Novel Withanolides from Tubocapsicum anomalum Suppress Triple-Negative Breast Cancer by Triggering Apoptosis and p53-ASCT2-SLC7A11-Mediated Ferroptosis.

Triple-negative breast cancer (TNBC) is a malignant breast cancer. There is an urgent need for effective drugs to be developed for TNBC. Tubocapsicum anomalum (T. anomalum) has been reported to have an anti-tumor effect, and six novel withanolides were isolated from it and designated as TAMEWs. However, its anti-TNBC effect is still unknown. The results of an MTT assay indicated a higher sensitivity of TNBC cells to TAMEWs compared to other cells. TAMEWs induced apoptosis via mitochondrial dysfunction. They caused increased levels of lipid ROS and Fe2+, with downregulation of GSH and cystine uptake, and it has been confirmed that TAMEWs induced ferroptosis. Additionally, the results of Western blotting indicate that TAMEWs significantly decrease the expressions of ferroptosis-related proteins. Through further investigation, it was found that the knockdown of the p53 gene resulted in a significant reversal of ferroptosis and the expressions of its associated proteins SLC7A11, ASCT2, and GPX4. In vivo, TAMEWs suppressed TNBC growth with no obvious damage. The IHC results also showed that TAMEWs induced apoptosis and ferroptosis in vivo. Our findings provide the first evidence that TAMEWs suppress TNBC growth through apoptosis and ferroptosis.

A Homodimer of Withaferin A Formed by Base-Promoted Elimination of Acetic Acid from 27-O-Acetylwithaferin A Followed by a Diels-Alder Reaction.

Treatment of 27-O-acetylwithaferin A (2) with the non-nucleophilic base, 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), afforded 5ß,6ß-epoxy-4ß-hydroxy-1-oxo-witha-2(3),23(24),25(27)-trienolide (3) and 4, a homodimer of withaferin A resulting from a Diels-Alder [4 + 2] type cycloaddition of the intermediate α,ß-dimethylene-δ-lactone (9). Structures of 3 and 4 were elucidated using HRMS and 1D and 2D NMR spectroscopic data. The structure of 4 was also confirmed by single crystal X-ray crystallographic analysis of its bis-4-O-p-nitrobenzoate (8). Formation of withaferin A homodimer (4) as the major product suggests regio- and stereoselectivity of the Diels-Alder [4 + 2] cycloaddition reaction of 9. Acetylation of 2-4 afforded their acetyl derivatives 5-7, respectively. Compounds 2-4 and 6-8 were evaluated for their cytotoxic activities against four prostate cancer (PC) cell lines (LNCaP, 22Rv1, DU-145, and PC-3) and normal human foreskin fibroblast (HFF) cells. Significantly, 4 exhibited improved activity compared to the other compounds for most of the tested cell lines.

Withanolides from the active extract of Physalis angulate and their anti-hepatic fibrosis effects.

ETHNOPHARMACOLOGICAL RELEVANCE: Physalis angulata L., a traditional Chinese medicine called "Kuzhi" in China, was used traditionally to treat liver diseases (eg. icterus, hepatitis) as well as malaria, asthma, and rheumatism. AIM OF THE STUDY: Our study aimed to investigate the withanolides with anti-hepatic fibrosis effect from P. angulate. MATERIALS AND METHODS: Withanolides were obtained from the EtOH extract of P. angulate by bioassay-molecular networking analysis-guided isolation using column chromatography and normal/reversed-phase semipreparative HPLC. The structures of new withanolides were elucidated by combinations of spectroscopic techniques with NMR and ECD calculations. MTT cell viability assay, AO/EB staining method, cell wound healing assay, ELISA and Western blot experiments were employed to evaluate the anti-hepatic fibrosis activity and to uncover related mechanism. Molecular docking analysis and cellular thermal shift assay were used to evaluate and verify the interaction between the active withanolides and their potential targets. RESULTS: Eight unreported withanolides, withagulides A-H (1-8), along with twenty-eight known ones were obtained from P. angulate. Withanolides 6, 9, 10, 24, 27, and 29-32 showed marked anti-hepatic fibrosis effect with COL1A1 expression inhibition above 50 %. Physalin F (9), the main component in the active fraction, significantly decreased the TGF ß1-stimulated expressions of collagen I and α-SMA in LX-2 cells. Mechanism study revealed that physalin F exerted its anti-hepatic fibrosis effect via the PI3K/AKT/mTOR signaling pathway. CONCLUSION: This study suggested that withanolides were an important class of natural products with marked anti-hepatic fibrosis effect. The main withanolide physalin F might be a promising candidate for hepatic fibrosis treatment. The work provided experimental foundation for the use of P. angulate to treat hepatic fibrosis.

