A classic withanolide is defined as a highly oxygenated C28 ergostane-type steroid that is characterized by a C22-hydroxy-C26-oic acid δ-lactone in the nine-carbon side chain. Analysis of the reported 13C NMR data of classic withanolides with hydroxy groups (C-14, C-17, and C-20) revealed that (1) a hydroxy (C-14 or C-17) substituent significantly alters the chemical shifts (C-7, C-9, C-12, and C-21) via the γ-gauche effect; (2) the chemical shift values (C-9, C-12, and C-21) reflect the orientation (α or β) of the hydroxy moiety (C-14 or C-17); (3) a double-bond positional change in ring A (Δ2 to Δ3), or hydroxylation (C-27), results in a minuscule effect on the chemical shifts of carbons in rings C and D (from C-12 to C-18); and (4) the 13C NMR γ-gauche effect method is more convenient and reliable than the traditional approach (1H NMR shift comparisons in C5D5N versus CDCl3) to probe the orientation of the hydroxy substituent (C-14 and C-17). Utilization of these rules demonstrated that the reported 13C NMR data of withanolides 1a–29a were inconsistent with their published structures, which were subsequently revised as 1–16 and 12 and 18–29, respectively. When combined, this strongly supports the application of these methods to determine the relative configuration of steroidal substituents.

•Six new pregnane glycosides were isolated from Asclepias sullivantii.•Structure elucidation of new compounds was conducted with NMR and HRMS experiments.•This work represents the first phytochemical study of Asclepias sullivantii.Phytochemical investigation of the above-ground biomass of Asclepias sullivantii L. (Asclepiadaceae) afforded six new pregnane glycosides, named sullivantosides A-F (1–6). The structures of 1–6 were elucidated through a variety of spectroscopic and spectrometric techniques (1D and 2D NMR; HRESIMS). To the best of our knowledge, this work represents the first phytochemical study of this species.

Four withanolides (1–4) and two sucrose esters (5, 6) were isolated from the aerial parts of Physalis neomexicana. The structures of 1–6 were elucidated through a variety of spectroscopic techniques. Cytotoxicity studies of the isolates revealed that 2 inhibited human breast cancer cell lines (MDA-MB-231 and MCF-7) with IC50 values of 1.7 and 6.3 μM, respectively.

•Eight withanolides 1–8 were isolated from Physalis coztomatl (Solanaceae).•Physacoztolides 1–6 are highly oxygenated.•The structure of withanolide 1 was confirmed by X-ray crystallographic analysis.•Coagulansin A reported in the literature contains a 17α-hydroxy moiety rather than the reported 17β-hydroxy functionality.Six withanolides (1–6), as well as two known withanolides (physachenolide D 7 and withanoside VI 8), were isolated from the aerial parts of Physalis coztomatl (Solanaceae). Structural elucidations of 1–6 were achieved through 2D NMR and other spectroscopic techniques, while the structure of 1 was confirmed by X-ray crystallographic analysis. In addition, the stereochemical orientation of the 17-hydroxy group in withanolides was discussed in relation to 13C NMR shifts of C-12, 13, 14 and 16. Such analysis established that coagulansin A contains a 17α-hydroxy moiety rather than the reported 17β-hydroxy functionality, and has been revised accordingly.Six withanolides were isolated from Physalis coztomatl (Solanaceae). The structure of 1 was confirmed by X-ray crystallography.

Nine new withanolides (1–9), withahisolides A–I, were isolated along with nine known compounds (10–18) from the aerial parts of Physalis hispida. The structures of 1–9 were elucidated through a variety of spectroscopic techniques, while the structures of 1 and 2 were confirmed by X-ray crystallographic analysis. Compounds 1–3 are the first withanolides with nonaromatic six-membered ring D moieties. In addition, withanolide 8 represents a novel withanolide skeleton due to the absence of a C-13–C-17 bond within the steroidal nucleus.

•Four withanolides 1–4 were isolated from Jaborosa caulescens var. bipinnatifida (Solanaceae).•2,3-Dihydrotrechonolide A (1) and 2,3-dihydro-21-hydroxytrechonolide A (2) are uncommon.•The structure of withanolide 1 was confirmed by X-ray crystallographic analysis.Withanolides 2,3-dihydrotrechonolide A (1) and 2,3-dihydro-21-hydroxytrechonolide A (2) were isolated along with two known withanolides trechonolide A (3) and jaborosalactone 39 (4) from Jaborosa caulescens var. bipinnatifida (Solanaceae). The structures of 1–2 were elucidated through 2D NMR and other spectroscopic techniques. In addition, the structure of withanolide 1 was confirmed by X-ray crystallographic analysis.Withanolides (1 and 2) were isolated from Jaborosa caulescens var. bipinnatifida (Solanaceae). The structure of 1 was confirmed by X-ray crystallography.

