Biosynthesis of withanolides in Acnistus breviflorus: biogenetic relationships among the main withanolides

To excised leaves and 15-day-old seedlings of Acnistus breviflorus sodium [1-¹⁴C]acetate, [2-¹⁴C]mevalonolactone and [¹⁴C-methyl]methionine were administered in separate experiments. From the absolute incorporation values of withaferin A (1), jaborosalactone A (2) and jaborosalactone D (3) isolated at different times after administration of the tracers, it was deduced that compound 2 is a precursor of both 1 and 3 and that the withanolides are later biodegraded to unknown products. Inoculation of [¹⁴C]jaborosalactone A confirmed its transformation into 1 and 3.     

2,3-dihydrojaborosalactone A,a withanolide from Acnistus breviflorus

From Acnistus breviflorus the new 27-hydroxy-5β,6β-epoxy-1-oxo-(22R)-witha-24-enolide (2,3-dihydrojaborosalactone A) as well as seven known withanolides, withaferin A, 2,3-dihydrowithaferin A, 6α-chloro-5β-hydroxywithaferin A, 5,6-deoxywithaferin A, jaborosalactone A, jaborosalactone D and jaborosalactone E were isolated and characterized by means of spectroscopic (¹H NMR, ¹³ C NMR and mass spectral) methods. Depending on the extraction solvent (methanol or ethanol), a known artifact (3β-methoxy-2,3-dihydrowithaferin A) and the new 5α-methoxy-4,5-dihydrojaborosalactone B and 5α-ethoxy-4,5-dihydrojaborosalactone B were also isolated and characterized.     

Jaborosalactone L,a withanolide from Jaborosa leucotricha

From the plant Jaborosa leucotricha a new withanolide 17α-hydroxy-5β,6β-epoxy-1-oxo-22R-witha-2,24-dienolide has been isolated and fully characterized.     

Trechonolide A,a new withanolide type from Trechonaetes laciniata

The structure of a new withanolide type isolated from Trechonaetes laciniata, having a hemiacetal ring system and a 5-member ring lactone has been elucidated by X-ray analysis as (23R)-5β,6β-epoxy-12β,17β-dihydroxy-1-oxo-12,22-hemiacetal-ergosta-2,24-dien-23,26-olide (trechonolide A). A second compound (trechonolide B) having at C-12 a β-methoxy group, has also been isolated and assumed to be an artifact since, by heating trechonolide A in methanol with a trace of acid, the methoxy derivative was produced.     

Karyological studies in Jaborosa (Solanaceae)

Sixteen species of Jaborosa (Solanaceae), including eight endemic to Argentina, were studied karyologically. The numbers n  = 12 and/or 2n  = 24 were found in all species, the majority of the cases being new reports. Mitotic chromosomes were small- to medium-sized, the average length varying from 2.95 to 4.93 µm. All species had one to three chromosome pairs with satellites. The karyotypes, obtained for 13 species, were slightly asymmetrical: A ₁ ranged from 0.228 to 0.483, A ₂ ranged from 0.095 to 0.182, and Paszko's asymmetry index ranged from 0.84 to 3.47. In all species, metacentric chromosomes were the most common, followed by submetacentrics, but subtelocentrics were rare. Morphological similarities and sectional arrangements were not reflected in either a principal components analysis plot or asymmetry index plot, but the species could be singled out by their karyotype formulae and the different karyotype parameters taken. In Jaborosa , a notably diversified genus, exo-morphological evolution has taken place, together with evident chromosome rearrangements, whose disposition is different and not as clear as in related genera.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 156 , 467–478.     

Intraspecific variation in the pollen exine sculpture of Jaborosa runcinata Lam. (Solanaceae)

The variability of ectexine sculpture in the pollen of Jaborosa runcinata (Solanaceae) was studied with light and scanning electron microscopy. Three main ectexine variants are recognized: incomplete reticulate, a combination of incomplete reticulate and gemmate‐granular, and gemmate‐granular. A range of transient conditions were also noted between the two extremes. Apertures vary from a basically triporate‐aspidate arrangement to pollen grains which appear to have a rudimentary colpus. The sculptural variability of the ectexine, and a tendency towards a colpus, in J. runcinata are interesting as intraspecific variation does not appear to be common within Solanaceae.     

Biosynthesis of steroidal withanolides in Withania somnifera

Administration of 24-methylene-cholesterol-[28-³H] to Withania somnifera, yielded [³H] radioactivity in the isolated withaferin A and withanolide D, whereas administered 24-(R,S)-methyl-cholesterol-[28-³H] was not incorporated into these compounds. 24-Methylene-cholesterol is, therefore, proposed as a sterol precursor of the withanolides. A novel procedure is described for the isolation of withanolides from W. somnifera. This method in conjunction with an improved procedure for administration of labelled sterols and mevalonolactone produces a greatly increased yield of labelled withanolides.     

