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Recently, the isolation and elucidation of a series of polyhydroxyanthraquinones were reported from an organic extract of a solid phase culture of an endophytic fungus, Penicillium restrictum (strain G85). One of these compounds, ω-hydroxyemodin (1), showed promising quorum-sensing inhibition against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) in both in vitro and in vivo models. The initial supply of 1 was 19 mg, and this amount needed to be scaled by a factor of 30 to 50 times, in order to generate material for further in vivo studies. To do so, improvements were implemented to enhance both the fermentation of the fungal culture and the isolation of this compound, with the target of generating > 800 mg of study materials in a period of 13 wk. Valuable insights, both regarding chemistry and mycology, were gained during the targeted production of 1 on the laboratory-scale. In addition, methods were modified to make the process more environmentally friendly by judicious choice of solvents, implementing procedures for solvent recycling, and minimizing the use of halogenated solvents.

Post-traumatic stress disorder (PTSD) is a debilitating psychopathological disease that is triggered by exposure to traumatic events. It is usually associated with substantial comorbidities, such as cognitive impairment, anxiety, and depression. Silymarin has been recently reported to exert neuroprotective activities against neurodegenerative diseases such as Alzheimerʼs and Parkinsonʼs diseases. Herein, the beneficial effects of silymarin in ameliorating PTSD-like symptoms such as memory impairments, anxiety, and depression were evaluated using a single-prolonged stress (SPS) rat model of PTSD. Male Wistar rats were randomly assigned into four groups: control, silymarin, SPS, or SPS + silymarin. Rats were administrated silymarin, 100 mg/kg i. p. for 4 wk. Rats in all groups were tested for short- and long-term memory in the radial arm water maze (RAWM), for anxiety-like behaviors using the open field test (OFT) and elevated plus maze (EPM) test, and for depression-like symptoms using the tail suspension test (TST). Conventional analyses of the RAWM, EPM, OFT, and TST were conducted using analysis of variance. Additionally, the anxiety-related behavior parameters of the EPM and OFT were entered to principal component analysis. Regression scores based on the first two extracted components, which accounted for 61% of the variance, were indicative of the anxiolytic activity of silymarin. Collectively, the results suggest that silymarin treatment prevents SPS-induced long-term memory impairments, anxiety, and depressive-like symptoms in rat models.

In research focused on the discovery of new chemical diversity from freshwater fungi, a peak library was built and evaluated against a prostate cancer cell line, E006AA-hT, which was derived from an African American, as this population is disproportionately affected by prostate cancer. The chemical study of the bioactive sample accessioned as G858 (Delitschia sp.) led to the isolation of eight new α-pyrone derivatives (1 – 7, and 11), as well as the new 3S*,4S*-7-ethyl-4,8-dihydroxy-3,6-dimethoxy-3,4-dihydronaphthalen-1(2H)-one (15). In addition, the known compounds 5-(3-S-hydroxybutyl)-4-methoxy-6-methyl-2H-pyran-2-one (8), 5-(3-oxobutyl)-4-methoxy-6-methyl-2H-pyran-2-one (9), pyrenocine I (10), 5-butyl-6-(hydroxymethyl)-4-methoxy-2H-pyran-2-one (12), sporidesmin A (13), 6-ethyl-2,7-dimethoxyjuglone (14), artrichitin (16), and lipopeptide 15G256ε (17) were also obtained. The structures of the new compounds were elucidated using a set of spectroscopic (NMR) and spectrometric (HRMS) methods. The absolute configuration of the most abundant member of each subclass of compounds was assigned through a modified Mosherʼs ester method. For 15, the relative configuration was assigned based on analysis of 3 J values. Compounds 1, 2, 5 – 14, 16, and 17 were evaluated against the cancer cell line E006AA-hT under hypoxic conditions, where compound 13 inhibited cell proliferation at a concentration of 2.5 µM.

