Chemodivergent Photocatalyzed Heterocyclization of Hydrazones and Isothiocyanates for the Selectivity Synthesis of 2-Amino-1,3,4-thiadiazoles and 1,2,4-Triazole-3-thiones.

A photocatalytic chemodivergent reaction for the selectivity formation of C-S and C-N bonds in a controlled manner was proposed. The reaction medium, either neutral or acidic, is critical to dictate the formation of 2-amino-1,3,4-thiadiazoles and 1,2,4-triazole-3-thiones from isothiocyanates and hydrazones. This is a practical protocol to achieve the chemoselectivity under mild and metal-free conditions.

The generally useful estimate of solvent systems method facilitates off-line two-dimensional countercurrent chromatography for isolating compositions from Siraitia grosvenorii roots.

Solvent system selection is a crucial and the most time-consuming step for successful countercurrent chromatography separation. A thin-layer chromatography-based generally useful estimate of solvent systems method has been developed to simplify the solvent system selection. We herein utilized the method to select a solvent system for off-line two-dimensional countercurrent chromatography to separate chemical compositions from a complex fraction of the Siraitia grosvenorii root extract. The first-dimensional countercurrent separation using chloroform/methanol/water (10:5.5:4.5, v/v/v) yielded four compounds with high purity and three mixture fractions (Fr I, III, and VII). The second-dimensional countercurrent separation conducted on Fr I, III, and VII using the hexane/ethyl acetate/methanol/water (4:6:6:4, 3:7:3:7, v/v/v) and chloroform/methanol/water (10:9:6, v/v/v) solvent systems, respectively, produced another four compounds. Four triterpenoids and four lignans were finally isolated, including two novel compounds. Hence, the generally useful estimate of solvent systems method is a feasible and efficient approach for selecting an applicable solvent system for separating complex samples. In addition, the off-line two-dimensional countercurrent chromatography method can improve both the peak resolution and the capacity of countercurrent chromatography.

Iridoid-glycoside isolation and purification from Premna fulva leaves.

Premna fulva Craib, rich in iridoid glycosides, is widely used to treat periarthritis, osteoproliferation, pain, and other diseases. However, no studies have reported effective purification methods for obtaining iridoid glycosides as active materials. This paper describes an efficient strategy for separating iridoid glycosides from Premna fulva leaves using high-speed counter-current chromatography and preparative high-performance liquid chromatography. A two-phase solvent system, ethyl acetate/n-butanol/water (7.5:2.5:10, v/v), was selected for high-speed counter-current chromatography separation. The proposed method effectively separated and purified four iridoid glycosides and four lignans, including three new iridoid glycosides (4-6) and five known compounds (1-3, 7, 8), from Premna fulva leaves, indicating that high-speed counter-current chromatography combined with prep-HPLC can efficiently isolate catalpol derivatives from the genus Premna. Additionally, the in vitro anti-inflammatory activities of all isolated compounds were analyzed using lipopolysaccharide-stimulated RAW 264.7 cells, and the results indicated that six compounds (1 and 3-7) exhibited potential anti-inflammatory activities.

Synthesis, Antiproliferative Evaluation, and Molecular Docking Study of Novel Longifolene-Derived Tetraline Fused Thiazole-Amide Derivatives.

Twenty novel longifolene-derived tetraline fused thiazole-amide compounds were synthesized from longifolene, a renewable natural resource. Their structures were characterized by FT-IR, NMR, ESI-MS, and elemental analysis. The in vitro antiproliferative activity of these compounds against SK-OV-3 ovarian cancer cell lines, MCF-7 human breast cancer cell lines, HepG2 human liver cancer cell lines, A549 human lung adenocarcinoma cell lines, and T-24 human bladder cancer cell lines was tested by MTT assay. Compounds 6a-6c displayed significant and broad-spectrum antiproliferative activity against almost the tested cancer cell lines with IC50 in the range of 7.84 to 55.88 µM, of which compound 6c exhibited excellent antiproliferative activities with 7.84 µM IC50 against SKOV-3, 13.68 µM IC50 against HepG2, 15.69 µM IC50 against A549, 19.13 µM IC50 against MCF-7, and 22.05 µM IC50 against T-24, showing better and broad-spectrum antiproliferative effect than that of the positive control 5-FU. Furthermore, the action model was analyzed by the molecular docking study. Some intriguing structure-activity relationships were found and discussed herein by DFT theoretical calculation.

