided fractionation. The ethanol extract was exposed to silica gel chromatography followed by reversed phase HPLC to isolate the most effective fraction against V. parahaemolyticus. Using further UHPLC-orbitrap-ion trap mass spectrometry, five compounds were isolated with broad-spectrum potency against a range of Vibrio species viz. V. parahaemolyticus, V. alginolyticus, V. harveyi, V. vulnificus and V. anguillarum. The IC50 values for the compounds ranged from 8 to 48 µg/mL against the most sensitive species V. vulnificus and 58 to >400 µg/mL against V. alginolyticus. The results of the toxicity tests demonstrated that the compounds were not harmful for shrimp. The study's findings indicate that S. cumini leaf extract may contain bioactive molecules that are able to be substituted for antibiotics to treat vibriosis in shrimp farming.

Compound isolation through bioassay-guided fractionation of Tectona grandis leaf extract against Vibrio pathogens in shrimp.

Tectona grandis Linn, commonly known as teak, is traditionally used to treat a range of diseases, including the common cold, headaches, bronchitis, scabies, diabetes, inflammation, and others. The present study was conducted with the purpose of isolating and identifying the active compounds in T. grandis leaf against a panel of Vibrio spp., which may induce vibriosis in shrimp, using bioassay-guided purification. The antimicrobial activity was assessed using the microdilution method, followed by the brine shrimp lethality assay to determine toxicity. Following an initial screening with a number of different solvents, it was established that the acetone extract was the most effective. The acetone extract was then exposed to silica gel chromatography followed by reversed-phase HPLC and further UHPLC-orbitrap-ion trap mass spectrometry to identify the active compounds. Three compounds called 1-hydroxy-2,6,8-trimethoxy-9,10-anthraquinone, deoxyanserinone B, and khatmiamycin were identified with substantial anti-microbial action against V. parahaemolyticus, V. alginolyticus, V. harveyi, V. anguillarum, and V. vulnificus. The IC50 values of the three compounds viz. 1-hydroxy-2,6,8-trimethoxy-9,10-anthraquinone, deoxyanserinone B, and khatmiamycin varied between 2 and 28, 7 and 38, and 7 and 56 µg/mL, respectively, which are as good as the standard antibiotics such as amoxicillin and others. The in vivo toxicity test revealed that the compounds were non-toxic to shrimp. The results of the study suggest that T. grandis leaf can be used as a source of bioactive compounds to treat Vibrio species in shrimp farming.

Metabolic profiling of Chimonanthus grammatus via UHPLC-HRMS-MS with computer-assisted structure elucidation and its antimicrobial activity.

Chimonanthus grammatus is used as Hakka traditional herb to treat cold, flu, etc. So far, the phytochemistry and antimicrobial compounds have not been well investigated. In this study, the orbitrap-ion trap MS was used to characterize its metabolites, combined with a computer-assisted structure elucidation method, and the antimicrobial activities were assessed by a broth dilution method against 21 human pathogens, as well as the bioassay-guided purification work to clarify its main antimicrobial compounds. A total of 83 compounds were identified with their fragmentation patterns, including terpenoids, coumarins, flavonoids, organic acids, alkaloids, and others. The plant extracts can strongly inhibit the growth of three Gram-positive and four Gram-negative bacteria, and nine active compounds were bioassay-guided isolated, including homalomenol C, jasmonic acid, isofraxidin, quercitrin, stigmasta-7,22-diene-3ß,5α,6α-triol, quercetin, 4-hydroxy-1,10-secocadin-5-ene-1,10-dione, kaempferol, and E-4-(4,8-dimethylnona-3,7-dienyl)furan-2(5H)-one. Among them, isofraxidin, kaempferol, and quercitrin showed significant activity against planktonic Staphylococcus aureus (IC50 = 13.51, 18.08 and 15.86 µg/ml). Moreover, their antibiofilm activities of S. aureus (BIC50 = 15.43, 17.31, 18.86 µg/ml; BEC50 = 45.86, ≥62.50, and 57.62 µg/ml) are higher than ciprofloxacin. The results demonstrated that the isolated antimicrobial compounds played the key role of this herb in combating microbes and provided benefits for its development and quality control, and the computer-assisted structure elucidation method was a powerful tool for chemical analysis, especially for distinguishing isomers with similar structures, which can be used for other complex samples.

