Zhongze Ma

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.

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.

Huperzine A regulates amyloid precursor protein processing via protein kinase C and mitogen-activated protein kinase pathways in neuroblastoma SK-N-SH cells over-expressing wild type human amyloid precursor protein 695

Alpha-secretase (alpha-secretase), cleaves the amyloid precursor protein (APP) within the amyloid-beta (Abeta) sequence, resulting in the release of a secreted fragment of APP (alphaAPPs) and precluding Abeta generation. We investigated the effects of the acetylcholinesterase inhibitor, huperzine A (Hup A), on APP processing and Abeta generation in human neuroblastoma SK-N-SH cells overexpressing wild-type human APP695. Hup A dose-dependently (0-10 microM) increased alphaAPPs release. Therefore, we evaluated two alpha-secretase candidates, a disintegrin and metalloprotease (ADAM) 10 and ADAM17 in Hup A-induced non-amyloidogenic APP metabolism. Hup A enhanced the level of ADAM10, and the inhibitor of tumor necrosis factor-alpha converting enzyme (TACE)/ADAM17 inhibited the Hup A-induced rise in alphaAPPs levels, further suggesting Hup A directed APP metabolism toward the non-amyloidogenic alpha-secretase pathway. Hup A had no effect on Abeta generation in this cell line. The steady-state levels of full-length APP and cell viability were unaffected by Hup A. Alpha-APPs release induced by Hup A treatment was significantly reduced by muscarinic acetylcholine receptor antagonists (particularly by an M1 antagonist), protein kinase C (PKC) inhibitors, GF109203X and calphostin C, and the mitogen-activated kinase kinase (MEK) inhibitors, U0126 and PD98059. Furthermore, Hup A markedly increased the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase, which was blocked by treatment with U0126 and PD98059. In addition, Hup A inhibited acetylcholinesterase activity by 20% in neuroblastoma cells. Our results indicate that the activation of muscarinic acetylcholine receptors, PKC and MAP kinase may be involved in Hup A-induced alphaAPPs secretion in neuroblastoma cells and suggest multiple pharmacological mechanisms of Hup A regarding the treatment of Alzheimers disease (AD).

Effects of huperzine A on amyloid precursor protein processing and β-amyloid generation in human embryonic kidney 293 APP swedish mutant cells

The amyloid precursor protein (APP) is cleaved enzymatically by nonamyloidogenic and amyloidogenic pathways. α‐Secretase (α‐secretase), cleaves APP within the β‐amyloid (Aβ) sequence, resulting in the release of a secreted fragment of APP (αAPPs) and precluding Aβ generation. In this study, we investigated the effects of an acetylcholinesterase inhibitor, huperzine A (Hup A), on APP processing and Aβ generation in human embryonic kidney 293 cells transfected with human APP bearing the Swedish mutation (HEK293 APPsw). Hup A dose dependently (0–10 μM) increased αAPPs release and membrane‐coupled APP CTF‐C83, suggesting increased APP metabolism toward the nonamyloidogenic α‐secretase pathway. The metalloprotease inhibitor TAPI‐2 inhibited the Hup A‐induced increase in αAPPs release, further suggesting a modulatory effect of Hup A on α‐secretase activity. The synthesis of full‐length APP and cell viability were unchanged after Hup A incubation, whereas the level of AβTotal was significantly decreased, suggesting an inhibitory effect of Hup A on Aβ production. Hup A‐induced αAPPs release was significantly reduced by the protein kinase C (PKC) inhibitors GF109203X and Calphostin C. These data, together with the finding that the PKCα level was enhanced prior to the increase of αAPPs secretion, indicate that PKC may be involved in Hup A‐induced αAPPs secretion by HEK293 APPsw cells. Our data suggest alternative pharmacological mechanisms of Hup A relevant to the treatment of Alzheimers disease.

The isoflavone puerarin reduces alcohol intake in heavy drinkers: A pilot study

BACKGROUND Isoflavone compounds naturally occurring in the root of the kudzu plant have been used historically to treat alcohol-related problems. A pilot study was conducted to assess the effects of one primary isoflavone--puerarin--for its ability to modify alcohol intake in humans. METHODS Ten (10) healthy adult volunteers were administered puerarin (1200 mg daily) in a double-blind, placebo-controlled, crossover design experiment for one week prior to an afternoon drinking session lasting 1.5h. Participants had access to up to six bottles of their preferred brand of beer in addition to juice and water. A time course of drinking, sip volumes, and total amount consumed were recorded. RESULTS Participants consumed on average 3.5 (±0.55) beers when treated with placebo and 2.4 (±0.41) beers when treated with puerarin. In contrast to drinking following placebo treatment when 3 participants drank 5 beers and 1 participant drank all 6 beers, none drank 5 or 6 beers when treated with puerarin. Drinking topography also changed. When treated with puerarin, participants decreased sip size, took more sips to finish a beer, and took longer to consume each beer. Additionally, after finishing a beer, latency to opening the next beer was increased. CONCLUSIONS This study is the first demonstration that a single isoflavone found in the kudzu root can alter alcohol drinking in humans. These results suggest that alcohol consumption patterns are influenced by puerarin administration and this botanical medication may be a useful adjunct in the treatment of excessive alcohol intake.

Anti-hyperalgesic and anti-inflammatory effects of the modified Chinese herbal formula Huo Luo Xiao Ling Dan (HLXL) in rats.

