Effect of Extract and Synthesized Derivatives of Isolated Compound from Symplocos chinensis f. Pilosa Ohwi on Neuropathic Pain in Mice.

Neuropathic pain is described as the "most terrible of all tortures that a nerve wound may inflict." The aim of the present study was to demonstrate the antinociceptive effect of Symplocos chinensis f. pilosa Ohwi water extract (SCW) and synthesized derivatives of the isolated compound. The antinociceptive effect was tested using the acetic acid-induced writhing and 5% formalin tests. Antinociceptive effects on neuropathic pain were evaluated using the von Frey test with chronic constriction injury (CCI) and surgical nerve injury (SNI) models and tail-flick test with a vincristine-induced pain model. An Ames test was also conducted. 5-hydroxymethylfurfural (5-HMF) was isolated and derivatives were synthesized with various acid groups. Among the plant water extracts, SCW showed significantly effective activity. Additionally, SCW presented antinociceptive effects in the neuropathic pain models. The SCW water fraction resulted in fewer writhes than the other fractions, and isolated 5-HMF was identified as an effective compound. Because 5-HMF revealed a positive response in the Ames test, derivatives were synthesized. Among the synthesized derivations, 5-succinoxymethylfurfural (5-SMF) showed the best effect in the neuropathic pain model. Our data suggest that SCW and the synthesized compound, 5-SMF, possess effective antinociceptive activity against neuropathic pain.

Antinociceptive effect of chrysin in diabetic neuropathy and formalin-induced pain models.

Chrysin, a natural flavonoid, is the main ingredient of many medicinal plants, which shows potent pharmacological properties. In the present study, the antinociceptive effects of chrysin were examined in ICR mice. Chrysin orally administered at the doses of from 10 to 100 mg/kg exerted the reductions of formalin-induced pain behaviors observed during the second phase in the formalin test in a dose-dependent manner. In addition, the antinociceptive effect of chrysin was further characterized in streptozotocin-induced diabetic neuropathy model. Oral administration chrysin caused reversals of decreased pain threshold observed in diabetic-induced peripheral neuropathy model. Intraperitoneally (i.p.) pretreatment with naloxone (a classic opioid receptor antagonist), but not yohimbine (an antagonist of α2-adrenergic receptors) or methysergide (an antagonist of serotonergic receptors), effectively reversed chrysin-induced antinociceptive effect in the formalin test. Moreover, chrysin caused a reduction of formalin-induced up-regulated spinal p-CREB level, which was also reversed by i.t. pretreated naloxone. Finally, chrysin also suppressed the increase of the spinal p-CREB level induced by diabetic neuropathy. Our results suggest that chrysin shows an antinociceptive property in formalin-induced pain and diabetic neuropathy models. In addition, spinal opioid receptors and CREB protein appear to mediate chrysin-induced antinociception in the formalin-induced pain model.

Anti-Nociceptive Effect and Standardization from Mixture of Agrimonia pilosa Ledeb and Salvia miltiorrhiza Bunge Extracts.

Agrimonia pilosa Ledeb has been previously reported to produce an anti-nociceptive effect in ICR mice in both tail-flick and hot-plate tests. Studies have shown that Salvia miltiorrhiza Bunge, also renowned in traditional Chinese medicine, is an effective anti-inflammatory treatment. Among the extraction solvents investigated, a 50% ethanol (EtOH) extract of A. pilosa produced the highest anti-nociceptive effect in monosodium uric acid-induced gout pain models and the greatest yield. The 80% EtOH extract of S. miltiorrhiza moderately inhibited lipopolysaccharide-induced nitric oxide release from RAW 264.7 murine macrophages and exhibited outstanding yield. The mixture of optimized A. pilosa and S. miltiorrhiza extracts were evaluated for enhanced anti-nociceptive effects in gout arthritis treatment. To control extract quality, four marker compounds were determined using an HPLC-DAD method. A 1:1 mixture of A. pilosa 50 and S. miltiorrhiza 80% EtOH extracts of produced better results than when the extracts were administered individually.