Bioassay-Guided Isolation of Anthelmintic Components from Semen pharbitidis, and the Mechanism of Action of Pharbitin.

Parasitic helminths continue to pose problems in human and veterinary medicine, as well as in agriculture. Semen pharbitidis, the seeds of Pharbitis nil (Linn.) Choisy (Convolvulaceae), is a well-known traditional Chinese medicinal botanical preparation widely used for treating intestinal parasites in China owing to its desirable efficacy. However, the anthelmintic compounds in Semen pharbitidis and their mechanism of action have not been investigated yet. This study aimed to identify the compounds active against helminths from Semen pharbitidis, and to establish the mechanism of action of these active compounds. Bioassay-guided fractionation was used to identify the anthelmintic compounds from Semen pharbitidis. The anthelmintic assay was performed by monitoring Caenorhabditis elegans (C. elegans) motility with a WMicrotracker instrument. Active compounds were identified by high-resolution mass spectrometry. Several (analogues of) fragments of the anthelmintic compounds were purchased and tested to explore the structure-activity relationship, and to find more potent compounds. A panel of C. elegans mutant strains resistant to major currently used anthelmintic drugs was used to explore the mechanism of action of the active compounds. The bioassay-guided isolation from an ethanol extract of Semen pharbitidis led to a group of glycosides, namely pharbitin (IC50: 41.0 ± 9.4 µg/mL). Hit expansion for pharbitin fragments yielded two potent analogues: 2-bromohexadecanoic acid (IC50: 1.6 ± 0.7 µM) and myristoleic acid (IC50: 35.2 ± 7.6 µM). One drug-resistant mutant ZZ37 unc-63 (x37) demonstrated a ~17-fold increased resistance to pharbitin compared with wild-type worms. Collectively, we provide further experimental scientific evidence to support the traditional use of Semen pharbitidis for the treatment of intestinal parasites. The anthelmintic activity of Semen pharbitidis is due to pharbitin, whose target could be UNC-63 in C. elegans.

Essential Oils as Novel Anthelmintic Drug Candidates.

Helminths, with an estimated 1.5 billion annual global infections, are one of the major health challenges worldwide. The current strategy of the World Health Organization to prevent helminth infection includes increasing hygienic awareness, providing better sanitation and preventative anthelmintic drug therapy in vulnerable populations. Nowadays, anthelmintic drugs are used heavily in livestock, both in case of infection and as a preventative measure. However, this has led to the development of resistance against several of the most common drugs, such as levamisole, ivermectin and thiabendazole. As many as 70% of the livestock in developed countries now has helminths that are drug resistant, and multiple resistance is common. Because of this, novel anthelmintics are urgently needed to help combat large-scale production losses. Prior to this review, no comprehensive review of the anthelmintic effects of essential oils and their components existed. Multiple review articles have been published on the uses of a single plant and its extracts that only briefly touch upon their anthelmintic activity. This review aims to provide a detailed overview of essential oils and their components as anthelmintic treatment against a wider variety of helminths.

Identification of a Hydroxygallic Acid Derivative, Zingibroside R1 and a Sterol Lipid as Potential Active Ingredients of Cuscuta chinensis Extract That Has Neuroprotective and Antioxidant Effects in Aged Caenorhabditis elegans.

We examined the effects of the extracts from two traditional Chinese medicine plants, Cuscuta chinensis and Eucommia ulmoides, on the healthspan of the model organism Caenorhabditis elegans. C. chinensis increased the short-term memory and the mechanosensory response of aged C. elegans. Furthermore, both extracts improved the resistance towards oxidative stress, and decreased the intracellular level of reactive oxygen species. Chemical analyses of the extracts revealed the presence of several bioactive compounds such as chlorogenic acid, cinnamic acid, and quercetin. A fraction from the C. chinensis extract enriched in zingibroside R1 improved the lifespan, the survival after heat stress, and the locomotion in a manner similar to the full C. chinensis extract. Thus, zingibroside R1 could be (partly) responsible for the observed health benefits of C. chinensis. Furthermore, a hydroxygallic acid derivative and the sterol lipid 4-alpha-formyl-stigmasta-7,24(241)-dien-3-beta-ol are abundantly present in the C. chinensis extract and its most bioactive fraction, but hardly in E. ulmoides, making them good candidates to explain the overall healthspan benefits of C. chinensis compared to the specific positive effects on stress resistance by E. ulmoides. Our findings highlight the overall anti-aging effects of C. chinensis in C. elegans and provide first hints about the components responsible for these effects.