Chinese herbal medicine has been used for thousands of years in China and other Asian countries to treat a variety of inflammatory diseases. The classic Chinese herbal formula, Huo Luo Xiao Ling Dan (HLXL) is commonly used in traditional Chinese herbal medicine for the treatment of joint pain and other symptoms of arthritis. The present study is an investigation of the effects of a modified HLXL extract on persistent hyperalgesia and edema in rats with peripheral inflammation. Inflammation was induced by injecting complete Freunds adjuvant (CFA) into one hind paw. Four dosages of the extract were compared to a vehicle control. Each was administered intragastrally (i.g.) daily for seven days beginning one day before CFA. Hyperalgesia was assessed using a paw withdrawal latency (PWL) test and edema was determined by measuring paw thickness at pre-CFA and 2 hours, 24 hours, and 5 days post-CFA. Immunohistochemistry was performed 2 hours post-CFA to determine spinal Fos protein expression. Adverse effects of the extract were monitored by observing the animals closely for unusual behavioral changes. Compared to the control, HLXL at the two lower dosages (0.575 g/kg and 1.15 g/kg) were effective in the later stage (day 5) of inflammatory hyperalgesia and edema, while the two higher dosages (2.3 g/kg and 4.6 g/kg) alleviated early stage hind paw inflammation and hyperalgesia and facilitated recovery from paw edema and hyperalgesia during the late stage. HLXL at 2.30 g/kg significantly suppressed Fos expression in laminae I-II, III-IV and V-VI ipsilaterally and in III-IV contralaterally. No significant signs of toxicity or adverse effects were observed. The data suggest that HLXL dosage-dependently attenuates CFA-induced inflammation and hyperalgesia, at least in part by inhibiting noxious transmission at the dorsal horn of the spinal cord.

Suppression of ongoing experimental arthritis by a Chinese herbal formula (Huo-Luo-Xiao-Ling Dan) involves changes in antigen-induced immunological and biochemical mediators of inflammation

Rheumatoid arthritis (RA) is one of the major autoimmune diseases of global prevalence. The use of the anti-inflammatory drugs for the treatment of RA is associated with severe adverse reactions and toxicity. This limitation has necessitated the search for novel therapeutic products. We report here a traditional Chinese medicine-based herbal formula, Huo luo xiao ling dan (HLXL), which has potent antiarthritic activity as validated in the rat adjuvant-induced arthritis (AA) model. HLXL (2.3 g/Kg) was fed to Lewis (RT.11) rats daily by gavage beginning at the onset of arthritis and then continued through the observation period. HLXL inhibited the severity of ongoing AA. This suppression of arthritis was associated with significant alterations in the T cell proliferative and cytokine responses as well as the antibody response against the disease-related antigen, mycobacterial heat-shock protein 65 (Bhsp65). There was a reduction in the level of the proinflammatory cytokines IL-17 and IL-1β but enhancement of the anti-inflammatory cytokine IL-10 level. In addition, there was inhibition of both the anti-Bhsp65 antibody response and the serum level of nitric oxide. Thus, HLXL is a promising CAM modality for further testing in RA patients.

l-Isocorypalmine reduces behavioral sensitization and rewarding effects of cocaine in mice by acting on dopamine receptors

BACKGROUND We previously reported isolation of l-isocorypalmine (l-ICP), a mono-demethylated analog of l-tetrahydropalmatine (l-THP), from the plant Corydalis yanhusuo. Here we characterized its in vitro pharmacological properties and examined its effects on cocaine-induced behaviors in mice. METHODS Receptor binding, cAMP and [(35)S]GTPγS assays were used to examine pharmacological actions of l-ICP in vitro. Effects of l-ICP on cocaine-induced locomotor hyperactivity and sensitization and conditioned place preference (CPP) in mice were investigated. HPLC was employed to analyze metabolites of l-ICP in mouse serum. RESULTS Among more than 40 targets screened, l-ICP and l-THP bound only to dopamine (DA) receptors. l-ICP was a high-affinity partial agonist of D1 and D5 receptors and a moderate-affinity antagonist of D2, D3 and D4 receptors, whereas l-THP bound to only D1 and D5 receptors, with lower affinities than l-ICP. At 10mg/kg (i.p.), l-ICP inhibited spontaneous locomotor activity for a shorter time than l-THP. Pretreatment with l-ICP reduced cocaine-induced locomotor hyperactivities. Administration of l-ICP before cocaine once a day for 5 days reduced cocaine-induced locomotor sensitization on days 5 and 13 after 7 days of withdrawal. Pretreatment with l-ICP before cocaine daily for 6 days blocked cocaine-induced CPP, while l-ICP itself did not cause preference or aversion. HPLC analysis showed that l-ICP was the main compound in mouse serum following i.p. injection of l-ICP. CONCLUSIONS l-ICP likely acts as a D1 partial agonist and a D2 antagonist to produce its in vivo effects and may be a promising agent for treatment of cocaine addiction.

Synthesis and biological evaluation of C-12 triazole and oxadiazole analogs of salvinorin A

Salvinorin A (1), the main active ingredient of Salvia divinorum, is a potent and selective kappa-opioid receptor (KOPR) agonist. A series of C-12 triazole analogs and the oxadiazole (4) analog of 1 are synthesized and screened for binding affinity at kappa, mu (MOPR), or delta (DOPR). Surprisingly, all triazole analogs have shown negligible binding affinity at opioid receptors and the oxadiazole 4, a reported MOPR and KOPR antagonist, exhibits very low affinities to opioid receptors and no antagonism in our binding assays. These results suggest that electronic factors that may affect either the electron density of hydrogen bond acceptor at C-12 or hydrophobic interactions between C-12 moiety and KOPR are critical to C-12 analogs affinity for KOPR.