David Y.-W. Lee

Identification of hepatoprotective flavonolignans from silymarin

Silymarin, also known as milk thistle extract, inhibits hepatitis C virus (HCV) infection and also displays antioxidant, anti-inflammatory, and immunomodulatory actions that contribute to its hepatoprotective effects. In the current study, we evaluated the hepatoprotective actions of the seven major flavonolignans and one flavonoid that comprise silymarin. Activities tested included inhibition of: HCV cell culture infection, NS5B polymerase activity, TNF-α-induced NF-κB transcription, virus-induced oxidative stress, and T-cell proliferation. All compounds were well tolerated by Huh7 human hepatoma cells up to 80 μM, except for isosilybin B, which was toxic to cells above 10 μM. Select compounds had stronger hepatoprotective functions than silymarin in all assays tested except in T cell proliferation. Pure compounds inhibited JFH-1 NS5B polymerase but only at concentrations above 300 μM. Silymarin suppressed TNF-α activation of NF-κB dependent transcription, which involved partial inhibition of IκB and RelA/p65 serine phosphorylation, and p50 and p65 nuclear translocation, without affecting binding of p50 and p65 to DNA. All compounds blocked JFH-1 virus-induced oxidative stress, including compounds that lacked antiviral activity. The most potent compounds across multiple assays were taxifolin, isosilybin A, silybin A, silybin B, and silibinin, a mixture of silybin A and silybin B. The data suggest that silymarin- and silymarin-derived compounds may influence HCV disease course in some patients. Studies where standardized silymarin is dosed to identify specific clinical endpoints are urgently needed.

Silymarin Inhibits In Vitro T-Cell Proliferation and Cytokine Production in Hepatitis C Virus Infection

BACKGROUND & AIMS Silymarin, an extract from the seeds of the milk thistle plant Silybum marianum, has been used for centuries for the treatment of chronic liver diseases. Despite common use by patients with hepatitis C in the United States, its clinical efficacy remains uncertain. The goal of this study was to determine whether silymarin has in vitro effects on immune function that might have implications for its potential effect on hepatitis C virus (HCV)-induced liver disease. METHODS Freshly isolated peripheral blood mononuclear cells (PBMC) and T cells from HCV-infected and uninfected subjects were tested in vitro for responses to nonspecific and antigenic stimulation in the presence and absence of a standardized preparation of silymarin (MK001). RESULTS Minimal MK001 toxicity on PBMC was found at concentrations between 5 and 40 microg/mL. MK001 dose dependently inhibited the proliferation and secretion of tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and interleukin (IL)-2 by PBMC stimulated with anti-CD3. In addition, MK001 inhibited proliferation by CD4(+) T cells to HCV, Candida, and tetanus protein antigens and by HLA-A2/HCV 1406-1415-specific CD8(+) T cells to allogeneic stimulation. MK001 inhibited T-cell TNF-alpha and IFN-gamma cytokine secretion to tetanus and Candida protein antigens. Finally, MK001 inhibited nuclear factor-kappaB transcriptional activation after T-cell receptor-mediated stimulation of Jurkat T cells, consistent with its ability to inhibit Jurkat T-cell proliferation and secretion of IL-2. CONCLUSIONS Silymarins ability to inhibit the proliferation and proinflammatory cytokine secretion of T cells, combined with its previously described antiviral effect, suggests a possible mechanism of action that could lead to clinical benefit during HCV infection.

The role of NR2B containing NMDA receptor in place preference conditioned with morphine and natural reinforcers in rats

It has been reported that N-methyl-D-aspartate (NMDA) receptor is implicated in drug addiction and antagonists of the NMDA receptor complex can inhibit the development and expression of conditioned place preference (CPP) induced by several addictive drugs, implying that this class of compounds might be considered as candidate for the treatment of substance abuse. To explore this possibility, it is important to evaluate whether the inhibitory effect of NMDA receptor antagonists would be confined to behaviors produced by drugs of abuse only, but not by natural reinforcers. According to the quantitative changes of NMDA receptor subunits, including NR1, NR2A, and NR2B, induced by diverse types of reinforcers, we chose NR2B subunit as the target of research. Experimental results showed that (1) an augmented expression of NR2B subunit was revealed by Western blotting in the nucleus accumbens (NAc) and the hippocampus in rats with CPP induced by morphine, but not by natural rewards such as food, novel environment and social interaction. (2) Ifenprodil, an antagonist highly selective for NR2B subunit of the NMDA receptor, produced a dose-dependent reduction in CPP induced by morphine and novel environment, but not that by food consumption and social interaction. Taking together, these findings suggested that NR2B containing NMDA receptor may be more involved with morphine reward rather than natural rewards, and that antagonism of NR2B may have a potential for the treatment of morphine abuse.