CDK12 orchestrates super-enhancer-associated CCDC137 transcription to direct hepatic metastasis in colorectal cancer.

BACKGROUND: Hepatic metastasis is the primary and direct cause of death in individuals with colorectal cancer (CRC) attribute to lack of effective therapeutic targets. The present study aimed to identify potential druggable candidate targets for patients with liver metastatic CRC. METHODS: The transcriptional profiles of super-enhancers (SEs) in primary and liver metastatic CRC were evaluated in publicly accessible CRC datasets. Immunohistochemistry of human CRC tissues was conducted to determine the expression level of CDK12. Cellular proliferation, survival and stemness were examined upon CDK12 inhibition by shCDK12 or a selective CDK12 inhibitor named SR-4835 with multiple in vitro and in vivo assays. RNA sequencing and bioinformatics analyses were carried out to investigate the mechanisms of CDK12 inhibition in CRC cells. RESULTS: We identified CDK12 as a driver gene for direct hepatic metastasis in CRC. Suppression of CDK12 led to robust inhibition of proliferation, survival and stemness. Mechanistically, CDK12 intervention preferentially repressed the transcription of SE-associated genes. Integration of the SE landscape and RNA sequencing, BCL2L1 and CCDC137 were identified as SE-associated oncogenic genes to strengthen the abilities of cellular survival, proliferation and stemness, eventually increasing liver metastasis of CRC. CONCLUSIONS: Our data highlight the potential of CDK12 and SE-associated oncogenic transcripts as therapeutic targets for patients with liver metastatic CRC.

Ethnobotanical study of Hakka traditional medicine in Ganzhou, China and their antibacterial, antifungal, and cytotoxic assessments.

BACKGROUND: Traditional herbs played a crucial role in the health care of the Hakka people. However, studies to identify these traditional herbs are few. Here we document and assess the potential of these plants for treating microbial infections. Many herbs used by the Hakka people could potentially be a novel medicinal resource. METHODS: Local herb markets were surveyed via semi-structured interviews, complemented by direct observations to obtain information on herbal usage. For each herb selected for this study, extracts in four different solvents were prepared, and tested for activity against 20 microorganisms, as well as cancerous and noncancerous cells. All data were subjected to cluster analysis to discover relationships among herbs, plant types, administration forms, solvents, microorganisms, cells, etc., with the aim to discern promising herbs for medicine. RESULTS: Ninety-seven Hakka herbs in Ganzhou were documented from 93 plants in 62 families; most are used for bathing (97%), or as food, such as tea (32%), soup (12%), etc. Compared with the Chinese Pharmacopoeia and Chinese Materia Medica, 24 Hakka medicines use different plant parts, and 5 plants are recorded here for the first time as traditional medicines. The plant parts used were closely related with the life cycle: annual and perennial herbs were normally used as a whole plant, and woody plants as (tender) stem and leaf, indicating a trend to use the parts that are easily collected. Encouragingly, 311 extracts (94%) were active against one or more microorganisms. Most herbs were active against Gram-positive bacteria, such as Staphylococcus aureus (67%), Listeria innocua (64%), etc. Cytotoxicity was often observed against a tumor cell, but rarely against normal cells. Considering both antimicrobial activity and cytotoxicity, many herbs reported in this study show promise as medicine. CONCLUSION: Hakka people commonly use easily-collected plant parts (aerial parts or entire herb) as medicine. External use of decoctions dominated, and may help combating microbial infections. The results offer promising perspectives for further research since little phytopharmacology and phytochemistry has been published to date.

Flavonoid-Rich Fractions of Bauhinia holophylla Leaves Inhibit Candida albicans Biofilm Formation and Hyphae Growth.