In previously reported studies [1], our team performed the gram-scale isolation of four key flavonolignan diastereoisomers (silybin A, silybin B, isosilybin A, and isosilybin B) from milk thistle [Silybum marianum (L.) Gaertn. (Asteraceae)]. Presently, these materials are undergoing evaluation, both in vitro and in vivo, for their prostate cancer chemopreventive, antiproliferative, and pro-apoptotic properties [2-5]. In that earlier work, over 250 g of milk thistle extract [termed ‘silymarin’; see [6] for a review on the nomenclature of milk thistle components] were processed through several rounds of chromatography to yield 3 to 4 grams of each diastereoisomer [1]. Based on the distinctive UV profile of this class of flavonolignans, and retention times that were longer than the most retained compound, isosilybin B, in reverse phase HPLC, we hypothesized that some of the side fractions contained constituents of a similar structural type present in low yields. As isosilybin B and, to a lesser extent, isosilybin A seem to be the most potent in various prostate cancer chemopreventive and antiproliferative activity assays [2-5, 7] the evaluation of these new compounds in vitro could expand our knowledge of the structure-activity relationships of flavonolignans against this type of neoplasm. Compound 1 was isolated as a yellow film. The molecular formula, determined by HRESIMS, was C25H22O10, giving an index of hydrogen deficiency of 15. This is the same molecular formula of flavonolignan diastereoisomers from milk thistle [8, 9]. The 1D NMR data of 1 showed signals typical of a trans-3-hydroxyflavanone moiety with hydroxyl substitutions at positions C-5 and C-7 (Table 1). Additional aromatic proton and carbon signals, as well as two oxygenated methines, an oxymethylene, and a methoxy signal also indicated a flavonolignan structural type [8, 9]. However, the lignan-derived third aromatic ring in 1 displayed a 1,3,5-substitution pattern, as evidenced by coupling constants (J2″=J4″=J6″=2 Hz), positioning a hydroxyl group at C-5″ and a methoxy group at C-3″. Alternatively, the major flavonolignans, silybin A (3), silybin B (4), isosilybin A (5), and isosilybin B (6) (Figure 1), have a 1,3,4- substitution pattern. HMBC data revealed that the regiochemistry of 1 (Figure 2) was similar to that of compounds 5 and 6. Figure 1 Structures of flavonolignans from milk thistle. Figure 2 Key HMBC correlations in 1 Table 1 NMR data of compounds 1 and 2 in DMSO-d6 The spectroscopic and spectrometric data of 2 were nearly identical to those of 1. The major differences were discernable in the CD (Figure 3) and optical rotation data, where 1 and 2 had values of equal magnitude but opposite sign. The trivial names isosilybin C (2) and isosilybin D (1) were assigned to these new compounds, given their stereochemical similarities to isosilybin A (5) and isosilybin B (6), respectively. Figure 3 CD data of compounds 1 and 2; the CD data for compounds 3-6 has been published previously [8]. Compounds 1-6 were evaluated for antiproliferative/cytotoxic activity against a panel of three prostate cancer cell lines. Compounds 1 and 2 had decreased activity, relative to the well studied compounds 3-6 [2-4, 8]. This suggested that an ortho relationship for the hydroxyl and methoxy substituents (in 3-6) was more favorable than the meta relationship for the same substituents (in 1 and 2), although other structural features may also be important. The configuration that was most similar to isosilybin B (6), being compound 1, was more potent, as we predicted. Activity for the multicomponent mixture from which these compounds were isolated, silymarin, was also examined as a positive control. Medicinal chemistry studies are ongoing to probe the structure-activity relationships in the flavonolignan series further.