Phytochemical Analysis of Agrimonia pilosa Ledeb, Its Antioxidant Activity and Aldose Reductase Inhibitory Potential.

The aim of this study was to determine aldose reductase (AR) inhibitory activity and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity of compounds from Agrimonia pilosa Ledeb (AP). We isolated agrimoniin (AM), four flavonoid glucosides and two flavonoid glucuronides from the n-butanol fraction of AP 50% methanol extract. In addition to isolated compounds, the AR-inhibitory activity and the DPPH free radical scavenging activity of catechin, 5-flavonoids, and 4-flavonoid glucosides (known components of AP) against rat lens AR (RLAR) and DPPH assay were measured. AM showed IC50 values of 1.6 and 13.0 µM against RLAR and DPPH scavenging activity, respectively. Additionally, AM, luteolin-7-O-glucuronide (LGN), quercitrin (QU), luteolin (LT) and afzelin (AZ) showed high inhibitory activity against AR and were first observed to decrease sorbitol accumulation in the rat lens under high-sorbitol conditions ex vivo with inhibitory values of 47.6%, 91.8%, 76.9%, 91.8% and 93.2%, respectively. Inhibition of recombinant human AR by AM, LGN and AZ exhibited a noncompetitive inhibition pattern. Based on our results, AP and its constituents may play partial roles in RLAR and oxidative radical inhibition. Our results suggest that AM, LGN, QU, LT and AZ may potentially be used as natural drugs for treating diabetic complications.

Aldose reductase inhibitory compounds from Xanthium strumarium.

As part of our ongoing search for natural sources of therapeutic and preventive agents for diabetic complications, we evaluated the inhibitory effects of components of the fruit of Xanthium strumarium (X. strumarium) on aldose reductase (AR) and galactitol formation in rat lenses with high levels of glucose. To identify the bioactive components of X. strumarium, 7 caffeoylquinic acids and 3 phenolic compounds were isolated and their chemical structures were elucidated on the basis of spectroscopic evidence and comparison with published data. The abilities of 10 X. strumarium-derived components to counteract diabetic complications were investigated by means of inhibitory assays with rat lens AR (rAR) and recombinant human AR (rhAR). From the 10 isolated compounds, methyl-3,5-di-O-caffeoylquinate showed the most potent inhibition, with IC50 values of 0.30 and 0.67 µM for rAR and rhAR, respectively. In the kinetic analyses using Lineweaver-Burk plots of 1/velocity and 1/substrate, methyl-3,5-di-O-caffeoylquinate showed competitive inhibition of rhAR. Furthermore, methyl-3,5-di-O-caffeoylquinate inhibited galactitol formation in the rat lens and in erythrocytes incubated with a high concentration of glucose, indicating that this compound may be effective in preventing diabetic complications.

Anti-inflammatory effect of visnagin in lipopolysaccharide-stimulated BV-2 microglial cells.

Visnagin, which is found in Ammi visnaga, has biological activity as a vasodilator and reduces blood pressure by inhibiting calcium influx into the cell. The present study demonstrates the anti-inflammatory effect of visnagin on lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. When cells were treated with visnagin prior to LPS stimulation, production of nitric oxide and expression of iNOS were attenuated in a dose-dependent manner. Visnagin also caused a significant decrease of mRNA expression and release of TNF-α, IL-1ß and IFNγ. In addition, visnagin reduced LPS-induced IL-6 and MCP-1 mRNA level. We further found that visnagin dose-dependently inhibited LPS-induced AP-1 and NF-κB luciferase activities. Taken together, our results for the first time suggest that the anti-inflammatory effect of visnagin might result from the inhibition of transcription factors, such as AP-1 and NF-κB.

Possible involvement of the hypothalamic pro-opiomelanocortin gene and beta-endorphin expression on acute morphine withdrawal development.