Lindenane-Type Sesquiterpene Dimers Mitigate Lipopolysaccharide-Induced Inflammation by Inhibiting Toll-Like Receptor Signaling.

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and trigger an inflammatory response via the myeloid differential factor 88 (MyD88)-dependent and toll-interleukin-1 receptor domain-containing adapter-inducing interferon-ß (TRIF)-dependent pathways. Lindenane type sesquiterpene dimers (LSDs) are characteristic metabolites of plants belonging to the genus Sarcandra (Chloranthaceae). The aim of this study was to evaluate the potential anti-inflammatory effects of the LSDs shizukaol D (1) and sarcandrolide E (2) on lipopolysaccharides (LPS)-stimulated RAW264.7 macrophages in vitro, and explore the underlying mechanisms. Both LSDs neutralized the LPS-induced morphological changes and production of nitric oxide (NO), as determined by CCK-8 assay and Griess assay, respectively. Furthermore, shizukaol D (1) and sarcandrolide E (2) downregulated interferon ß (IFNß), tumor necrosis factor α (TNFα) and interleukin-1ß (IL-1ß) mRNA levels as measured by reverse transcription polymerase chain reaction (RT-PCR), and inhibited the phosphorylation of nuclear factor kappa B p65 (p65), nuclear factor kappa-Bα (IκBα), Jun N-terminal kinase (JNK), extracellular regulated kinase (ERK), mitogen-activated protein kinase p38 (p38), MyD88, IL-1RI-associated protein kinase 1 (IRAK1), and transforming growth factor-ß-activated kinase 1 (TAK1) proteins in the Western blotting assay. In conclusion, LSDs can alleviate the inflammatory response by inhibiting the TLR/MyD88 signalling pathway.

Identification and Isolation of α-Glucosidase Inhibitors from Siraitia grosvenorii Roots Using Bio-Affinity Ultrafiltration and Comprehensive Chromatography.

The discovery of bioactive compounds from medicinal plants has played a crucial role in drug discovery. In this study, a simple and efficient method utilizing affinity-based ultrafiltration (UF) coupled with high-performance liquid chromatography (HPLC) was developed for the rapid screening and targeted separation of α-glucosidase inhibitors from Siraitia grosvenorii roots. First, an active fraction of S. grosvenorii roots (SGR2) was prepared, and 17 potential α-glucosidase inhibitors were identified based on UF-HPLC analysis. Second, guided by UF-HPLC, a combination of MCI gel CHP-20P column chromatography, high-speed counter-current countercurrent chromatography, and preparative HPLC were conducted to isolate the compounds producing active peaks. Sixteen compounds were successfully isolated from SGR2, including two lignans and fourteen cucurbitane-type triterpenoids. The structures of the novel compounds (4, 6, 7, 8, 9, and 11) were elucidated using spectroscopic methods, including one- and two-dimensional nuclear magnetic resonance spectroscopy and high-resolution electrospray ionization mass spectrometry. Finally, the α-glucosidase inhibitory activities of the isolated compounds were verified via enzyme inhibition assays and molecular docking analysis, all of which were found to exhibit certain inhibitory activity. Compound 14 exhibited the strongest inhibitory activity, with an IC50 value of 430.13 ± 13.33 µM, which was superior to that of acarbose (1332.50 ± 58.53 µM). The relationships between the structures of the compounds and their inhibitory activities were also investigated. Molecular docking showed that the highly active inhibitors interacted with α-glucosidase through hydrogen bonds and hydrophobic interactions. Our results demonstrate the beneficial effects of S. grosvenorii roots and their constituents on α-glucosidase inhibition.