Chlorinated withanolides from Withania somnifera and Acnistus breviflorus

The structure of a new chlorinated withanolide isolated from the hybrid plants of Withania somnifera, chemotypes III (Israel) by Indian I, is established as the chlorohydrin of withanolide D (6α-chloro-4β,5β,20α_F-trihydroxy-l-oxo-22R-witha-2,24-dienolide). Two other known chlorinated withanolides had been isolated from different sources and additional data are provided, including the X-ray diffraction study of 4-deoxyphysalolactone (chlorohydrin of withanolide E). All available data are used for comparative analysis of the six known chlorinated withanolides. The origin of the chlorine atom in these compounds in the plants has been determined by carrying out a simple reaction of withanolide D with NaCl on silica gel.     

An in vitro study of apoptotic like death in Leishmania donovani promastigotes by withanolides

The aim of this study was to isolate and evaluate the withanolides in inducing apoptotic like death in Leishmania donovani in vitro. Withanolides were fractionated and isolated from the leaves of Withania somnifera and LC-MS/MS analysis of two fractions namely, F5 and F6 of ethanolic extracts, obtained through column chromatography with silica gel, was performed. The antileishmanial effect of withanolides on L. donovani promastigotes was assessed in vitro using PI dye exclusion test. The effect of withanolides on promastigote morphology was determined by scanning electron microscopy. To understand their mode of action against L. donovani, DNA fragmentation, quantification of parasites at sub G₀/G₁ phase, determination of phosphatidylserine externalization, measurement of reactive oxygen species (ROS) and mitochondrial membrane potential (Ψ_m) were done. Results showed that LC–MS/MS analysis confirmed the presence of withanolides in isolated fractions. Treatment with withanolides resulted in morphological alterations from spindle to round shape and loss of flagella/cell integrity in promastigotes. Moreover, it induced DNA nicks, cell cycle arrest at sub G₀/G₁ phase and externalization of phosphatidylserine in dose and time dependent manner via increase in ROS and decrease in Ψ_m. Results of this study indicate that withanolides induce apoptotic like death through the production of ROS from mitochondria and disruption of Ψ_m in promastigotes of L donovani.     

Virginols A–C,three new withanolides from Physalis virginiana

Virginols A–C are three new withanolides containing an epoxylactol at the side chain. They were isolated from the aerial parts of Physalis virginiana Miller. Structures of these compounds were determined by analyses of their spectroscopic data, including 1D and 2D NMR. The structure of virginol A was confirmed by X-ray diffraction analysis. The anti-inflammatory activity of virginols was evaluated in the TPA-induced ear mouse edema model.     

New withanolides from a cross of a South African chemotype by chemotype II (Israel) in Withania somnifera

Three new withanolides have been isolated from hybrids obtained by crossing a chemotype of Withania somnifera received from South Africa and chemotype II originating in Israel. The compounds have been characterized as 4β,20α-dihydroxy-1-oxo-5β,6β-epoxy-20R,22R-witha-24-enolide, 20α-hydroxy-1,4-dioxo-5β,6β-epoxy-20R,22R-witha-2,24-dienolide, and 20α-hydroxy-1,4-dioxo-5β,6β-epoxy-20R,22R-witha-2-enolide. The major steroid of the plant is withanolide D, while the other known withanolides present are 4β,20α-dihydroxy-1-oxo-5β,6β-epoxy-20R,22R,24S,25R-witha-2-enolide and withaferin A. The structures assigned to the new compounds are based on spectral evidence, analysis of their fragmentation under electron impact, and on chemical correlation with known compounds. The formation of these withanolides in this new hybrid is discussed briefly.     

Cytotoxic withanolides from the aerial parts of Tubocapsicum anomalum

Ten new withanolides (1–10) and three artificial withanolides (11–13) were isolated from the aerial parts of Tubocapsicum anomalum, together with five known analogues (14–18). Their structures were determined on the basis of extensive spectroscopic and chemical methods. They include seven acnistin–type (1–4, 11, 14 and 15), three withajardin–type (5–7), and eight normal–type (8–10, 12, 13 and 16–18) withanolides. Of normal–type withanolides, a chemical conversion from the 16α,17α–epoxywithanolide (16) to Δ^13,14–16α–hydroxywithanolide (18) was achieved by Wagner–Meerwein rearrangement. All isolates were evaluated for their cytotoxicity against four human tumor cell lines (HCT–116, HepG2, MCF–7 and A375). Among them, compounds 1–3, 6–8, 14, 16–18 showed cytotoxic activity with IC₅₀ values of 0.24–8.71 μM.     