6α,7α-Epoxy-5α-hydroxy-1-oxo-22R-witha-2,24-dienolide (C28H38O5), 5β,6β-epoxy-4β,20-dihydroxy-1-oxo-22R-witha-2,24-dienolide (C28H38O6), and 4β,27-dihydroxy-1-oxo-22R-witha-2,5,24-trienolide (C28H38O5), were isolated from Withania somnifera roots. The structures of these withanolides were established by spectroscopic analysis and X-ray diffraction studies as withanolide B, withanolide D, and 5,6-deoxywithaferin A, respectively. All three compounds crystallize in the orthorhombic space group P212121 with unit cell parameters: a = 9.1012(3) Å, b = 12.2728(4) Å, c = 21.5396(7) Å, Z = 4 for withanolide B; a = 7.6569(5) Å, b = 9.9426(7) Å, c = 33.767(2) Å, Z = 4 for withanolide D; and a = 6.9310(3) Å, b = 10.6745 (4) Å, c = 34.667(2) Å, Z = 4 for 5,6-deoxywithaferin A, respectively. The crystal structures have been solved by direct methods and refined to R1 = 0.024 for 4,290 unique reflections (withanolide B), R1 = 0.042 for 4,601 unique reflections (withanolide D), and R1 = 0.033 for 4,626 unique reflections (5,6-deoxywithaferin A), respectively. Rings B, C, D of the steroid skeletons are trans connected while ring C adopts a chair conformation, five-membered ring D adopts a half-chair, and the δ-lactone ring E in the side chain adopts a sofa conformation. For withanolide B, rings A and B are trans connected and both rings A and B exist in a half-chair conformation; for withanolide D, rings A and B are cis connected, and ring A has a sofa conformation while ring B has a distorted boat conformation; for 5,6-deoxywithaferin A, ring A has a half chair conformation and ring B exists as a boat conformation.

Methanol solutions of the main withanolides (6–8) naturally present in Physalis longifolia yielded five artificial withanolides (1–5), including three new compounds (1–3). Withanolides 1 and 2 were identified as intramolecular Michael addition derivatives, while withanolides 3–5 were the result of intermolecular Michael addition. A comprehensive literature investigation was conducted to identify potential withanolide Michael addition artifacts isolated from Solanaceous species to date.

A new withanolide, named withawrightolide (1), and four known withanolides (2–5) were isolated from the aerial parts of Datura wrightii. The structure of compound 1 was elucidated through 2D NMR and other spectroscopic techniques. In addition, the structure of withametelin L (2) was confirmed by X-ray crystallographic analysis. Using MTS viability assays, withanolides 1–5 showed antiproliferative activities against human glioblastoma (U251 and U87), head and neck squamous cell carcinoma (MDA-1986), and normal fetal lung fibroblast (MRC-5) cells with IC50 values in the range between 0.56 and 5.6 μM.

Phytochemical investigation of the dried biomass of Asclepias syriaca afforded five new compounds (1–5), along with 19 known structures. Overall, the secondary metabolites isolated and identified from this plant showed a wide structural diversity including pentacyclic triterpenes, cardiac glycosides, flavonoid glycosides, lignans, a phenylethanoid, and a glycosylated megastigmane. In addition, the isolates were tested against the cancer breast cell line Hs578T, and those showing IC50 values lower than 50 μM (1 and 6–9) were further investigated in three additional breast cancer cell lines (MCF-7, T47D, and Sk-Br-3) and the normal breast cell line Hs578Bst.

As part of our ongoing effort to explore the chemical diversity of plants of the United States Midwest region, the isolation and identification of 13 pregnane glycosides named verticillosides A–M from Asclepias verticillata L. are reported. The structures of these compounds were elucidated by various spectroscopic techniques, including 1D and 2D NMR, IR, UV, and HRMS. The cytotoxicity of the isolates was evaluated against paired breast cell lines Hs578T (cancer) and Hs578Bst (normal), however, no significant growth inhibition was observed.Graphical abstractIsolation and identification of 13 pregnane glycosides named verticillosides A–M from Asclepias verticillata L.Highlights► Verticillosides A–M, 13 pregnane glycosides, were isolated from Asclepias verticillata L. ► 800 MHz NMR experiments and X-ray crystallography were employed for structure elucidation. ► Isolates did not show significant cytotoxicity against breast cancer cell line Hs578T.