Recent advances in aryl C-glycoside synthesis.

Aryl C-glycosides are stable analogs of the corresponding O-glycosides. Because of their favorable pharmacological profiles attributed primarily to the C-glycosyl moiety, aryl C-glycosides have gained increasing popularity as drug candidates. In this review we focus on the synthesis of aryl C-glycosides including puerarin and kendomycin. This review is organized based on the type of chemistry used in the assembly of the C-glycosides with the following sub-sections: electrophilic reaction, cross-coupling reaction, free radical reaction, cyclization, intramolecular O-C rearrangement, umpolung, and miscellaneous reactions.

Discovery of cyclooxygenase inhibitors from medicinal plants used to treat inflammation

Eleven authenticated botanicals used in the traditional Chinese medicine Huo-Luo-Xiao-Ling Dan were screened for ligands to cyclooxygenase (COX) using pulsed ultrafiltration liquid chromatography-mass spectrometry, and a mass spectrometry-based enzyme assay was used to determine the concentration of each of 17 ligands that inhibited COX-1 or COX-2 by 50% (IC(50)). Acetyl-11-keto-beta-boswellic acid, beta-boswellic acid, acetyl-alpha-boswellic acid, acetyl-beta-boswellic acid, and betulinic acid were COX-1 selective inhibitors with IC(50) values of approximately 10 microM. Senkyunolide O and cryptotanshinone were COX-2 selective inhibitors with IC(50) values of 5 microM and 22 microM, respectively. Roburic acid and phenethyl-trans-ferulate inhibited COX-1 and COX-2 equally. COX inhibition and the IC(50) values of most of these natural product ligands have not been reported previously.

Extract of the Chinese herbal formula Huo Luo Xiao Ling Dan inhibited adjuvant arthritis in rats

ETHNOPHARMACOLOGICAL RELEVANCE The herbal formula Huo Luo Xiao Ling Dan (HLXL) and its modifications have been used in traditional Chinese medicine for about one hundred years to alleviate pain and inflammation. AIM To investigate the effects of HLXL on complete Freunds adjuvant (CFA)-induced multiple-joint arthritis in rats. MATERIALS AND METHODS Male Lewis rats, 190-210 g, were immunized subcutaneously at the base of the tail with 200 microl of heat-killed Mycobacterium tuberculosis in mineral oil (5 mg/ml). HLXL (2.30 and 4.60 g/kg) or vehicle control (n=8 per group) was administered orally (i.g.) once a day between days 16 and 25 post-CFA injection. The rats were observed for signs of arthritis with arthritic changes (erythema, edema, induration) being scored on a scale of 0-4 of increasing severity using a standard scoring system. The maximum arthritis score per rat was 16. A plethysmometer was used to measure edema volume in each paw. Adverse effects of HLXL were monitored by closely observing the animals for unusual behavioral changes. Levels of tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) in local tissue were measured by enzyme-linked immunosorbent assay on day 25 post-CFA. RESULTS HLXL significantly decreased arthritis scores between days 23-25 in the 2.30 g/kg group and 21-25 in the 4.60 g/kg group (p<0.05). It reduced paw edema on days 22 and 24 in the 2.30 g/kg group and on days 20, 22 and 24 in the 4.60 g/kg group compared to control (p<0.05). Local tissue TNF-alpha and IL-1beta levels on day 25 post-CFA injection were significantly (p<0.05) lower in rats treated with HLXL than in control rats. No observable adverse effects were found. CONCLUSION The data suggest that HLXL produces significant anti-arthritic effects that may be mediated by suppressing pro-inflammatory cytokines, and it appears to be safe.

Synthesis and in vitro pharmacological studies of new C(2) modified salvinorin A analogues.

Salvinorin A is the most potent naturally occurring opioid agonist yet discovered with high selectivity and affinity for kappa-opioid receptor. To explore its structure and activity relationships, a series of salvinorin A derivatives modified at the C2 position were prepared and studied. These salvinorin A derivatives were screened for binding and functional activities at the human kappa-opioid receptor. Compound 4, containing a methoxymethyl group at the 2-position, was a full kappa-agonist with an EC50 value at 0.6 nM, which is about 7 times more potent than salvinorin A.