We studied the effects of supraspinally administered morphine on the expression of the hypothalamic pro-opiomelanocortin (POMC) gene and beta-endorphin. Mice were administered morphine intracerebroventricularly (i.c.v.) either once or 5 times for 5 days (once/day). A single morphine administration significantly increased the hypothalamic POMC gene and beta-endorphin expression at 2h after application in dose-dependent fashion; however, repeated morphine administration had no effect on the hypothalamic POMC gene and beta-endorphin expression. In the immunoblot and immunohistochemical study, the increase of beta-endorphin was observed in the arcuate nucleus of the hypothalamus. Moreover, the expressions of c-Fos, phosphorylated calcium/calmodulin-dependent protein kinase-IIalpha (pCaMK-IIalpha), and phosphorylated cAMP response element-binding protein (pCREB) were increased by a single i.c.v. morphine injection at various time points, but the expressions of phosphorylated extracellular signal-regulated protein kinase1/2 (pERK1/2) and phosphorylated IkappaB (pIkappaB) were not. We also found that the expressions of c-Fos, pCaMKIIalpha, and pCREB were co-localized with the POMC expression. Meanwhile, naloxone as well as muscimol and baclofen significantly attenuated the increases of the POMC gene expression induced by a single morphine administration. Furthermore, the pretreatment of muscimol and baclofen 10 min before morphine injection robustly attenuated the withdrawal behavior induced by a single morphine administration. These results imply that the hypothalamic POMC gene and beta-endorphin expression may play an important role in the development of an acute physical dependency of morphine. In that, GABAergic neurotransmission appear to be involved in the regulation of the hypothalamic POMC gene expression induced by supraspinal morphine administration.

The analgesic effect of decursinol.

Although decursinol, which is one of the coumarins purified from the dried roots of Angelica gigas Nakai, was previously demonstrated to have antinociceptive effects on various mouse pain models such as tail-flick, hot-plate, formalin, writhing, and several cytokine-induced pain tests, the possible involvement of its analgesic effects and non-steroidal anti-inflammatory drugs (NSAIDs) has not been clearly elucidated yet. In this study, we characterized the possible interaction between decursinol and aspirin or acetaminophen in the writhing test. The antinociceptive effects of decursinol were observed at an orally-administered dose of 50 mg/kg but not at 25 or 10 mg/kg. In addition, the analgesic effects of aspirin (ASA) and acetaminophen (APAP) were shown at an orally-administered dose of 200 mg/kg but not at 50 or 100 mg/kg. We examined the effects of decursinol on the ASA or APAP at sub-analgesic doses. Although the co-administration of decursinol and ASA did not show any differences at doses of 10 or 25 mg/kg and 50 or 100 mg/kg, respectively, synergistic effects between decursinol and APAP were observed in the group of decursinol (25 mg/kg) and APAP (100 mg/kg) co-administration. These results indicated that the analgesic effect of decursinol might be involved in supraspinal cyclooxygenase regulation that might be overlapped with APAP-induced analgesic mechanisms rather than systemic or peripheral prostaglandin modulation.

Formalin pretreatment attenuates tail-flick inhibition induced by beta-endorphin administered intracerebroventricularly or intrathecally in mice.

We examined the effect of the subcutaneous (s.c.) pretreatment of formalin into both hind paws of mice on the antinociception induced by the intracerebroventricularly (i.c.v.) or intrathecally (i.t.) administration of beta-endorphin using the tail-flick test. Pretreatment with formalin (5%) for 5 h had no affect on the i.c.v. administered beta-endorphin-induced tail-flick response. However, pretreatment with formalin for 40 h attenuated the tail-flick inhibition induced by i.c.v. administered beta-endorphin. This antinociceptive tolerance to i.c.v. beta-endorphin continued up to 1 week, but to a lesser extent. Pretreatment with formalin for 5 and 40 h significantly reduced the i.t. beta-endorphin-induced inhibition of the tail-flick response, which continued up to 1 week. The s.c. formalin treatment increased the hypothalamic pro-opiomelanocortin (POMC) mRNA level at 2 h, but this returned to the basal level after 40 h. Our results suggest that the increase in the POMC mRNA level in the hypothalamus appears to be involved in the supraspinal or spinal beta-endorphin-induced antinociceptive tolerance in formalin-induced inflammatory pain.

Effect of Zen Meditation on serum nitric oxide activity and lipid peroxidation.

