"Quantity-effect" research strategy for comparison of antioxidant activity and quality of Rehmanniae Radix and Rehmannia Radix Praeparata by on-line HPLC-UV-ABTS assay.

BACKGROUND: Quantitation analysis and chromatographic fingerprint of multi-components are frequently used to evaluate quality of herbal medicines but fail to reveal activity of the components. It is necessary to develop a rational approach of chromatography coupled with activity detection for quality assessment of herbal medicines. METHODS: An on-line HPLC-ultraviolet detection-2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) free radical scavenging (HPLC-UV-ABTS) method was developed to obtain the chromatographic fingerprints and ABTS+• inhibition profiles (active fingerprints) of Rehmanniae Radix (Dihuang) and Rehmannia Radix Praeparata (Shu Dihuang). Eighteen compounds showing ABTS+• inhibition activity were identified by HPLC-fourier-transform mass spectrometry (HPLC-FTMS). Verbascoside was used as a positive control to evaluate the total activities of the samples and the contribution rate of each compound. The similarities of the chromatographic and active fingerprints were estimated by the vectorial angle cosine method. RESULTS: The results showed that the HPLC-UV-ABTS method could efficiently detect antioxidant activity of the herbal medicine samples. The antioxidants were different between the two herbs and several new antioxidants were identified in Shu Dihuang. A function equation was generated in terms of the negative peak area (x) and the concentrations of verbascoside (y, µg/mL), y = 2E-07 × 4 - 8E-05 × 3 + 0.0079 × 2 + 0.5755x + 1.4754, R2 = 1. Iridoid glycosides were identified as main antioxidants and showed their higher contributions to the total activity of the samples. The total contributions of the three main active components in the Dihuang and Shu Dihuang samples to the total activity, such as echinacoside, verbascoside and an unknown compound, were 39.2-58.1% and 55.9-69.4%, respectively. The potencies of the main active components in the Shu Dihuang samples were two to ten times those in the Dihuang samples. Similarity values for S12 in the chromatographic fingerprints and S03, S12 and P03 in the active fingerprints were less than 0.9. The three batches of samples might show their different quality with the other samples. CONCLUSIONS: The results suggested that the combination of "quantity-effect" research strategy and the HPLC-UV-ABTS analysis method could comprehensively evaluate the active components and quality of Dihuang and Shu Dhuang.

Phosphorylation of CaMKII in the rat dorsal raphe nucleus plays an important role in sleep-wake regulation.

The Ca(2+) modulation in the dorsal raphe nucleus (DRN) plays an important role in sleep-wake regulation. Calmodulin-dependent kinase II (CaMKII) is an important signal-transducing molecule that is activated by Ca(2+) . This study investigated the effects of intracellular Ca(2+) /CaMKII signaling in the DRN on sleep-wake states in rats. Maximum and minimum CaMKII phosphorylation was detected at Zeitgeber time 21 (ZT 21; wakefulness state) and ZT 3 (sleep state), respectively, across the light-dark rhythm in the DRN in rats. Six-hour sleep deprivation significantly reduced CaMKII phosphorylation in the DRN. Microinjection of the CAMKII activation inhibitor KN-93 (5 or 10 nmol) into the DRN suppressed wakefulness and enhanced rapid-eye-movement sleep (REMS) and non-REM sleep (NREMS). Application of a high dose of KN-93 (10 nmol) increased slow-wave sleep (SWS) time, SWS bouts, the mean duration of SWS, the percentage of SWS relative to total sleep, and delta power density during NREMS. Microinjection of CaCl2 (50 nmol) in the DRN increased CaMKII phosphorylation and decreased NREMS, SWS, and REMS. KN-93 abolished the inhibitory effects of CaCl2 on NREMS, SWS, and REMS. These data indicate a novel wake-promoting and sleep-suppressing role for the Ca(2+) /CaMKII signaling pathway in DRN neurons. We propose that the intracellular Ca(2+) /CaMKII signaling in the dorsal raphe nucleus (DRN) plays wake-promoting and sleep-suppressing role in rats. Intra-DRN application of KN-93 (CaMKII activation inhibitor) suppressed wakefulness and enhanced rapid-eye-movement sleep (REMS) and non-REMS (NREMS). Intra-DRN application of CaCl2 attenuated REMS and NREMS. We think these findings should provide a novel cellular and molecular mechanism of sleep-wake regulation.

Glucocorticoid receptors in the locus coeruleus mediate sleep disorders caused by repeated corticosterone treatment.