Bioassay-guided fractionation of a CH2Cl2−MeOH extract of the aerial parts of Albizia inundata resulted in the isolation of two new natural oleanane-type triterpene saponins {3-O-[α-l-arabinopyranosyl(1→6)]-2-acetamido-2-deoxy-β-d-glucopyranosyl oleanolic acid (1) and 3-O-[α-l-arabinopyranosyl(1→2)-α-l-arabinopyranosyl(1→6)]-2-acetamido-2-deoxy-β-d-glucopyranosyl acacic acid lactone (2)} along with seven known saponins {3-O-[α-l-arabinopyranosyl(1→6)]-2-acetamido-2-deoxy-β-d-glucopyranosyl echinocystic acid (3), 3-O-[β-d-xylopyranosyl (l→2)-α-l-arabinopyranosyl(l→6)]-2-acetamido-2-deoxy-β-d-glucopyranosyl acacic acid lactone (concinnoside D) (4), 3-O-[β-d-glucopyranosyl(l→2)]-β-d-glucopyranosyl oleanolic acid (5), 3-O-[α-l-arabinopyranosyl(1→2)-α-l-arabinopyranosyl(l→6)]-β-d-glucopyranosyl oleanolic acid (6), 3-O-[β-d-xylopyranosyl(1→2)-α-l-arabinopyranosyl(l→6)]-β-d-glucopyranosyl oleanolic acid (7), 3-O-[α-l-arabinopyranosyl(l→2)-α-l-arabinopyranosyl(1→6)-[β-d-glucopyranosyl(l→2)]-β-d-glucopyranoside echinocystic acid (8), and 3-O-[β-d-xylopyranosyl(l→2)-α-l-arabinopyranosyl(1→6)-[β-d-glucopyranosyl(l→2)]-β-d-glucopyranoside echinocystic acid (9)}. The structures of 1 and 2 were established on the basis of extensive 2D NMR (1H−1H COSY or DQF-COSY, HSQC, HMBC, TOCSY, and HSQC-TOCSY) spectroscopic, ESIMS, and chemical methods. Saponins 1, 3, 6, and 7 showed cytotoxicity against human head and neck squamous cells (JMAR, MDA1986) and melanoma cells (B16F10, SKMEL28) with IC50 values in the range 1.8−12.4 μM, using the MTS assay.

Fourteen new withanolides, 1–14, named withalongolides A–N, respectively, were isolated from the aerial parts of Physalis longifolia together with eight known compounds (15–22). The structures of compounds 1–14 were elucidated through spectroscopic techniques and chemical methods. In addition, the structures of withanolides 1, 2, 3, and 6 were confirmed by X-ray crystallographic analysis. Using a MTS viability assay, eight withanolides (1, 2, 3, 7, 8, 15, 16, and 19) and four acetylated derivatives (1a, 1b, 2a, and 2b) showed potent cytotoxicity against human head and neck squamous cell carcinoma (JMAR and MDA-1986), melanoma (B16F10 and SKMEL-28), and normal fetal fibroblast (MRC-5) cells with IC50 values in the range between 0.067 and 9.3 μM.

A chlorinated withanolide, 6α-chloro-5β,17α-dihydroxywithaferin A (1), and nine known withanolides, 6α-chloro-5β-hydroxywithaferin A (2), (22R)-5β-formyl-6β,27-dihydroxy-1-oxo-4-norwith-24-enolide, withaferin A, 2,3-dihydrowithaferin A, 3-methoxy-2,3-dihydrowithaferin A, 2,3-didehydrosomnifericin, withanone, withanoside IV and withanoside X, were isolated from Withania somnifera (Solanaceae). All structures were elucidated on the basis of spectroscopic methods (IR, HRESIMS, 1D/2D NMR). X-ray crystallography confirmed the absolute configuration of 1.Graphical abstractTen withanolides including one previously unknown chlorinated compound (1) were isolated from Withania somnifera (Solanaceae). Spectroscopic methods and X-ray crystallography elucidated the absolute configuration of 1.Highlights► From Withania somnifera, one new chlorinated withanolide, 6α-chloro-5β,17α-dihydroxywithaferin A (1), was isolated together with nine known ones. ► Full NMR assignments for two chlorinated withanolides (1) and (2) were described. ► X-ray crystallography confirmed the absolute configuration of 1.

Three unprecedented purine-containing compounds, named [6]-, [8]-, and [10]-zingerines as they are 5-(6-amino-9H-purin-9-yl) analogs of [6]-, [8]-, and [10]-gingerols, respectively, were isolated from a methanolic extract of ginger rhizomes using a phase trafficking-based method that utilizes solid phase reagents allowing for fast and selective simultaneous separation of basic, acidic, and neutral components of natural products extracts.Graphical abstractIsolation and structural determination of the zingerines, unprecedented purine-containing compounds from ginger rhizomes (Zingiber officinale Roscoe), using a phase-trafficking approach..Highlights► We describe the zingerines, new purine-containing compounds obtained from ginger. ► Zingerines contain a purine ring attached to gingerol-type carbon skeleton. ► Isolation of zingerines was facilitated by using phase-trafficking approach.