This study evaluated the effect of the extract and fractions of Bauhinia holophylla on Candida albicans planktonic growth, biofilm formation, mature biofilm, and hyphae growth. Three C. albicans strains (SC5314, ATCC 18804, and ATCC 10231) were tested. The crude extract and the fractions were obtained by exhaustive percolation and liquid-liquid partition, respectively. Phytochemical analyses of B. holophylla extract and fractions were performed using high-performance liquid chromatography coupled with a diode-array detector and mass spectrometry (HPLC-DAD-MS). A microdilution assay was used to evaluate the effect of the B. holophylla extract and fractions on C. albicans planktonic growth, and crystal violet staining was used to measure the total biomass of the biofilm. Hyphae growth was analyzed using light microscopy. Thirteen flavonoids were identified, with a predominance of the flavonol-3-O-glycoside type based on quercetin, myricetin, and kaempferol. Flavonoid-rich fractions of B. holophylla leaves displayed antifungal activity and inhibited both biofilm formation and hyphae growth in all the tested strains, but were not effective on C. albicans planktonic growth and mature biofilm. This study indicates that flavonoid-rich fractions from B. holophylla leaves interfere with the virulence of Candida species and support the use of Bauhinia spp. in folk medicine to treat infections.

Identification of healthspan-promoting genes in Caenorhabditis elegans based on a human GWAS study.

To find drivers of healthy ageing, a genome-wide association study (GWAS) was performed in healthy and unhealthy older individuals. Healthy individuals were defined as free from cardiovascular disease, stroke, heart failure, major adverse cardiovascular event, diabetes, dementia, cancer, chronic obstructive pulmonary disease (COPD), asthma, rheumatism, Crohn's disease, malabsorption or kidney disease. Six single nucleotide polymorphisms (SNPs) with unknown function associated with ten human genes were identified as candidate healthspan markers. Thirteen homologous or closely related genes were selected in the model organism C. elegans for evaluating healthspan after targeted RNAi-mediated knockdown using pathogen resistance, muscle integrity, chemotaxis index and the activity of known longevity and stress response pathways as healthspan reporters. In addition, lifespan was monitored in the RNAi-treated nematodes. RNAi knockdown of yap-1, wwp-1, paxt-1 and several acdh genes resulted in heterogeneous phenotypes regarding muscle integrity, pathogen resistance, chemotactic behaviour, and lifespan. Based on these observations, we hypothesize that their human homologues WWC2, CDKN2AIP and ACADS may play a role in health maintenance in the elderly.

Ethnomedicinal, Phytochemical and Pharmacological Investigations of Tetradenia riparia (Hochst.) Codd (Lamiaceae).

Tetradenia riparia Hochsteter codd. (Lamiaceae) in its native African continent, is considered one of the most popular aromatic medicinal plants. In folk medicine it may be used as an infusion to treat respiratory problems, cough, headache, stomach pain, diarrhea, fever, malaria, and dengue; and in the form of compresses it is applied for the relief of headaches and toothaches. The species T. riparia has been researched for decades to isolate and identify chemical constituents present in extracts or essential oil obtained from the leaves, floral buds, or stems of this plant. The present study reviews the scientific literature on ethnomedicinal, phytochemical, and pharmacological aspects of T. riparia. We discuss issues related to the botanical and geographical description of the species, ethnobotanical uses, phytochemical studies on its essential oil and extracts, and biological activities of T. riparia. Several compounds have already been isolated from leaves, such as ibozol, 7α-hydroxyroileanone, 1',2'-dideacetylboronolide, 8(14),15-sandaracopimaradiene-7α,18-diol; 5,6-dihydro-α-pyrone and α-pyrone. Terpenes predominated in the essential oil, comprising monoterpenes, sesquiterpenes, diterpenes, hydrocarbons, and oxygenates. Most phytocompounds were isolated from the leaves and flower buds, namely fenchone, 14-hydroxy-9-epi (E)-caryophyllene, 9ß, 13ß-epoxy-7-abietene, and 6,7-dehydroroileanone. These compounds provide the species a high pharmacological potential, with antimicrobial, antioxidant, antitumor, analgesic, anti-leishmania, anti-tuberculosis, and anti-parasitic activities. Therefore, this species is a promising herbal medicine.

Corrigendum: Bioassay-Guided Interpretation of Antimicrobial Compounds in Kumu, a TCM Preparation From Picrasma quassioides' Stem via UHPLC-Orbitrap-Ion Trap Mass Spectrometer Combined With Fragmentation and Retention Time Calculation.

[This corrects the article DOI: 10.3389/fphar.2021.761751.].

Optimization of a locomotion-based zebrafish seizure model.