Identification of Unstable Ellagitannin Metabolites in the Leaves of Quercus dentata by Chemical Derivatization.

The identification of unstable metabolites of ellagitannins having ortho-quinone structures or reactive carbonyl groups is important to clarify the biosynthesis and degradation of ellagitannins. Our previous studies on the degradation of vescalagin, a major ellagitannin of oak young leaves, suggested that the initial step of the degradation is regioselective oxidation to generate a putative quinone intermediate. However, this intermediate has not been identified yet. In this study, young leaves of Quercus dentata were extracted with 80% acetonitrile containing 1,2-phenylenediamine to trap unstable ortho-quinone metabolites, and subsequent chromatographic separation afforded a phenazine derivative of the elusive quinone intermediate of vescalagin. In addition, phenylenediamine adducts of liquidambin and dehydroascorbic acid were obtained, which is significant because liquidambin is a possible biogenetic precursor of C-glycosidic ellagitannins and ascorbic acid participates in the production of another C-glycosidic ellagitannin in matured oak leaves.

The Triterpenoids from Munronia pinnata and Their Anti-Proliferative Effects.

Six new tirucallane-type triterpenoids, named munropenes A-F (1-6), were extracted from the whole plants of Munronia pinnata using a water extraction method. Their chemical structures were determined based on detailed spectroscopic data. The relative configurations of the acyclic structures at C-17 of munropenes A-F (1-6) were established using carbon-proton spin-coupling constants (2,3JC,H) and inter-proton spin-coupling constants (3JH,H). Furthermore, the absolute configurations of munropenes A-F (1-6) were determined through high-performance liquid chromatography (HPLC), single-crystal X-ray diffraction, and electronic circular dichroism (ECD) analyses. The antiproliferative effects of munropenes A-F were evaluated in five tumor cell lines: HCT116, A549, HepG2, MCF7, and MDAMB. Munropenes A, B, D, and F (1, 2, 4, and 6) inhibited proliferation in the HCT116 cell line with IC50 values of 40.90, 19.13, 17.66, and 32.62 µM, respectively.

Design and synthesis of mogrol derivatives modified on a ring with anti-inflammatory and anti-proliferative activities.

A class of novel mogrol derivatives modified on A ring were synthesized. The screening result showed that indole-fused derivatives exhibited lower toxicity and better anti-inflammatory activity in LPS-induced RAW 264.7 cells model than mogrol and other compounds. Derivative B8 exerted superior inhibitory result of NO production (IC50 = 5.05 µM) and inhibitory ability of TNF-α and IL-6 secretion to mogrol through iNOS/NF-κB pathway. Besides, the CCK8 assay was performed to evaluate their anti-proliferative activity against non-small cell lung cancer including A549, NCI-H460, H1299 and H1975 cells. Compared with mogrol, compound B8 showed moderate anti-proliferative activities against A549 and H1975 cells, while derivatives bearing α, ß-unsaturated ketone scaffold displayed broad-spectrum growth inhibition against four cell lines. Among them, compound A9 showed 12-fold higher activity than mogrol against H1299 and H1975 cells. The suppressive effect on expression level of p-p65 might account for the compound A9-induced growth inhibition and cell cycle arrest at G1 phase.

Preparative isolation of diterpenoids from Salvia bowleyana Dunn roots by high-speed countercurrent chromatography combined with high-performance liquid chromatography.