Withanolides from Physalis angulata var. villosa and the Relative Configurational Revision of Some Known Analogs.

Physalis angulata var. villosa is a plant possessing abundant withanolides, but in-depth research is lacking. In our ongoing study of P. angulata var. villosa, 15 previously undescribed withanolides (1-15), along with 21 known analogs (16-36), were isolated from the whole plant. The structures of the withanolides (1-15) were elucidated based on analysis of their 1D and 2D NMR, HRESIMS, and ECD data. Additionally, the application of γ-gauche effects with the help of ROESY correlations led to the formulation of empirical rules for withanolides with 14-OH/15-OAc to rapidly determine the 14-OH orientations, making it possible to propose configurational revisions of 19 previously reported analogs (1'-19'). Withanolides 1, 4-6, and 10 showed potent cytotoxic activities against three human cancer cell lines (HCT-116, MDA-MB-231, and A549).

Cytotoxic withanolides from the leaves of Datura stramonium L.

Six novel withanolides, along with nine known related compounds were isolated from the leaves of Datura stramonium L. The structures and absolute configurations of the new withanolides were elucidated by employing various spectral techniques and comparing them with those previously reported in the literature. In addition, four withanlides demonstrated interesting cytotoxic activity on LN229 cells with IC50 <20 µM.

Target Separation and Potential Anticancer Activity of Withanolide-Based Glucose Transporter Protein 1 Inhibitors from Physalis angulata var. villosa.

The glucose transporter 1 (GLUT1) protein is involved in the basal-level absorption of glucose in tumor cells. Inhibiting GLUT1 decreases tumor cell proliferation and induces tumor cell damage. Natural GLUT1 inhibitors have been studied only to a small extent, and the structures of known natural GLUT1 inhibitors are limited to a few classes of natural products. Therefore, discovering and researching other natural GLUT1 inhibitors with novel scaffolds are essential. Physalis angulata L. var. villosa is a plant known as Mao-Ku-Zhi (MKZ). Withanolides are the main phytochemical components of MKZ. MKZ extracts and the components of MKZ exhibited antitumor activity in recent pharmacological studies. However, the antitumor-active components of MKZ and their molecular mechanisms remain unknown. A cell membrane-biomimetic nanoplatform (CM@Fe3O4/MIL-101) was used for target separation of potential GLUT1 inhibitors from MKZ. A new withanolide, physagulide Y (2), together with six known withanolides (1, 3-7), was identified as a potential GLUT1 inhibitor. Physagulide Y was the most potent GLUT1 inhibitor, and its antitumor activity and possible mechanism of action were explored in MCF-7 human cancer cells. These findings advance the development of technologies for the targeted separation of natural products and identify a new molecular framework for the investigation of natural GLUT1 inhibitors.

UPLC-Q-Orbitrap-MS/MS-Guided Isolation of Bioactive Withanolides from the Fruits of Physalis angulata.

Physalis angulata Linn. is an exotic Amazonian fruit that is commonly recognized as wild tomato, winter cherry, and gooseberry. While its fruit is known to contain many nutrients, such as minerals, fibers, and vitamins, few papers have investigated withanolide derivatives from its fruits. UPLC-Q-Orbitrap-MS/MS, which produces fragmentation spectra, was applied for the first time to guide the isolation of bioactive withanolide derivatives from P. angulata fruits. As a result, twenty-six withanolide derivatives, including two novel 1,10-secowithanolides (1 and 2) and a new derivative (3), were obtained. Compounds 1 and 2 are rare rearranged 1,10-secowithanolides with a tetracyclic 7/6/6/5 ring system. All structures were assigned through various spectroscopic data and quantum chemical calculations. Nine withanolide derivatives exhibited significant inhibitory effects on three tumor cell lines with IC50 values of 0.51-13.79 µM. Moreover, three new compounds (1-3) exhibited potential nitric oxide inhibitory effects in lipopolysaccharide-stimulated RAW264.7 cells (IC50: 7.51-61.8 µM). This investigation indicated that fruits of P. angulata could be applied to treat and prevent cancer and inflammatory-related diseases due to their potent active withanolide derivatives.

Antibacterial activity of withanolides and their structure-activity relationship.