BACKGROUND: Locomotor assays in zebrafish have emerged as a screening test in early drug discovery for antiseizure compounds. However, parameters differ considerably between published studies, which may explain some discrepant results with (candidate) antiseizure medications. NEW METHOD: We optimized a locomotor-based seizure assay in zebrafish with pentylenetetrazol (PTZ) as the pharmacological proconvulsant to generate a therapeutic window in which proconvulsant-treated zebrafish larvae could be discriminated from a non-treated control. To generate a reliable control, exposure time and concentration of valproate (VPA, anticonvulsant) was optimized. RESULTS: Wells with one or three larvae show a similar PTZ dose-dependent increase in locomotion with less variability in motility for the latter. Zebrafish immersed in 10 mM PTZ showed a significant increase in movement with a sustained effect, without any indication of toxicity. Animals treated with 3 mM VPA showed the strongest reduction of PTZ-induced movement without toxicity. The decrease in PTZ-induced locomotion was greater after 18 h versus 2 h. COMPARISON WITH EXISTING METHOD(S): For the larval zebrafish PTZ-induced seizure model, varying experimental parameters have been reported in literature. Our results show that PTZ is often used at toxic concentrations, and we provide instead reliable conditions to quantify convulsant behaviour using an infrared-beam motility assay. CONCLUSIONS: We recommend using three zebrafish larvae per well to quantify locomotion in 96-multiwell plates. Larvae should preferably be exposed to 10 mM PTZ for 1 h, consisting of 30 min acclimation and 30 min subsequent recording. As positive control for anticonvulsant activity, we recommend exposure to 3 mM VPA for 18 h before administration of PTZ.

Anticancer Activities of Mushrooms: A Neglected Source for Drug Discovery.

Approximately 270 species of mushrooms have been reported as potentially useful for human health. However, few mushrooms have been studied for bioactive compounds that can be helpful in treating various diseases. Like other natural regimens, the mushroom treatment appears safe, as could be expected from their long culinary and medicinal use. This review aims to provide a critical discussion on clinical trial evidence for mushrooms to treat patients with diverse types of cancer. In addition, the review also highlights the identified bioactive compounds and corresponding mechanisms of action among the explored mushrooms. Furthermore, it also discusses mushrooms with anticancer properties, demonstrated either in vitro and/or in vivo models, which have never been tested in clinical studies. Several mushrooms have been tested in phase I or II clinical trials, mostly for treating breast cancer (18.6%), followed by colorectal (14%) and prostate cancer (11.6%). The majority of clinical studies were carried out with just 3 species: Lentinula edodes (22.2%), Coriolus versicolor, and Ganoderma lucidum (both 13.9%); followed by two other species: Agaricus bisporus and Grifola frondosa (both 11.1%). Most in vitro cell studies use breast cancer cell lines (43.9%), followed by lung (14%) and colorectal cancer cell lines (13.1%), while most in vivo animal studies are performed in mice tumor models (58.7%). Although 32 species of mushrooms at least show some promise for the treatment of cancer, only 11 species have been tested clinically thus far. Moreover, most clinical studies have investigated fewer numbers of patients, and have been limited to phase III or IV. Therefore, despite the promising preclinical and clinical data publication, more solid scientific efforts are required to clarify the therapeutic value of mushrooms in oncology.

Identification of novel topoisomerase II alpha inhibitors by virtual screening, molecular docking, and bioassay.

Breast cancer is one of the most common tumors, and its treatment still leaves room for improvement. Topoisomerase II alpha is a potential target for the treatment of human diseases such as breast cancer. In this article, we attempted to discover a novel anticancer drug. We have used the topoisomerase II alpha protein-Homo sapiens (Human) to hierarchically screen the Maybridge database. Based on their docking score, the top hit compounds have been assayed for inhibition in a topoisomerase II pBR322 DNA relaxation assay in vitro. Candidate compound 6 (CP6) was found to have the best inhibitory effect for topoisomerase II among the 20 tested compounds. In addition, CP6 had potent cytotoxicity against eight tested tumor cell lines. At the same time, CP6 was shown to have potential anti-multidrug resistance capabilities. This study identifies CP6, which can contribute to the development of new topoisomerase II inhibitors as anticancer agents.

Medicinal mushrooms: Clinical perspective and challenges.