Stress induced constant increase of cortisol level may lead to sleep disorder, but the mechanism remains unclear. Here we described a novel model to investigate stress mimicked sleep disorders induced by repetitive administration of corticosterone (CORT). After 7 days treatment of CORT, rats showed significant sleep disturbance, meanwhile, the glucocorticoid receptor (GR) level was notably lowered in locus coeruleus (LC). We further discovered the activation of noradrenergic neuron in LC, the suppression of GABAergic neuron in ventrolateral preoptic area (VLPO), the remarkable elevation of norepinephrine in LC, VLPO and hypothalamus, as well as increase of tyrosine hydroxylase in LC and decrease of glutamic acid decarboxylase in VLPO after CORT treatment. Microinjection of GR antagonist RU486 into LC reversed the CORT-induced sleep changes. These results suggest that GR in LC may play a key role in stress-related sleep disorders and support the hypothesis that repeated CORT treatment may decrease GR levels and induce the activation of noradrenergic neurons in LC, consequently inhibit GABAergic neurons in VLPO and result in sleep disorders. Our findings provide novel insights into the effect of stress-inducing agent CORT on sleep and GRs' role in sleep regulation.

WS0701: a novel sedative-hypnotic agent acting on the adenosine system.

To characterize the sedative and hypnotic profile of the novel adenosine derivative ((3S,4R,5R)-3,4-dihydroxy-5-(6-((4-hydroxy-3-methoxybenzyl)amino)-9H-purin-9-yl)tetrahydrofuran-2-yl) methyl diaconate (WS0701), we performed a variety of behavioural tests and investigated the influence of WS0701 on various sleep stages. In mice, WS0701 significantly increased the number of entries and time spent in open arms in the elevated plus maze test, indicating an anxiolytic effect. WS0701 decreased locomotor activity counts and head dips in the hole-board test and enhanced sodium pentobarbital-induced hypnosis. However, WS0701 did not induce the loss of the righting reflex or amnesic effects in behavioural models. In rats, WS0701 exerted a sedative effect and markedly prolonged the time spent in non-rapid-eye-movement sleep, especially slow-wave sleep, but reduced the time spent in rapid-eye-movement sleep (REMS). Pretreatment with the selective adenosine A2a receptor antagonist SCH58261 attenuated the sedative and hypnotic effects of WS0701. WS0701 did not protect mice against picrotoxin-induced seizures, but inhibited adenosine deaminase activity and increased adenosine levels in the frontal cortex and hypothalamus of mice. In conclusion, WS0701 shows anxiolytic, sedative as well as sleep stage alterative effects, which may be related to the adenosine system.

Tetrandrine, an antihypertensive alkaloid, improves the sleep state of spontaneously hypertensive rats (SHRs).

ETHNOPHARMACOLOGICAL RELEVANCE: Radix of Stephania tetrandrae S. Moore has been used since antiquity in China as an antirheumatic, antihypertension, analgesic and antipyretic agent. Tetrandrine is the major component of Stephania tetrandrae. This study aims to evaluate the antihypertensive and hypnotic effect of tetrandrine on spontaneously hypertensive rats (SHR) and the possible mechanisms. MATERIALS AND METHODS: Electroencephalography (EEG) and electromyography (EMG) were recorded in freely moving rats and the sleep parameters were analyzed with SleepSign software. The levels of serotonin (5-HT), norepinephrine (NE), dopamine (DA) and their metabolites were examined to investigate the underlying mechanisms by using HPLC-ECD. Blood pressure was measured by noninvasive blood pressure tail cuff test. RESULTS: Tetrandrine (100mg/kg, i.g.) significantly suppressed blood pressure of SHR rats day by day during three days treatment. Meanwhile, tetrandrine remarkably improved the sleep efficiency by increasing total sleep time, rapid eye movement (REM) sleep and non-REM (NREM) sleep (including deep sleep and light sleep) time from the first day. Three days treatment of tetrandrine induced 5-HT concentration decrease in DRN, 5-HIAA concentration increase in LC and 5-HIAA/5-HT ratio increase in VTA and LC. In contrast, no changes in NE and DA concentrations in the DRN, VTA and LC occurred in SHR after tetrandrine treatment. These results indicate that modulation of 5-HT, its metabolite 5-HIAA and the 5-HIAA/5-HT ratio in DRN, VTA and LC are likely the mechanism of antihypertensive and hypnotic effects of tetrandrine at least in part. CONCLUSION: This is the first observation that tetrandrine possesses both anti-hypertension and hypnotic effects in SHR and suggested that tetrandrine may be useful for the treatment of hypertension patients who accompanied with short sleep time and poor sleep efficiency.

In vitro antioxidant and in vivo anti-inflammatory activities of Ophioglossum thermale.