Herbal Remedies for Alcoholism: Promises and Possible Pitfalls

This review summarizes the findings of the effects on alcohol intake in alcohol-preferring rats of extracts or purified compounds from two of the most promising herbs: kudzu (Pueraria lobata) and St. Johns Wort (Hypericum perforatum). It is a summary of a symposium presented at the 2002 RSA meeting in San Francisco. The meeting organizers/co-chairs were David Overstreet and Wing-Ming Keung. The presentations were (1) Introduction to the symposium, by David Y. W. Lee and David H. Overstreet; (2) Effects of daidzin on alcohol intake-search for mechanisms of action, by Wing-Ming Keung; (3) Long-term suppressive effects of puerarin on alcohol drinking in rats, by David Overstreet and David Y. W. Lee; (4) St. Johns Wort extract reduces alcohol intake in FH and P rats, by Amir Rezvani and David Overstreet; and (5) extracts reduce alcohol intake in Marchigian Sardinian alcohol-preferring rats, by Maurizio Massi.

Peripheral electrical stimulation reversed the cell size reduction and increased BDNF level in the ventral tegmental area in chronic morphine-treated rats

Chronic morphine administration induces functional and morphological alterations in the mesolimbic dopamine system (MLDS), which is believed to be the neurobiological substrate of opiate addiction. Our previous studies have demonstrated that peripheral electrical stimulation (PES) can suppress morphine withdrawal syndrome and morphine-induced conditioned place preference (CPP) in rats. The present study was designed to investigate if PES could reverse the cell size reduction induced by chronic morphine treatment in the ventral tegmental area (VTA), which is an important area of the MLDS. Immunohistochemical observations showed that the cell size of dopaminergic neurons in the VTA reduced significantly in the chronic morphine-treated rats with a concomitant decrease in the number of BDNF-positive cells compared to the saline-treated rats. A much milder morphological change, accompanying with an increased number of BDNF-positive cells, was observed in dopaminergic neurons in the rats that received repeated 100 Hz PES after morphine withdrawal. In another experiment, enzyme-linked immunosorbent assay (ELISA) reconfirmed a significant up-regulation of BDNF protein level in the VTA in the rats received 100 Hz PES after morphine abstinence. These results indicate that PES could facilitate the morphological recovery of the VTA dopaminergic cells damaged by chronic morphine treatment and up-regulate the BDNF protein level in the VTA. Activation of endogenous BDNF by PES may play a role in the recovery of the injured dopaminergic neurons in the morphine addictive rats.

2-Methoxymethyl-salvinorin B is a potent kappa opioid receptor agonist with longer lasting action in vivo than salvinorin A.

Salvinorin (Sal) A is a naturally occurring, selective κ opioid receptor (KOPR) agonist with a short duration of action in vivo. Pharmacological properties of a C(2) derivative, 2-methoxymethyl (MOM)-Sal B, were characterized. MOM-Sal B bound to KOPR with high selectivity and displayed ∼3-fold higher affinity than U50,488H [(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methanesulfonate] and Sal A. It acted as a full agonist at KOPR in guanosine 5′-O-(3-[35S]thio)triphosphate binding and was ∼5- and ∼7-fold more potent than U50,488H and Sal A, respectively. In Chinese hamster ovary cells stably expressing KOPR, all three κ agonists internalized or down-regulated KOPR to similar extents, with MOM-Sal B being the most potent. In mice, MOM-Sal B (0.05–1 mg/kg s.c.) caused immediate and dose-dependent immobility lasting ∼3 h, which was blocked by norbinaltorphimine. In contrast, ambulation in a Y-maze was increased when rats received MOM-Sal B (1–5 mg/kg s.c.). In addition, MOM-Sal B (0.5–5 mg/kg i.p.) produced antinociception (hot-plate test) and hypothermia in a dose-dependent manner in rats. MOM-Sal B was more potent than U50,488H in both tests and more efficacious than U50,488H in the hot-plate test. These latter two in vivo effects were blocked by norbinaltorphimine, indicating KOPR-mediated actions. Sal A at 10 mg/kg elicited neither antinociception nor hypothermia 30 min after administration to rats. In summary, MOM-Sal B is a potent and efficacious KOPR agonist with longer lasting in vivo effects than Sal A.