Two new withanolides, (17R,20S,22R)-4ß-acetoxy-5ß,6ß-epoxy-19,27-dihydroxy-1-oxo-witha-2,24-dienolide (withalongolide A 4-acetate (5) and (17R,20S,22R)-5ß,6ß-epoxy-27-hydroxy-1,4-dioxo-witha-24-enolide (9), and seven known withanolides with normal structure (1-4, 6-8) were isolated from aerial parts of Cuatresia colombiana. Several semisynthetic derivatives were prepared from the natural metabolites withaferin A and jaborosalactone 38. The compounds were fully characterized by a combination of spectroscopic methods (1D and 2D NMR and MS). The compounds isolated from C. colombiana, sixteen withanolides previously isolated from different Solanaceae species with different skeletons and semisynthetic derivatives were evaluated for their antibacterial activity against a selected panel of Gram-positive and Gram-negative bacteria. According to the bioactivity against S. aureus and E. faecalis, the compounds evaluated were divided into three groups: compounds with high activity (MIC 0.063 mM), compounds with moderate activity (0.5 mM > MIC > 0.125 mM) and non-active compounds (MIC ≥1 mM); in addition, some structure-activity relationship keys could be inferred.

A critical assessment of the whole plant-based phytotherapeutics from Withania somnifera (L.) Dunal with respect to safety and efficacy vis-a-vis leaf or root extract-based formulation.

Withania somnifera (L.) Dunal, popularly known as Ashwagandha or Indian ginseng, is well acclaimed for its health-enhancing effects, including its potent immunomodulatory, anti-inflammatory, neuroprotective, and anti-tumorigenic properties. The prime biological effectors of these attributes are a diverse group of ergostane-based steroidal lactones termed withanolides. Withanones and withanosides are distributed differentially across the plant body, whereas withanolides and withanones are known to be more abundant in leaves, while withanosides are found exclusively in the roots of the plants. Standardized W. somnifera extract is Generally Recognized as Safe (GRAS)-affirmed, however, moderate to severe toxic manifestations may occur at high dosages. Withaferin A, which also happens to be the primary bioactive ingredient for the effectiveness of this plant. There have been contrasting reports regarding the distribution of withaferin A in W. somnifera. While most reports state that the roots of the plant have the highest concentrations of this phytochemical, several others have indicated that leaves can accumulate withaferin A in proportionately higher amounts. A comprehensive survey of the available reports suggests that the biological effects of Ashwagandha are grossly synergistic in nature, with many withanolides together mediating the desired physiological effect. In addition, an assorted formulation of withanolides can also neutralize the toxic effects (if any) associated with withaferin A. This mini-review presents a fresh take on the recent developments regarding the safety and toxicity of the plant, along with a critical assessment of the use of roots against leaves as well as whole plants to develop therapeutic formulations. Going by the currently available scientific evidence, it is safe to infer that the use of whole plant formulations instead of exclusively root or leaf recipes may present the best possible option for further exploration of therapeutic benefits from this novel medicinal plant.HighlightsTherapeutic potential of withanolides owes to the presence of α,ß unsaturated ketone which binds to amines, alcohols, and esters and 5ß, 6ß epoxy group which react with side chain thiols of proteins.At concentrations above NOAEL (no observed adverse effect level), the same mechanisms contribute towards toxicity of the molecule.Although withanosides are found exclusively in roots, whole plants have higher contents of withanones and withanolides.Whole plant-based formulations have other metabolites which can nullify the toxicity associated with roots.Extracts made from whole plants, therefore can holistically impart all therapeutic benefits as well as mitigate toxicity.

Withanolides from Datura ceratocaula and Datura discolor and their acetylcholinesterase inhibitory activity.

The investigation of the chemical constituents of Datura ceratocaula and D. discolor allowed to isolate three new withanolides, datucerolide A (1) from the first species, and datudiscolides A (8) and B (9) from the second. In addition, seven known withanolides and five ubiquitous compounds were isolated from these plants, along with 27-O-ß-d-glucopyranosyl dinnoxolide A (5), which was obtained as the tetraacetyl derivative 4. All the structures were elucidated by analyses of their spectroscopic and spectrometric data and that of dinnoxolide A (6) was confirmed by X-ray diffraction analysis. The structure 4 was assigned earlier to daturametelin G-Ac and that of 5 to datinolide B, therefore, it will be discussed whether these assignments are correct. On the other hand, the structure of datudiscolide A (8) was previously assigned to the aglycone of dinoxin B (14), however, a revision of its reported NMR data showed inconsistencies with the proposed structure. The inhibitory activity of withanolides 2, 3, 6-8, 12, and 13 against acetylcholinesterase enzyme (AChE) was evaluated. Compounds 6, 7, 12, and 13 exhibited the best activity with IC50 values ranging from 2.8 to 21.5 µM.