The root of Salvia bowleyana Dunn (Lamiaceae) is used as a traditional Chinese medicine that has multiple therapeutic effects. In this study, an efficient strategy was developed to separate diterpenoid compounds, which are the main active ingredients in Salvia bowleyana Dunn roots, from complex crude extracts by high-speed countercurrent chromatography combined with preparative high-performance liquid chromatography. A two-phase solvent system comprising n-hexane-ethyl acetate-methanol-water (7:3:7:3, v/v/v/v) was selected for high-speed countercurrent chromatographic separation. Three major diterpenoids, 6α-hydroxysugiol (7), sugiol (8), and 6, 12-dihydroxyabieta-5,8,11,13-tetraen-7-one (9) were obtained at purities of 98.9, 95.4, and 96.2%, respectively, and minor diterpenoids were enriched via one-step separation. The enriched minor diterpenoids were further purified by continuous preparative high-performance liquid chromatography to yield two new norabietanoids (1, 6) and four known compounds (2-5). The structures of these new compounds were determined using NMR spectroscopy, high-resolution electrospray ionization mass spectrometry, and electronic circular dichroism spectroscopy. The results suggest that high-speed countercurrent chromatography combined with preparative high-performance liquid chromatography efficiently isolates diterpenoids, including minor components, from complex natural products.

Synthesis and Antiproliferative Activity of Ester Derivatives of Mogrol through JAK2/STAT3 Pathway.

In attempt to enhance the antiproliferative activity of mogrol, two series of ester derivatives modified at C3 -OH and C11 -OH were designed and synthesized. The activity against human cancer cells including A549, NCI-H460 and CNE1 was screened by Cell Counting Kit-8 (CCK8) assay. According to the results, modifications of the mogrol core through introduction of different ester scaffolds drastically improved the cytotoxicity, and some of the derivatives exhibited even higher activity than the positive drug. Among them, compound M2h exhibited nearly 4 times more cytotoxic than 5-Fu against CNE1 cells, derivative M6c showed ten times higher activity with the IC50 value of 10.59 µM than mogrol against NCI-H460 cells, and compound M6a which contained one 1,2,3-triazole motif showed the strongest activity with an three folds lower IC50 value than mogrol. Furthermore, the most potent compound M2h could lead to cell cycle arrest at G2 phase on CNE1 cell lines and M6a induced G1 phase arrest on A549 cell lines. It was noteworthy that both M2h and M6a regulated signal transducer and activator of transcription 3 (STAT3) signal pathway through inhibiting phosphorylation of Janus Kinase 2 (JAK2) and STAT3, and simultaneously increasing the protein level of downstream cyclin p21.

Rhodomollosides A and B, glycosides of methyl everninate from the aerial parts of Rhododendron molle.

Two new glycosides of methyl everninate, rhodomollosides A (1) and B (2), were isolated from the aerial parts of a medicinal plant Rhododendron molle. The structures of 1 and 2 were elucidated on the basis of detailed spectroscopic analyses as well as HPLC analyses for thiazolidine derivatives of their sugar moieties. The sugar moiety of rhodomolloside A (1) was elucidated to be a rare monosaccharide, D-allose, while rhodomolloside B (2) was assigned as a D-glucoside of methyl everninate. Furthermore, they were evaluated for their cytotoxicity against RAW264.7 cells, and for their inhibitory effects with a lipopolysaccharide (LPS)-stimulated murine macrophages RAW 264.7 cells model.

Structural Characterization and Assessment of Anti-Inflammatory Activities of Polyphenols and Depsidone Derivatives from Melastoma malabathricum subsp. normale.