Antioxidant fractions from Ophioglossum thermale were extracted with five different polar solvents using a Soxhlet type extractor. The total phenolic content of the extracts was determined by the Folin-Ciocalteu method. The ethyl acetate fraction of O. thermale was found to contain maximum phenolics. The dried fractions were screened for their antioxidant activity potential using in vitro model systems such as 1,1-diphenyl-2-picryl hydrazyl (DPPH), nitroblue tetrazolium (NBT) and lipid-peroxidation reduction at different concentrations. Results revealed that the EtOAc fraction exhibited the best performance in the DPPH assay, NBT assay and lipid peroxidation. All fractions showed more potent antioxidant capacity than green tea extract, a well-known antioxidant. Furthermore, the EtOAc fraction has the highest total phenolic content (475.65 mg of EGCG/g). In addition, the EtOAc fraction at 0.005% and 0.01% (g/100 ml) also significantly inhibited UVB irradiation-induced ROS generation in human dermal fibroblasts (HDFs). In a carrageenan-induced edema model, the EtOAc fraction showed an inhibitory effect (21.5%, p < 0.05) at 200 mg/kg (p.o.) after 300 min administration. Consequently, 3-O-methylquercetin (3MQ) was also isolated from the antioxidative EtOAc fraction. The data obtained using the above in vitro and in vivo tests suggest that the antioxidant activity of O. thermale and its anti-inflammatory effect on carrageenan-induced acute inflammation can be attributed to its ameliorating effect on oxidative damage, and thus it has great potential as a source for natural health products. To the best of our knowledge, this is the first report on the antioxidant activity of different polar extracts from O. thermale.

Augmentative effect of spinosin on pentobarbital-induced loss of righting reflex in mice associated with presynaptic 5-HT1A receptor.

OBJECTIVES: This study investigated whether spinosin potentiates pentobarbital-induced loss of righting reflex (LORR) in mice via 5-HT(1A) receptors. METHODS: Our primary endpoint for sedation was LORR. In addition, the basal rectal temperature was measured. KEY FINDINGS: The results demonstrated that the 5-HT(1A) agonist 8-OH-DPAT (s.c.) induced reductions in duration of LORR at 0.1, 0.5 and 1.0 mg/kg (P < 0.01), and prolongation of LORR latency at 0.5 and 1.0 mg/kg (s.c., P < 0.01) in pentobarbital (45 mg/kg, i.p.)-treated mice. This effect of 8-OH-DPAT was antagonized either by 5-HT(1A) antagonist p-MPPI (5 mg/kg, i.p.) or by spinosin (15 mg/kg, i.g.) with significance, respectively. Co-administration of spinosin and p-MPPI both at ineffective doses (spinosin at 5.0 mg/kg, i.g. and p-MPPI at 1.0 mg/kg, i.p.) showed significant augmentative effects in reducing latency to LORR, and increasing LORR duration (P < 0.01) in pentobarbital-treated mice. On the other hand, spinosin inhibited 8-OH-DPAT-induced hypothermia, which has been generally attributed to the activation of somatodendritic 5-HT(1A) autoreceptors in mice. CONCLUSIONS: Based on our previous results and the present data, it should be presumed that presynaptic 5-HT(1A) autoreceptor mechanisms may be involved in the inhibitory effect of spinosin on 8-OH-DPAT-induced hypothermia and also in the potentiating effect of spinosin on pentobarbital-induced LORR in mice.

Extract of Ganoderma lucidum prolongs sleep time in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Ganoderma lucidum (Ling Zhi) is a basidiomycete white-rot macrofungus that has been used as a tranquilizing agent (i.e., An-Shen effect) for the treatment of restlessness, insomnia, and palpitation in China for hundreds of years. AIM OF THE STUDY: The present study aimed to investigate whether Ganoderma lucidum extract (GLE) influences the sleep of freely moving rats and the potential mechanism. MATERIALS AND METHODS: Ganoderma lucidum extract was extracted from fruiting bodies of Ganoderma lucidum. Rats were treated with GLE orally for 3 days, and on the third day, electroencephalographic and electromyographic recordings were made for 6h from 9:00 p.m. to 3:00 a.m. in freely moving rats. Sleep parameters were analyzed using SleepSign software. Tumor necrosis factor-α (TNF-α) levels were measured using the enzyme-linked immunosorbent assay. RESULTS: Three-day administration of GLE significantly increased total sleep time and non-rapid eye movement (NREM) sleep time at a dose of 80 mg/kg (i.g.) without influencing slow-wave sleep or REM sleep in freely moving rats. TNF-α levels were significantly increased concomitantly in serum, the hypothalamus, and dorsal raphe nucleus. The hypnotic effect of GLE (80 mg/kg, i.g.) was significantly inhibited by intracerebroventricular injection of TNF-α antibody (2.5 µg/rat). Co-administration of GLE (40 mg/kg, i.g.) and TNF-α (12.5 ng/rat, i.c.v.), both at ineffective doses, revealed an additive hypnotic effect. CONCLUSION: These results suggest that GLE has hypnotic effects in freely moving rats. The mechanism by which the extract promoted sleep remains unclear, but this effect appears to be primarily related to the modulation of cytokines such as TNF-α. Furthermore, these data at least partially support the ethnomedical use of Ganoderma lucidum.