Extracts of milk thistle ( Silybum marianum, Asteraceae), termed "silymarin," are used worldwide, primarily for hepatoprotective applications and recently for prostate cancer chemoprevention. Silymarin is a mixture of at least eight compounds, and four major constituents are a group of structurally related flavonolignans: silybin A, silybin B, isosilybin A, and isosilybin B. The initiation of IN VIVO studies to compare the respective preclinical activities of each compound required that gram quantities of these diastereoisomers be prepared. Procedures were developed and optimized to produce multigram-scale quantities of each of these in > 97 % purity. A hybrid chromatographic/precipitative technique was developed, whereby mixtures were chromatographed at high concentrations so as to induce formation of a precipitate in the column fractions, yielding samples that were more enriched in the desired compounds than would be obtained solely by the chromatographic steps alone.

A phytochemical investigation of the CHCl (3)-soluble extract of the entire plant of Egletes viscosa (Asteraceae), collected in Peru, afforded two new labdane glycosides, 13-hydroxy-7-oxolabda-8,14-diene 13( R)- O-alpha- L-arabinopyranoside and 13-hydroxylabda-7,14-diene 13( R)- O-alpha- L-arabinopyranoside, along with four known compounds. The structures of the new compounds were elucidated by spectroscopic and chemical methods.

Cranberry juice is used routinely, especially among women and the elderly, to prevent and treat urinary tract infections. These individuals are likely to be taking medications concomitantly with cranberry juice, leading to concern about potential drug-dietary substance interactions, particularly in the intestine, which, along with the liver, is rich in expression of the prominent drug metabolizing enzyme, cytochrome P450 3A (CYP3A). Using a systematic in vitro-in vivo approach, a cranberry juice product was identified recently that elicited a pharmacokinetic interaction with the CYP3A probe substrate midazolam in 16 healthy volunteers. Relative to water, a cranberry juice inhibited intestinal first-pass midazolam metabolism. In vitro studies were initiated to identify potential enteric CYP3A inhibitors from cranberry via a bioactivity-directed fractionation approach involving dried whole cranberry [Vaccinium macrocarpon Ait. (Ericaceae)], midazolam, and human intestinal microsomes (HIM). Three triterpenes (maslinic acid, corosolic acid, and ursolic acid) were isolated. The inhibitory potency (IC50) of maslinic acid, corosolic acid, and ursolic acid was 7.4, 8.8, and

The medicinal plant goldenseal (Hydrastis canadensis) has been used historically to treat infection [1], and it is currently among the top ten best selling botanicals in the US. This plant has demonstrated activity in vitro against a number of bacteria, including multidrug-resistant Staphylococcus aureus (MRSA) [2]. Previous studies have implicated several alkaloids, including berberine, in the antibacterial activity of H. canadensis [2, 3]. Recent results from our laboratories, however, have led to the hypothesis that non-alkaloidal constituents also play an important role. An extract from the aerial portions of H. canadensis was separated into 12 fractions using flash chromatography. One of these fractions contained only very low (1000 x < MIC) concentrations of alkaloids, but it enhanced significantly the antibacterial activity of berberine. LC-MS analysis of all 12 of the fractions indicated three potential active compounds (thus far unidentified) that were present at the highest concentration in the active fraction. On the basis of experiments comparing antibacterial activity against wild type and efflux pump knockout mutants of S. aureus, the active constituents of the fraction appear to act as efflux pump inhibitors. Our studies indicate that goldenseal's mechanism of antibacterial activity may be more complex than previously reported, and suggest that H. canadensis could be useful for the treatment of drug resistant bacterial infections, particularly as a topical agent. In addition, we demonstrate the effectiveness of combining bioassay guided fractionation with comprehensive LC-MS analysis to identify potential potentiators/synergists in medicinal plants. Acknowledgements: Bill Burch, Bear Wallow Ginseng, Hendersonville, NC; Research Corporation Cottrell College Science Award CC6642; NSF-MRI Award 0420292. References:1. Upton, R., Goldenseal Root (2001) Hydrastis canadensis: Standards of analysis, quality control, and therapeutics. American Herbal Pharmacopoeia. Santa Cruz; 2. Scazzocchino, F. et. al. (2001) Planta Med. 67: 561–564 3. Gentry, E. J. et. al. (1998)J. Nat. Prod. 61:1187–1193.