The roots of Melastoma malabathricum subsp. normale (D. Don) Karst. Mey have been used in traditional ethnic medicine systems in China to treat inflammation-triggered ailments, such as trauma, toothache, and fever. Therefore, the aim of this study is to screen for compounds with anti-inflammatory activity in the title plant. The extract of M. malabathricum subsp. normale roots was separated using various chromatographic methods, such as silica gel, ODS C18, MCI gel, and Sephadex LH-20 column chromatography, as well as semi-preparative HPLC. One new complex tannin, named whiskey tannin D (1), and an undescribed tetracyclic depsidone derivative, named guanxidone B (2), along with nine known polyphenols (2-10) and three known depsidone derivatives (12-14) were obtained from this plant. The structures of all compounds were elucidated by extensive NMR and CD experiments in conjunction with HR-ESI-MS data. All these compounds were isolated from this plant for the first time. Moreover, compounds 1-4, 8, and 10-14 were obtained for the first time from the genus Melastoma, and compounds 1, 2, and 11-14 have not been reported from the family Melastomataceae. This is the first report of complex tannin and depsidone derivatives from M. malabathricum subsp. normale, indicating their chemotaxonomic significance to this plant. Compounds 1-12 were investigated for their anti-inflammatory activities on the production of the nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and compounds 1, 11, and 12 showed anti-inflammatory activities with IC50 values of 6.46 ± 0.23 µM, 8.02 ± 0.35 µM, and 9.82 ± 0.43 µM, respectively. The structure-activity relationship showed that the catechin at glucose C-1 in ellagitannin was the key to its anti-inflammatory activity, while CH3O- at C-16 of aromatic ring A in depsidone derivatives had little effect on its anti-inflammatory activity. The study of structure-activity relationships is helpful to quickly discover new anti-inflammatory drugs. The successful isolation and structure identification of these compounds, especially complex tannin 1, not only provide materials for the screening of anti-inflammatory compounds, but also provide a basis for the study of chemical taxonomy of the genus Melastoma.

Synthesis and anti-proliferation activity of mogrol derivatives bearing quinoline and triazole moieties.

A series of novel derivatives based on mogrol were designed and synthesized in attempt to improve anti-lung cancer activity. The cytotoxicity against human lung cancer cells including A549 and NCI-H460 were performed by Cell Counting Kit-8 (CCK8) assay in vitro. The screening result showed that compound 8f exhibited the strongest activity with an IC50 value of 4.47 µM against A549 cell, and could induce the cell apoptosis in a dose-dependent manner and arrest cell cycle at G0/G1 phase. Besides, compound 8f displayed anti-proliferation effect on A549 cell through inhibiting phosphorylation of signal transducer and activator of transcription 3 (STAT3). Furthermore, compared with morgol, compound 10a significantly improved the cytotoxicity against NCI-H460 with the IC50 value of 17.13 µM. The research stimulated the development of potential therapeutic agent for lung cancer from the natural mogrol.

Ellagitannin Digestion in Moth Larvae and a New Dimeric Ellagitannin from the Leaves of Platycarya strobilacea.

Ellagitannins (ETs) are plant polyphenols with various health benefits. Recent studies have indicated that the biological activities of ETs are attributable to their degradation products, including ellagic acid and its gut microflora metabolites, such as urolithins. Insect tea produced in the Guangxi region, China, is made from the frass of moth larvae that feed on the ET-rich leaves of Platycarya strobilacea. Chromatographic separation of the Guangxi insect tea showed that the major phenolic constituents are ellagic acid, brevifolin carboxylic acid, gallic acid, brevifolin, and polymeric polyphenols. Chemical investigation of the feed of the larvae, the fresh leaves of P. strobilacea, showed that the major polyphenols are ETs including pedunculagin, casuarictin, strictinin, and a new ET named platycaryanin E. The new ET was confirmed as a dimer of strictinin having a tergalloyl group. The insect tea and the leaves of P. strobilacea contained polymeric polyphenols, both of which were shown to be composed of ETs and proanthocyanidins by acid hydrolysis and thiol degradation. This study clarified that Guangxi insect tea contains ET metabolites produced in the digestive tract of moth larvae, and the metabolites probably have higher bioavailabilities than the original large-molecular ETs of the leaves of P. strobilacea.

Ellagitannins and Oligomeric Proanthocyanidins of Three Polygonaceous Plants.