This study was designed to investigate the effect of Zen Meditation on serum nitric oxide activity (NO) and oxidative stress (lipid peroxidation). The experimental group included 20 subjects who had practiced the Zen Meditation program in Meditation Center located in Seoul, South Korea. The control group included 20 subjects who did not practice any formal stress management technique and were age and sex matched with experimental group. To provide an assessment of nitric oxide production, the serum level of nitrate/nitrite was determined using the Griess reagent. Malondialdehyde (MDA) concentration was measured as a convenient index of lipid peroxidation by thiobarbituric acid (TBA) method. Meditation group showed a significant higher level of serum nitrate+nitrite concentration and a significant reduced level of serum malondialdehyde (MDA) than control group. A comprehensive randomized controlled trial should be performed to prove the causal relationship between meditation and level of nitric oxide or oxidative stress in reducing cardiovascular risk factors.

Antinociceptive profiles of platycodin D in the mouse.

Platycodin D (PD), one of several triterpene saponins, was isolated from roots of Platycodon grandiflorum. We previously reported that intracerebroventricular (i.c.v.) administration of PD showed an antinociceptive effect as measured by the tail-flick assay. However, its exact role in the regulation of antinociception in the various types of pain models has not yet been characterized. Thus, we attempted to find antinociceptive profiles of PD in various pain models. PD administered intraperitoneally (i.p.), i.c.v. or intrathecally (i.t.) showed antinociceptive effects in dose-dependent manners as measured by the tail-flick, writhing and formalin tests. In the tail-flick test, PD at the low doses reached the peak after 15 minutes and returned to the control level after 60 minutes. However, higher doses of PD showed a strong antinociception at least for 1 hour. PD administered i.t. showed stronger antinociception than that induced by i.c.v. administration PD in both tail-flick and writhing tests. In the formalin test, PD administered i.p., i.c.v. or i.t. showed antinociceptive effects during both the first (direct nociceptive stimulation) and second (late inflammatory) phases. Pretreatment with naltrexone i.p., i.c.v. or i.t. did not affect PD-induced inhibition of the tail-flick response. Our results suggest that PD shows a strong antinociceptive effect on the tail-flick, writhing and formalin tests, acting on central nervous system. However, PD-induced antinociception may not be mediated by the opioid receptors.

The inhibitory effect of ginseng saponins on the stress-induced plasma interleukin-6 level in mice.

The effect of ginseng saponins on plasma interleukin-6 (IL-6) in non-stressed and immobilization-stressed mice were investigated. Ginseng total saponins, ginsenosides Rb2, Rg1 and Rd administered intraperitoneally attenuated the immobilization stress-induced increase in plasma IL-6 level. But, intracerebroventricular injection of each ginsenoside did not affect plasma IL-6 level induced by immobilization stress. Ginsenosides Rb2, Rd and Rg1 significantly decreased norepinephrine and/or epinephrine-induced increase of IL-6 level in macrophage cell line (RAW 264.7). Thus, it can be suggested that the inhibitory action of ginseng saponins against the immobilization stress-induced increase of plasma IL-6 level would be in periphery; at least in part, mediated by blocking norepinephrine- and/or epinephrine-induced increase of IL-6 level in macrophage rather than in the brain. Ginseng saponins might be proposed as a possible candidate in the research or therapeutic modulation of stress-related disorders.

Antinociceptive profiles of crude extract from roots of Angelica gigas NAKAI in various pain models.

To characterize the antinociceptive profiles of Angelica gigas NAKAI (ANG; Korean angelica), methanol extract from the dried roots of ANG was made and mice were administered orally at the various doses (from 0.25 to 3 g/kg). ANG produced the increased latencies of the tail-flick and hot-plate paw-licking responses in a dose-dependent manner. In acetic acid-induced writhing test, ANG dose-dependently decreased writhing numbers. Moreover, the cumulative response time of nociceptive behaviors induced by intraplantar formalin injection was reduced during both the 1st and the 2nd phases in a dose-dependent manner in ANG-treated mice. Furthermore, oral administration of ANG did not cause licking, scratching and biting responses induced by TNF-alpha (100 pg), IFN-gamma (100 pg) or IL-1beta (100 pg) injected intrathecally (i.t.), especially at higher dose (3 g/kg). Additionally, in ANG treated mice, the cumulative nociceptive response time for i.t. administration of substance P or capsaicin was dose-dependently diminished. Finally, nociceptive responses elicited by i.t. injection of glutamate (20 microg), N-methyl-D-aspartic acid (60 ng), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (13 ng) or kainic acid (12 ng) were decreased by oral administration of ANG. Our results suggest that ANG produces antinociception via acting on the central nervous system and shows antinociceptive profiles in various pain models, especially inflammatory pain.