A novel simultaneous phase-trafficking approach using spatially separated solid-supported reagents for rapid separation of neutral, basic, and acidic compounds from organic plant extracts with minimum labor is reported. Acidic and basic ion-exchange resins were physically separated into individual sacks (“tea bags”) for trapping basic and acidic compounds, respectively, leaving behind in solution neutral components of the natural mixtures. Trapped compounds were then recovered from solid phase by appropriate suspension in acidic or basic solutions. The feasibility of the proposed separation protocol was demonstrated and optimized with an “artificial mixture” of model compounds. In addition, the utility of this methodology was illustrated with the successful separation of the alkaloid skytanthine from Skytanthus acutus Meyen and the main catechins and caffeine from Camellia sinensis L. (Kuntze). This novel approach offers multiple advantages over traditional extraction methods, as it is not labor intensive, makes use of only small quantities of solvents, produces fractions in adequate quantities for biological assays, and can be easily adapted to field conditions for bioprospecting activities.

As part of a program to discover drug leads from plant biodiversity, the present investigation was undertaken to explore the anticancer potential of compounds derived from selected Latin American plants. Bioassay-guided fractionation of a crude extract of the aerial parts of Vassobia breviflora led to the isolation of the withanolide-type steroidal lactone withaferin A (1). This compound was tested for antiproliferative activity against the head and neck squamous cell carcinoma (HNSCC) cell lines, MDA1986, JMAR, UM-SCC-2, and JHU011. The inhibitory concentrations to reduce cell viability to 50% (IC50) were determined by the MTS cytotoxicity assay, and 1 reduced cell viability with IC50 values in the range 0.5−2.2 μM. A mechanistic study showed that 1 induces apoptosis and cell death in HNSCC cells as well as a cell-cycle shift from G0/G1 to G2/M. Cells treated with 1 exhibited inactivation of Akt and a reduction in total Akt concentration. This investigation constitutes the first report of the antiproliferative activity of withaferin A (1) against head and neck squamous carcinoma.

Our research highlights the nutraceutical potential of the fruits of Physalis longifolia Nutt. (Solanaceae) in antitumor therapy. Recently we reported the isolation of withaferin A, along with 22 other withanolides from the aerial parts of P. longifolia. Herein, we present our biological, ethnobotanical, and phytochemical investigations on the edible fruit (known as “wild tomatillo” or “long leaf groundcherry”) of the species. Specifically, dried fruit extract was examined for its chemical composition and biological activity. Explorative chromatography analysis of the fruit extract indicated the presence of withaferin A, which was further confirmed by subsequent HPLC analysis. The therapeutic potential of orally administered fruit extract was also investigated, where gavage treatment induced a 60% volume reduction in triple negative breast carcinomas (MDA-MB-468LN) in an experimental mouse model. This encouraging data indicate the potential of P. longifolia fruits as a dietary supplement, while also supporting the previously reported pharmaceutical potency of semi-synthetic withalongolide derivatives as promising chemotherapeutic candidates for antitumor therapies.Download full-size image

•While anti-inflammatory effects of ginger are often attributed to its gingerols, each of its secondary metabolites, the essential oils and the gingerols, are joint protective in an experimental arthritis model.•Both of ginger’s secondary metabolites (essential oils and gingerols) have anti-arthritic effects in an experimental model.•Anti-arthritic effects of ginger essential oils (GEO) mirrored those of 17-β estradiol.•However, GEO had no effect on classic estrogen-responsive organs.•GEO were well tolerated.•GEO and gingerols may have additive joint protective effects in arthritis.Ginger and its extracts have been used traditionally as anti-inflammatory remedies, with a particular focus on the medicinal properties of its phenolic secondary metabolites, the gingerols. Consistent with these uses, potent anti-arthritic effects of gingerol-containing extracts were previously demonstrated by our laboratory using an experimental model of rheumatoid arthritis, streptococcal cell wall (SCW)-induced arthritis. In this study, anti-inflammatory effects of ginger’s other secondary metabolites, the essential oils (GEO), which contain terpenes with reported phytoestrogenic activity, were assessed in female Lewis rats with SCW-induced arthritis. GEO (28 mg/kg/d ip) prevented chronic joint inflammation, but altered neither the initial acute phase of joint swelling nor granuloma formation at sites of SCW deposition in liver. Pharmacologic doses of 17-β estradiol (200 or 600 μg/kg/d sc) elicited the same pattern of anti-inflammatory activity, suggesting that GEO could be acting as a phytoestrogen. However, contrary to this hypothesis, GEO had no in vivo effect on classic estrogen target organs, such as uterus or bone. En toto, these results suggest that ginger’s anti-inflammatory properties are not limited to the frequently studied phenolics, but may be attributable to the combined effects of both secondary metabolites, the pungent-tasting gingerols and as well as its aromatic essential oils.Download full-size image