The aim of this study was to characterize hydrolyzable tannins in Polygonaceous plants, as only a few plants have previously been reported to contain ellagitannins. From Persicaria chinensis, a new hydrolyzable tannin called persicarianin was isolated and characterized to be 3-O-galloyl-4,6-(S)-dehydrohexahydroxydiphenoyl-d-glucose. Interestingly, acid hydrolysis of this compound afforded ellagic acid, despite the absence of a hexahydroxydiphenoyl group. From the rhizome of Polygonum runcinatum var. sinense, a large amount of granatin A, along with minor ellagitannins, helioscpoinin A, davicratinic acids B and C, and a new ellagitannin called polygonanin A, were isolated. Based on 2D nuclear magnetic resonance (NMR) spectroscopic examination, the structure of polygonanin A was determined to be 1,6-(S)-hexahydroxydiphenoyl-2,4-hydroxychebuloyl-ß-d-glucopyranose. These are the second and third hydrolyzable tannins isolated from Polygonaceous plants. In addition, oligomeric proanthocyanidins of Persicaria capitatum and P. chinensis were characterized by thiol degradation. These results suggested that some Polygonaceous plants are the source of hydrolyzable tannins not only proanthocyanidins.

Structural Characterization and Assessment of Anti-Inflammatory and Anti-Tyrosinase Activities of Polyphenols from Melastoma normale.

Polyphenols, widely distributed in the genus Melastoma plants, possess extensive cellular protective effects such as anti-inflammatory, anti-tyrosinase, and anti-obesity, which makes it a potential anti-inflammatory drug or enzyme inhibitor. Therefore, the aim of this study is to screen for the anti-inflammatory and enzyme inhibitory activities of compounds from title plant. Using silica gel, MCI, ODS C18, and Sephadex LH-20 column chromatography, as well as semipreparative HPLC, the extract of Melastoma normale roots was separated. Four new ellagitannins, Whiskey tannin C (1), 1-O-(4-methoxygalloyl)-6-O-galloyl-2,3-O-(S)-hexahydroxydiphenoyl-ß-d-glucose (2), 1-O-galloyl-6-O-(3-methoxygalloyl)-2,3-O-(S)-hexahydroxydiphenoyl-ß-d-glucose (3), and 1-O-galloyl-6-O-vanilloyl-2,3-O-(S)-hexahydroxydiphenoyl-ß-d-glucose (4), along with eight known polyphenols were firstly obtained from this plant. The structures of all isolates were elucidated by HRMS, NMR, and CD analyses. Using lipopolysaccharide (LPS)-stimulated RAW2 64.7 cells, we investigated the anti-inflammatory activities of compounds 1-4, unfortunately, none of them exhibit inhibit nitric oxide (NO) production, their IC50 values are all > 50 µM. Anti-tyrosinase activity assays was done by tyrosinase inhibition activity screening model. Compound 1 showed weak tyrosinase inhibitory activity with IC50 values of 426.02 ± 11.31 µM. Compounds 2-4 displayed moderate tyrosinase inhibitory activities with IC50 values in the range of 124.74 ± 3.12-241.41 ± 6.23 µM. The structure-activity relationships indicate that hydroxylation at C-3', C-4', and C-3 in the flavones were key to their anti-tyrosinase activities. The successful isolation and structure identification of ellagitannin provide materials for the screening of anti-inflammatory drugs and enzyme inhibitors, and also contribute to the development and utilization of M. normale.

Classification of Flavonoid Metabolomes via Data Mining and Quantification of Hydroxyl NMR Signals.

Utilizing the distinct HMBC cross-peak patterns of lower-field range (LFR; 11.80-14.20 ppm) hydroxyl singlets, presented NMR methodology characterizes flavonoid metabolomes both qualitatively and quantitatively. It enables simultaneous classification of the structural types of 5-OH flavonoids and biogenetically related 2'-OH chalcones, as well as quantification of individual metabolites from 1H NMR spectra, even in complex mixtures. Initially, metabolite-specific LFR 1D 1H and 2D HMBC patterns were established via literature mining and experimental data interpretation, demonstrating that LFR HMBC patterns encode the different structural types of 5-OH flavonoids/2'-OH chalcones. Taking advantage of the simplistic multiplicity of the H,H-uncoupled LFR 5-/2'-OH singlets, individual metabolites could subsequently be quantified by peak fitting quantitative 1H NMR (PF-qHNMR). Metabolomic analysis of enriched fractions from three medicinal licorice (Glycyrrhiza) species established proof-of-concept for distinguishing three major structural types and eight subtypes in biomedical applications. The method identified 15 G. uralensis (GU) phenols from the six possible subtypes of 5,7-diOH (iso)flav(an)ones with 6-, 8-, and nonprenyl substitution, including the new 6-prenyl-licoisoflavanone (1) and two previously unknown compounds (4 and 7). Relative (100%) qNMR established quantitative metabolome patterns suitable for species discrimination and plant metabolite studies. Absolute qNMR with combined external and internal (solvent) calibration (ECIC) identified and quantified 158 GU metabolites. HMBC-supported qHNMR analysis of flavonoid metabolomes ("flavonomics") empowers the exploration of structure-abundance-activity relationships of designated bioactivity. Its ability to identify and quantify numerous metabolites simultaneously and without identical reference materials opens new avenues for natural product discovery and botanical quality control and can be adopted to other flavonoid- and chalcone-containing taxa.