Mushrooms are valued by humans worldwide as food, but also for their medicinal properties. Over 130 medicinal effects of mushrooms have been reported, including anti-diabetic, antioxidant, antimicrobial, anticancer, prebiotic, immunomodulating, anti-inflammatory and cardiovascular benefits. Several mushrooms have been tested in phase I, II, or III clinical trials for various diseases, including cancers, as well as to modulate immunity. Here, we review clinical studies on medicinal mushrooms or preparations (but not pure compounds) derived thereof. Overall, few phase III trials have been performed, and in many cases, these trials included a relatively small number of patients. Therefore, despite the promising published clinical data, especially on immune modulation, more work is required to clarify the therapeutic value of mushrooms.

The ß2-adrenergic receptor in the apical membrane of intestinal enterocytes senses sugars to stimulate glucose uptake from the gut.

The presence of sugar in the gut causes induction of SGLT1, the sodium/glucose cotransporter in intestinal epithelial cells (enterocytes), and this is accompanied by stimulation of sugar absorption. Sugar sensing was suggested to involve a G-protein coupled receptor and cAMP - protein kinase A signalling, but the sugar receptor has remained unknown. We show strong expression and co-localization with SGLT1 of the ß2-adrenergic receptor (ß 2-AR) at the enterocyte apical membrane and reveal its role in stimulating glucose uptake from the gut by the sodium/glucose-linked transporter, SGLT1. Upon heterologous expression in different reporter systems, the ß 2-AR responds to multiple sugars in the mM range, consistent with estimated gut sugar levels after a meal. Most adrenergic receptor antagonists inhibit sugar signaling, while some differentially inhibit epinephrine and sugar responses. However, sugars did not inhibit binding of I125-cyanopindolol, a ß 2-AR antagonist, to the ligand-binding site in cell-free membrane preparations. This suggests different but interdependent binding sites. Glucose uptake into everted sacs from rat intestine was stimulated by epinephrine and sugars in a ß 2-AR-dependent manner. STD-NMR confirmed direct physical binding of glucose to the ß 2-AR. Oral administration of glucose with a non-bioavailable ß 2-AR antagonist lowered the subsequent increase in blood glucose levels, confirming a role for enterocyte apical ß 2-ARs in stimulating gut glucose uptake, and suggesting enterocyte ß 2-AR as novel drug target in diabetic and obese patients. Future work will have to reveal how glucose sensing by enterocytes and neuroendocrine cells is connected, and whether ß 2-ARs mediate glucose sensing also in other tissues.

Bioassay-Guided Interpretation of Antimicrobial Compounds in Kumu, a TCM Preparation From Picrasma quassioides' Stem via UHPLC-Orbitrap-Ion Trap Mass Spectrometry Combined With Fragmentation and Retention Time Calculation.

The stem of Picrasma quassioides (PQ) was recorded as a prominent traditional Chinese medicine, Kumu, which was effective for microbial infection, inflammation, fever, and dysentery, etc. At present, Kumu is widely used in China to develop different medicines, even as injection (Kumu zhusheye), for combating infections. However, the chemical basis of its antimicrobial activity has still not been elucidated. To examine the active chemicals, its stem was extracted to perform bioassay-guided purification against Staphylococcus aureus and Escherichia coli. In this study, two types of columns (normal and reverse-phase) were used for speedy bioassay-guided isolation from Kumu, and the active peaks were collected and identified via an UHPLC-Orbitrap-Ion Trap Mass Spectrometer, combined with MS Fragmenter and ChromGenius. For identification, the COCONUT Database (largest database of natural products) and a manually built PQ database were used, in combination with prediction and calculation of mass fragmentation and retention time to better infer their structures, especially for isomers. Moreover, three standards were analyzed under different conditions for developing and validating the MS method. A total of 25 active compounds were identified, including 24 alkaloids and 1 triterpenoid against S. aureus, whereas only ß-carboline-1-carboxylic acid and picrasidine S were active against E. coli. Here, the good antimicrobial activity of 18 chemicals was reported for the first time. Furthermore, the spectrum of three abundant ß-carbolines was assessed via their IC50 and MBC against various human pathogens. All of them exhibited strong antimicrobial activities with good potential to be developed as antibiotics. This study clearly showed the antimicrobial chemical basis of Kumu, and the results demonstrated that HRMS coupled with MS Fragmenter and ChromGenius was a powerful tool for compound analysis, which can be used for other complex samples. Beta-carbolines reported here are important lead compounds in antibiotic discovery.