Inhibition of intracerebroventricular injection stress-induced plasma corticosterone levels by intracerebroventricularly administered compound K, a ginseng saponin metabolite, in mice.

Effects of major intestinal metabolites of ginsenosides, including compound K (IH-901, 20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol), compound Y (IH-902, 20-O-[alpha-L-arabinopyranosyl (1-->6)-beta-D-glucopyranosyl]-20(S)-protopanaxadiol), and ginsenoside Mc (IH-903, 20-O-[alpha-L-arabinofuranosyl (1-->6)-beta-D-glucopyranosyl]-20(S)-protopanaxadiol), on acute stress-induced plasma corticosterone levels were studied in mice. Intracerebroventricularly (i.c.v.) administered compound K (1 microg) attenuated the i.c.v. injection stress-induced increase in plasma corticosterone level, and this inhibitory effect was not affected by co-administered N(G)-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor. Compound K administered intraperitoneally affected neither the i.c.v. injection stress- nor the immobilization stress-induced increase in plasma corticosterone levels. Compound K and ginsenoside Mc did not affect plasma corticosterone levels induced by the two stress modalities used in this study.

Effect of ginsenoside Rd on nitric oxide system induced by lipopolysaccharide plus TNF-alpha in C6 rat glioma cells.

Effects of ginsenosides on nitric oxide (NO) production induced by lipopolysaccharide plus TNF-alpha (LNT) were examined in C6 rat glioma cells. Among several ginsenosides, ginsenoside Rd showed a complete inhibition against LNT-induced NO production. Ginsenoside Rd attenuated LNT-induced increased phosphorylation of ERK. Among several immediate early gene products, only Jun B and Fra-1 protein levels were increased by LNT, and ginsenoside Rd attenuated Jun B and Fra-1 protein levels induced by LNT. Furthermore, LNT increased AP-1 DNA binding activities, which were partially inhibited by ginsenoside Rd. Our results suggest that ginsenoside Rd exerts an inhibitory action against NO production via blocking phosphorylation of ERK, in turn, suppressing immediate early gene products such as Jun B and Fra-1 in C6 glioma cells.

Effects of ginseng saponin administered intraperitoneally on the hypothalamo-pituitary-adrenal axis in mice.

Intraperitoneal injection of ginseng total saponin (GTS; 5 and 20 mg/kg) raised plasma corticosterone levels in mice. However, interestingly, pretreatment of animals with the same doses of GTS (5 and 20 mg/kg) significantly attenuated the immobilization stress-induced increase in plasma corticosterone levels. Of the ginsenosides Rb(1), Rb(2), Rc, Rd, Re, Rf, Rg(1), 20(S)-Rg(3), and 20(R)-Rg(3) injected intraperitoneally at doses of 0.1-2 mg/kg, Rc (2 mg/kg) significantly inhibited the immobilization stress-induced increase in plasma corticosterone levels. GTS and Rc administered intraperitoneally did not affect the immobilization stress-induced elevation of plasma adrenocorticotropic hormone (ACTH) level. Pretreatment with GTS and Rc significantly attenuated the increase in plasma corticosterone levels induced by intraperitoneal injection of ACTH (30 microg/kg). These results suggest that GTS and Rc inhibit the immobilization stress-induced increase in plasma corticosterone levels by blocking ACTH action in the adrenal gland. Ginseng may be proposed to be useful for treatment of stress related disorders.

Effects of ginsenoside Rd and decursinol on the neurotoxic responses induced by kainic acid in mice.