Separation, synthesis, and cytotoxicity of a series of mogrol derivatives.

Four metabolites of mogrol were separated, identified and characterized. Their antitumor activity was evaluated, and the results showed side chain modification would probably enhance the cytotoxicity. Therefore, three types of amines, alcohols and rigid planar derivatives were synthesized. Compounds 20 and 21 containing a tetrahydro-ß-carboline structure at the end of the side chain exhibited IC50 values around 2-9 µM against A549 and CNE1 cell comparing with 80-90 µM of mogrol. Structure analysis suggested that the perhydrocyclopentanophenanthrene moiety and the tetrahydro-ß-carboline moiety could probably enhance the activity through an intramolecular synergistic effect.[Formula: see text].

Antitumor Activity and Mechanism of Robustic Acid from Dalbergia benthami Prain via Computational Target Fishing.

Dalbergia benthami Prain (D.benthami) is an important legume species of the Dalbergia family, due to the use of its trunk and root heart in traditional Chinese medicine (TCM). In the present study, we reported the isolation, characterization and pharmacological activities of robustic acid (RA) from the ethyl acetate extract of D. benthami Prain. The SwissADME prediction showed that the RA satisfied the Lipinski's rule of five (molecule weight (MW): 380.39 g/mol, lipid-water partition coefficient (log P): 3.72, hydrogen bond donors (Hdon): 1, hydrogen bond acceptors (Hacc): 6, rotatable bonds (Rbon): 3. Other chemical and pharmacological properties of this RA were also evaluated, including topological polar surface area (TPSA) = 78.13 Šand solubility (Log S) = -4.8. The probability values of the antineoplastic, anti-free radical activities and topoisomerase I (TopoⅠ) inhibitory activity were found to be 0.784, 0.644 and 0.379, respectively. The molecular docking experiment using the Surflex-Dock showed that the Total Score and C Score of RNA binding with the human DNA-Topo I complex were 7.80 and 4. The MTS assay experiment showed that the inhibitory rates of RA on HL-60, MT4, Hela, HepG2, SK-OV-3 and MCF-7 cells were 37.37%, 97.41%, 81.22%, 34.4%, 32.68% and 51.4%, respectively. In addition, RA exhibited an inhibitory effect on the angiogenesis of zebrafish embryo, a good TopoⅠ inhibitory activity at a 10 mM concentration and in a dose-dependent manner, excellent radical scavenging in the DPPH and ABTS assays, and the free radical scavenging rate was close to the positive control (BHT) at different concentrations (0.5-2.0 mg/mL). Furthermore, 18 potential targets were found for this RA by PharmMapper, including ANXA3, SRC, FGFR2, GSK3B, CSNK2B, YARS, LCK, EPHA2, MAPK14, RORA, CRABP2, PPP1CC, METAP2, MME, TTR, MET and KDR. The GO and KEGG pathway analysis revealed that the "protein tyrosine kinase activity", "rap1 signaling pathway" and "PI3K-Akt signaling pathway" were significantly enriched by the RA target genes. Our results will provide new insights into the pharmaceutical use of this species. More importantly, our data will expand our understanding of the molecular mechanisms of RA functions.