Golden berry leaf extract containing withanolides suppresses TNF-α and IL-17 induced IL-6 expression in HeLa Cells.

Inflammation, characterized by the overexpression of IL-6 in various tissues, has been reported as a symptom of coronavirus disease 2019. In this study, we established an experimental system for overexpression of IL-6 in HeLa cells stimulated by TNF-α and IL-17, along with identification of anti-inflammatory materials and components from local agricultural, forestry, and fishery resources. We constructed a library of extracts from natural sources, of which 111 samples were evaluated for their anti-inflammatory activities. The MeOH extract of Golden Berry (Physalis peruviana L) leaf was found to exhibit strong anti-inflammatory properties (IC50 = 4.97 µg/mL). Preparative chromatography identified two active constituents, 4ß-hydroxywithanolide E (4ß-HWE) (IC50 = 183 nM) and withanolide E (WE) (IC50 = 65.1 nM). Withanolides are known anti-inflammatory ingredients of Withania somnifera, an Ayurvedic herbal medicine. P. peruviana leaves containing 4ß-HWE and WE should be considered as useful natural resources for anti-inflammatory products.

Withanolides from Discopodium penninervium.

Discopodium penninervium is a medicinal plant endemic to Ethiopia. Its twigs extract led to the isolation of three new withanolides, discopodinolides A - C, and four known analogues. The structures of the compounds were elucidated using NMR, HRMS data analyses, and literature data. The relative configurations were defined by single-crystal X-ray crystallography and NOESY correlations. The antibacterial efficacy of the isolated compounds was evaluated against four commonly dispersed environmental strains of Escherichia coli, Bacillus subtilis, Bacillus pumilus, and Bacillus megaterium. Discopodinolides B and C exhibited moderate antibacterial activities against the pathogenic strains of E. coli, B. subtilis, and B. megaterium.

Withanolides from Physalin angulata and Their Inhibitory Effects on Nitric Oxide Production.

Six new withanolides, angulasteroidins A-F (1-6), along with twelve known analogs (7-18) were isolated from the whole plants of Physalis angulata. Their structures were elucidated by analysis of 1D and 2D NMR, ECD and IR spectra, HR-ESI-MS data, and ECD calculation. Compounds 1 and 6 were rare 1-10 seco withanolides. Compounds 2-4, 7-9, and 15 exhibited significant inhibitory activity on the production of nitric oxide in the LPS-activated RAW 264.7 mouse macrophage cell lines with IC50 values ranging from 0.23 to 9.06 µM.

Two new withanolides from the whole plants of Physalis peruviana.

Two new withaphysalin-type withanolides (18-O-ethylwithaphysalin R and 5-O-ethylphysaminimin C, 1 and 2), along with twelve known withanolides (3-14), were purified and identified from Physalis peruviana L. The chemical structures of these new isolates were elucidated through analyzing spectroscopic and HRESIMS data. All the obtained metabolites were appraised for their potential antiproliferative activity against the human breast cancer cell line MCF-7. Compound 7 was discovered to exhibit potent activity with an IC50 value of 3.51 µM and compounds 2, 6 and 14 showed weak cytotoxic effect.

Metabolomics combined to chemometrics reveals the putative α-glucosidase and α-amylase inhibitory metabolites of ground cherry (Physalis pruinosa L.).

In this work, metabolic profiling of the different parts of ground cherry (P. pruinosa) including fruits, calyces, leaves, stems and roots using UPLC-MS/MS analysis combined to chemometric analysis was attempted. A total of 82 chromatographic peaks belonging to different metabolite classes were identified including terpenes, flavonoids genin and glycosides, withanolides, physalins, sucrose esters, fatty acids and other miscellaneous compounds with withanolides being the most predominant class. Roots extracts possessed the highest relative content of the identified 5ß,6ß-epoxy withanolides and intermediate withanolides, while sucrose esters and flavonoidal glycosides were found in a great abundance in calyces extracts. Moreover, physalins were found in all extracts except for roots extracts. Studying the coefficients plots revealed that terpenes and physalins (physanicantriol, loliolide, physalisitin C) were responsible for discrimination of fruits extracts. Calyces, leaves and stems extracts were found to possess antioxidant activity and higher inhibition of α-glucosidase activity. In an attempt to identify the compounds responsible for the hypoglycemic activity using both α-amylase and α-glucosidase inhibition assays, OPLS models coefficient plots were constructed which indicated that physangulide B, physaperuvin G, neophysalin A, and acylsucroses were positively correlated to α-glucosidase inhibition, while guaiacyl-primeveroside, phyperunolide C, physalactone, physalolactone C and perulactone, were positively correlated to α-amylase inhibitory activity.