In silico evaluation of pharmacokinetics and acute toxicity of withanolides in Ashawagandha

Ashwagandha is a medicinal plant used in traditional Asian medicines as an adaptogen to promote both physical and mental health. Withanolides are the major bioactive phytochemicals in Ashwagandha; they are a group of naturally occurring C₂₈-steroidal lactones with an ergostane-based skeleton. Despite the broad use of Ashwagandha, data on the pharmacokinetic and toxicological properties of withanolides remain scarce. Here, 75 withanolides in Ashwagandha were identified in journal publications, databases, and monographs. In silico quantitative structure-activity relationship (QSAR) models were used to evaluate the physicochemical and pharmacokinetic properties as well as the acute toxicity of withanolides. Withanolides had high molecular weight and pKa, low aqueous solubility, and high lipophilicity. QSAR models also predicted high effective human jejunal permeability and plasma protein binding, tissue partitioning, extensive metabolism, hepatic uptake, and renal excretion for certain withanolides. In addition, two thirds of the withanolides evaluated had predicted median lethal dose (LD₅₀) under 100 mg/kg after oral administration in rats. These in silico results could be used to guide further testing and confirmed by in vitro and in vivo studies.     

Immunosuppressive Withanolides from Withania coagulans

Six new withanolides, withacoagulins A–F ( 1 – 6 , resp.), together with ten known withanolides, 7 – 16 , were isolated from the aerial parts of Withania coagulans. Their structures were determined by spectroscopic techniques including 1D‐ and 2D‐NMR (¹H, ¹³C, HMQC, and HMBC) and MS experiments. These compounds, including the crude extracts of this herb, exhibited strong inhibitory activities on the T‐ and B‐cell proliferation.     

A 12-deoxywithastramonolide-rich somaclonal variant in Withania somnifera (L.) Dunal—molecular cytogenetic analysis and significance as a chemotypic resource

Withania somnifera, commonly known as ashwagandha or Indian ginseng, is a valuable medicinal plant, synthesizing a wide array of pharmacologically active secondary metabolites known as withanolides. In this study, we investigated variation among 54 regenerated plants attained through indirect organogenesis from leaf explants. Organogenic calli were induced on Murashige and Skoog medium containing 2?mg?l^?1 kinetin and 1?mg?l^?1 indole-3-butyric acid. High-performance liquid chromatography was used for quantitative determination of the major withanolides in the somaclones. One somaclone (WS-R-1) showed significantly higher accumulation of 12-deoxywithastramonolide (WS-12D; 0.516%) compared to the explant donor mother plant (0.002%). The incidence of somaclonal variation at the cytological level was investigated by studying mitosis and meiosis in relation to chromosome number and structural organization. There were no alterations in chromosome phenotypes, somatic chromosome count, or meiotic behavior. Fidelity at genomic level was evaluated by random amplification of polymorphic DNA (RAPD) analyses, which revealed multiple genetic polymorphisms between the WS-12D over-producing somaclone and the explant donor mother plant. This study demonstrates the capability of inducing chemotypic variability for the development of high-yielding clones due to molecular instability in W. somnifera using an in vitro approach.     

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.     

Caffeoylquinic acid derivatives from the roots of Arctium lappa L. (burdock) and their structure–activity relationships (SARs) of free radical scavenging activities

Eight caffeoylquinic acid (CQA) derivatives have been identified from the roots of Arctium lappa L. (burdock), including three new derivatives (1–3) together with five known derivatives (4–8). Their chemical structures were determined by 1D and 2D NMR as well as HR-TOF-MS data. The free radical scavenging activities of each compound were determined experimentally and found to be significant, especially the tri-CQA (8) and di-CQAs (6–7). Besides, our data showed that the number of caffeoyl groups in the caffeoyl derivatives played a crucial role for these physiological functions.     

Further sesquiterpene lactones from the genus Dittrichia

The aerial parts of Dittrichia graveolens afforded in addition to compounds isolated previously five new sesquiterpene lactones, two benzoic acid derivatives while D. viscosa gave two further derivatives of costic acid. The structures were elucidated by high field NMR spectroscopy.     

The withanolides of Withania somnifera chemotypes I and II

Seven steroidal lactones of the withanolide series have been isolated as minor constituents of the leaves of Withania somnifera Dun. (Solanaceae) chemotype I, along with the major component withaferin A. Structures have been assigned to the new compounds: withanolide N (17α,27-dihydroxy-1-oxo-20R,22R-witha-2,5,14,24-tetraenolide) (6a) and withanolide O (4β,17α-dihydroxy-1-oxo-20R,22R-witha-2,5,8(14),24-tetraenolide) (7a). Similarly the leaves of W. somnifera chemotype II afforded three new withanolides along with the major component withanolide D (9a) and trace amounts of withanolide G (10). The new compounds are: 27-hydroxywithanolide D(4β,20α,27-trihydroxy-1-oxo-5β,6β-epoxy-20R,22R-witha-2,24-dienolide) (11a), 14α-hydroxywithanolide D (4β,14α,20α-trihydroxy-1-oxo-5β,6β-epoxy-20R,22R-witha-2,24-dienolide) (12a) and 17α-hydroxywithanolide D (4β,17β,20α-trihydroxy-1-oxo-5β,6β-epoxy-20S,22R-witha-2,24-dienolide) (13a). Whereas all the withanolides of chemotype I are unsubstituted at C-20 (20α-H), those of chemotype II possess an OH at this position (20α-OH).