Diltiazem potentiates pentobarbital-induced hypnosis via 5-HT1A and 5-HT2A/2C receptors: role for dorsal raphe nucleus.

It has been reported that the sedative component of pentobarbital is mediated by GABA receptors in an endogenous sleep pathway and the ventrolateral preoptic area (VLPO)-tuberomammillary nucleus (TMN) or VLPO-dorsal raphe nucleus (DRN) neural circuit is important in the sedative response to pentobarbital. Our previous findings indicated that the VLPO-TMN neuronal circuit may play crucial part in the augmentative effect of diltiazem on pentobarbital sleep and the serotonergic system may be involved. This study was designed to investigate the role of DRN and the serotonergic receptors 5-HT(1A) and 5-HT(2A/2C) in the augmentative effect of diltiazem on pentobarbital-induced hypnosis in rats. The results showed that diltiazem (5mg/kg, i.g.) significantly reversed pentobarbital-induced (35 mg/kg, i.p.) reduction of c-Fos expression in 5-HT neurons of DRNV (at -7.5mm Bregma), DRND, DRNVL and MRN (at -8.0mm Bregma). However it did not influence this reducing effect of pentobarbital on non-5-HT neurons either in DRN or in MRN. Moreover, the effect of diltiazem (1 or 2mg/kg, i.g.) on pentobarbital-induced (35 mg/kg, i.p.) hypnosis was significantly inhibited by 5-HT(1A) agonist 8-OH-DPAT (0.5mg/kg, i.p.) and 5-HT(2A/2C) agonist DOI (0.5mg/kg, i.p.), and potentiated by 5-HT(1A) antagonist p-MPPI (2mg/kg, i.p.) and 5-HT(2A/2C) antagonist ritanserin (2mg/kg, i.p.), respectively. From these results, it should be presumed that the augmentative effect of diltiazem on pentobarbital-induced sleep may be related to 5-HT(1A) and 5-HT(2A/2C) receptors, and DRN may be involved. In addition, it also suggested that the DRN may play a multi-modulating role in sleep-wake regulation rather than being recognized simply as arousal nuclei.

Ca²+ modulation in dorsal raphe plays an important role in NREM and REM sleep regulation during pentobarbital hypnosis.

Our previous studies indicated that L-type calcium channel blocker diltiazem could potentiate pentobarbital-induced hypnosis through serotonergic system. In view of the important role of dorsal raphe nucleus (DRN) on the sleep regulation and the pharmacological actions of calcium channel blocker, we presumed that Ca(2+) in the DRN may play an important role in sleep regulation in pentobarbital treated rats. Therefore, we investigated whether the Ca(2+) modulation in DRN by the microinjection of L-type Ca(2+) channel antagonist diltiazem, agonist BAY-K-8644, Ca(2+) chelator EGTA and CaCl(2) would alter the sleep parameters in pentobarbital treated rats. Results showed that perfusion of the agents attenuating Ca(2+) function, such as diltiazem (5 or 20 nmol) or EGTA (3 or 6 pmol) into DRN significantly increased pentobarbital (35 mg/kg, i.p.)-induced total sleep (TS), non-rapid eye movement (NREM) sleep and the slow wave sleep (SWS) ratio in NREM sleep. On the contrary, the DRN injection of the agents improving Ca(2+) function, such as BAY-K-8644 (10 nmol) or CaCl(2) (50 or 100 nmol) significantly reduced pentobarbital (35 mg/kg, i.p.)-induced TS, NREM sleep, rapid eye movement (REM) sleep and REM sleep ratio in TS without influence on SWS. These results suggested that the suppression of Ca(2+) function in DRN could increase NREM sleep including SWS, and the elevation of Ca(2+) function could reduce both NREM and REM sleep in pentobarbital treated rats.

[Optimization for formulation matrix proportion of xiangyu cataplasm by uniform design].

OBJECTIVE: To optimize the matrix formulation of cataplasm used to cure infantile diarrhea. METHODS: The optimum proportion of matrix for the preparation technology process of cataplasm was selected by uniform design and SPSS regression analysis. A check-up for adhibition , peeling strength, nonflowing, content of cream was founded. RESULTS: The best matrix's prescription gelatin: CMC-Na: PANA: kaolin: aluminum trichloride: citric acid: PVP K-30: PEG400: trimethylene glycol: tween-80 was 0.25 : 0.1 : 0.2 : 1.5 : 0.4 : 0.6 : 0.8 : 2 : 1 : 0.5. CONCLUSION: The preparation technique of cataplasm is feasible, and its quality is steerable, it is a safe and effective transdermal-drug delivery system.