Anti-inflammatory withajardins from the leaves of Athenaea velutina.

Withajardins, uncommon modified withanolide-type steroids, have been isolated exclusively from plants of the Solanaceae family so far. Two undescribed withajardins and the known tuboanosigenin were isolated from the hexane/EtOAc 1:1 extract from Athenaea velutina leaves. Their structures were established by an extensive analysis of 1D and 2D-NMR and HRMS data. The absolute configuration was determined by X-ray diffraction (withajardin L and tuboanosigenin) and circular dichroism (CD) analyses (withajardin M). The anti-inflammatory activity of compounds was evaluated through the inhibition of the lipopolysaccharide (LPS)-induced nitric oxide (NO), TNF-α, and IL-6 release in RAW264.7 cells. The cell viability effects to RAW 264.7 cells showed IC50 values of 74.4-354.4 µM. The compounds attenuated LPS-induced release of NO and decreased pro-inflammatory cytokines TNF-α and IL-6 in RAW264.7 cells.

Anti-inflammatory withanolides from the aerial parts of Physalis minima.

Eight undescribed and two known withanolides were obtained from the aerial parts of Physalis minima. The structures of these compounds were defined by spectroscopic methods including 1D and 2D NMR, HRESIMS, and electronic circular dichroism (ECD) data analysis. Physminin E was elucidated to be a rare 13,14-seco-withanolide. Inhibitory effects of these compounds on nitric oxide (NO) production were evaluated by using LPS-activated RAW264.7 macrophages, and physminin C was shown to be the most active with an IC50 value of 3.5 µM. The further mechanistic analysis of NO inhibition was performed by molecular docking and Western blotting.

Datinolides E-I, five new withanolides with anti-inflammatory activity from the leaves of Datura inoxia Mill.

Five new withanolides, datinolides E-I (1-5), and three known withanolides (6-8) were separated from Datura inoxia Mill. leaves, and datinolide E (1) was the first withanolide with C-27 connected to a nitrogen-containing group. Their structures were clarified by comprehensive spectroscopic analysis and comparison with literature. The anti-inflammatory of isolated compounds against RAW264.7 cells was investigated by the CCK8 assay.

Withaphysalins from Medicinal and Edible Physalis minima and Their Anti-inflammatory Activities.

Physalis minima is a medicinal and edible plant in China. In this study, 22 new withaphysalins, including a novel 1(10 → 6)abeo-14ß-hydroxy one (1) and other 15 unusual 14ß-hydroxy ones (3-4, 6-17, 19), were isolated from the whole herbs of P. minima together with two known analogues (23-24). Their structures were established by extensive analysis of high-resolution electrospray ionization mass spectrometry, IR, and 1D and 2D NMR spectroscopic data. Their absolute configurations were determined by electronic circular dichroism (ECD) spectra and single-crystal X-ray crystallographic analyses, together with DFT NMR calculations. All isolated compounds were evaluated for their anti-inflammatory activity via measuring the colorimetric reporter of the secreted embryonic alkaline phosphatase gene driven by an IFN-ß minimal promoter fused to five copies of the NF-κB consensus transcriptional response element and three copies of the c-Rel binding site in LPS-stimulated human THP1-Dual cells. Compounds 2, 5, 6, 9, 10, 11, and 20 showed significant anti-inflammatory effects with IC50 values in the range of 3.01-13.39 µM. Among them, compounds 2 and 10 showed better anti-inflammatory effects to inhibit the secretion of IL-6, IL-1ß, and TNF-α in LPS-stimulated THP1-Dual cells.

Four new withanolides with anti-inflammatory activity from Datura inoxia Mill. leaves.

Four previously undescribed withanolides, datinolides A-D (1-4) and eight known withanolides (5-12), were separated from the 70% ethyl alcohol extract of Datura inoxia Mill. leaves. All structures were clarified by comprehensive spectroscopic analysis. Furthermore, all withanolides were assessed for their anti-inflammatory activity and results showed that 1 exhibited a fairly good suppression against nitric oxide generation in lipopolysaccharide-stimulated RAW 264.7 cells (IC50 = 10.33 ± 1.53 µM).