In the present study, we examined the effects of ginsenoside Rd (G-Rd) and decursinol (DC) on various neurotoxic responses induced by kainic acid (KA) administered intracerebroventricularly ( i. c. v.) in ICR mice. Ginseng total saponin (GTS) inhibited the KA (0.5 microg)-induced lethal toxicity in a dose-dependent manner. Furthermore, G-Rd, a component of GTS, also attenuated the KA-induced lethal toxicity as well as DC pretreated orally for 30 min. In ICR mouse, neurotoxic damage induced by KA (0.1 microg) in the hippocampus was markedly concentrated in the CA3 pyramidal neurons. G-Rd and DC did not affect the pyramidal cell death in CA3 hippocampal region. In an immunohistochemical study, KA dramatically increased phospho-ERK and decreased phospho-CREB in the hippocampal area. G-Rd and DC attenuated, in part, the increased phospho-ERK and the decreased phospho-CREB protein levels. However, DC potentiated the increased c-Fos and c-Jun protein levels in the hippocampus induced by KA. Thus, our results suggest that the phosphorylation of ERK or the dephosphorylation of CREB protein may play a major role in the regulation of lethal toxicity induced by KA, whereas cell death in the hippocampal CA3 region induced by KA administered i. c. v. may not be directly mediated by ERK phosphorylation and CREB phosphorylation in the mouse.

Antinociceptive effects of ginsenosides injected intracerebroventricularly or intrathecally in substance P-induced pain model.

We have examined the effects of several ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1 and Rg3) administered intracerebroventricularly (i.c.v.) or intrathecally (i.t.) on the nociceptive behavior induced by substance P (0.7 microg) injected i.t. Among the several ginsenosides studied, Rb2, Rc, Rd, and Re, but not Rb1, Rf, Rg1 and Rg3, treated i.c.v. (50 microg) attenuated the nociceptive behavior induced by substance P injected i.t. On the other hand, we found that i.t. treatment with 50 microg of Rb1, Rb2, Rd, or Rf effectively attenuated the nociceptive behavior induced by i.t. injected substance P. However, the i.t. treatment with the same doses of Rc, Re, Rg1 or Rg3 was not effective for antagonizing i.t. injected substance P-induced nociceptive behavior. Our results show that ginsenosides Rb2, Rc, Rd, or R2 injected supraspinally exert a antinociceptive effect in the substance P-induced pain model. Furthermore, Rb1, Rb2, Rd, or Rf treated spinally produce antinociception in the substance P-induced pain model.

Antinociceptive mechanisms of platycodin D administered intracerebroventricularly in the mouse.

Platycodin D administered intracerebroventricularly (i.c.v.) showed an antinociceptive effect in a dose-dependent manner as measured by the tail-flick assay. The antinociception induced by platycodin D was maintained at least 1 h. MK-801 [(+/-)-5-methyl-10,11-dihydro-5 H-dibenzo[ a,d]cyclohepten-5,10-imine maleate], a competitive N-methyl- D-aspartic acid (NMDA) receptor antagonist, or CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), a non-NMDA receptor antagonist, muscimol (a GABA(A) receptor agonist), or baclofen (a GABA(B) receptor antagonist), or sulfated cholecystokinin (CCK-8 s; CCK A receptor agonist), injected i.c.v. significantly reduced the inhibition of the tail-flick response induced by platycodin D administered i.c.v. Additionally, intrathecal (i.t.) pretreatment with yohimbine (an alpha 2 -adrenergic receptor antagonist) or methysergide (a serotonin receptor antagonist) dose-dependently attenuated inhibition of the tail-flick response induced by i.c.v. administered platycodin D. However, naloxone (an opioid receptor antagonist) did not affect the inhibition of the tail-flick response induced by platycodin D. Our results suggest that platycodin D has an antinociceptive effect when it is administered supraspinally, and supraspinal GABA(A), GABA(B), NMDA and non-NMDA receptors are involved in platycodin D-induced antinociception. Furthermore, platycodin D administered supraspinally produces antinociception by stimulating descending noradrenergic and serotonergic, but not opioidergic, pathways.