[Research on chemical constituents of xuefu zhuyu decoction].

OBJECTIVE: To investigate the chemical constituents of Xufu Zhuyu decoction. METHODS: Silica gel column chromatography, Sephadex LH-20 chromatography and crystallization were employed for the isolation and purification. The structures were elucidated by physicochemical properties and spectral analysis. RESULTS: Eleven compounds were isolated and identified as follows: palmitic acid (1), stearic acid (2), beta-sitosterol (3), oleanolic acid (4), pregnenolone (5), tangeratin (6), 4-hydroxy-3-butylphthalide (7), sorhamnetin (8), friedelinol (9), beta-ecdysone (10), ferulic acid (11). CONCLUSION: All these compounds are isolated from Xuefu Zhuyu decoction for the first time.

Effects of evodiamine and rutaecarpine on the secretion of corticosterone by zona fasciculata-reticularis cells in male rats.

Evodiamine (EVO) and rutaecarpine (RUT) are two bioactive alkaloid isolated from Chinese herb named Wu-Chu-Yu. Previous studies have shown that EVO and RUT possess thermoregulation, vascular regulation, anti-allergic, anti-nociceptive and anti-inflammatory activities. The mechanisms of EVO and RUT effect on steroidogenesis are not clear. The goal of this study was to characterize the mechanism by which EVO and RUT affect corticosterone production in rat zona fasciculata-reticularis (ZFR) cells. ZFR cells were isolated from adrenal glands of male rats and incubated with adrenalcorticotropin (ACTH, 10(-9) M), forskolin (an adenylyl cyclase activator, 10(-5) M), 8-bromo-adenosine 3':5'-cyclic monophosphate (8-Br-cAMP, a permeable cAMP analog, 10(-4) M), or steroidogenic precursors including 25-hydroxycholesterol, pregnenolone, progesterone, and deoxycorticosterone, 10(-5) M each, in the presence or absence of EVO and RUT respectively (0-10(-3) M) at 37 degrees C for 1 h. The concentrations of corticosterone, pregnenolone and progesterone in the media were measured by radioimmunoassay. After administration of ZFR cells with EVO or RUT (10(-4) M) for 60 and 120 min, Western blot analysis was employed to explore the influence of EVO and RUT on the expression of cytochrome P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory protein (StAR). EVO and RUT reduced both basal and ACTH-, forskolin-, as well as 8-Br-cAMP-stimulated corticosterone production in rat ZFR cells. The enhanced corticosterone production caused by the administration of four steroidogenic precursors was decreased following EVO or RUT challenge. These results suggest that EVO and RUT inhibit corticosterone production in rat ZFR cells via a mechanism involving: (1) a decreased activity of cAMP-related pathways; (2) a decreased activity of the steroidogenic enzymes, that is, 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 11beta-hydroxylase (P450c11), during steroidogenesis; and (3) an inhibition of StAR protein expression.

[Determination of unconjugated neurosteroids in rat brain regions by liquid chromatography-negative atmospheric pressure ionization mass spectrometry].

AIM: To simultaneously determine three unconjugated neurosteroids, dehydroepiandrosterone (DHEA) , pregnenolone (PREG), allopregnenolone (AP), from several brain regions of the rat. METHODS: Neurosteroids were isolated separately in a two steps procedure by using ethyl acetate-n-hexane (90:10) as the first step to extract the unconjugated steroids, then the steroid fractions were further purified by SPE. All steroids were derivatized with 2-nitro-4-trifluoromethylphenylhydrazine (2-NFPH) and analyzed by HPLC-MS ( APCI) using selected-ion monitoring. Methyltestosterone was chosen as the internal standard. Results The linear calibration curve of DHEA was obtained in the concentration range of 0.030-2.00 microg x L(-1). The linear calibration curves of PREG and AP were obtained in the concentration range of 0.025-2.00 microg x L(-1). The concentrations of DHEA, PREG and AP in male rat brain regions were (0.70 +/- 0.23), (4.8 +/- 1.9), (1.1 +/- 0.6) ng x g(-1) for frontal cortex, (0.57 +/- 0.28), (6 +/- 3), (0.5 +/- 0.3) ng x g(-1) for hippocampus, (1.5 +/- 1.0), (9 +/- 5), (1.4 +/- 0.9) ng x g(-1) for amygdale, (0.52 +/- 0.14), (7.7 +/- 2.8), (0.5 +/- 0.6) ng x g(-1) for striatum, (2.9 +/- 1.6), (18 +/- 9), (1.6 +/- 1.3) ng x g(-1) for nucleus accumbens, (4.0 +/- 2.0), (27 +/- 12), (0.8 +/- 0.5) ng x g(-1) for pituitary gland, (1.7 +/- 1.2), ( 16 +/- 10), and (0. 8 +/- 0.7) ng x g(-1) for hypothalamus, respectively. CONCLUSION: Good linearity and accuracy were observed for each steroid. The procedure was suitable for measuring concentrations of the unconjugated steroids in rat brain simultaneously.

Cellular distribution and bioactivity of the key steroidogenic enzyme, cytochrome P450side chain cleavage, in sensory neural pathways.

Neurosteroids are steroids produced within the nervous system. Based on behavioural responses evoked in animals by synthetic steroid injections, several studies suggested neurosteroid involvement in important neurophysiological processes. These observations should be correlated only to neuroactive effects of the injected steroids. Neurosteroids mostly control the CNS activity through allosteric modulation of neurotransmitter receptors within concentration ranges used by neurotransmitters themselves. Therefore, neurosteroid production within pathways controlling a neurophysiological process is necessary to consider neurosteroid involvement in that process. Because of the increasing speculation about pain modulation by neurosteroids based on pharmacological observations, we decided to clarify the situation by investigating neurosteroidogenesis occurrence in sensory pathways, particularly in nociceptive structures. We studied the presence and activity of cytochrome P450side chain cleavage (P450scc) in rat pain pathways. P450scc-immunoreactive cells were localized in dorsal root ganglia (DRG), spinal cord (SC) dorsal horn, nociceptive supraspinal nuclei (SSN) and somatosensory cortex. Incubation of DRG, SSN or SC tissue homogenates with [3H]cholesterol yielded the formation of radioactive metabolites including [3H]pregnenolone of which the synthesis was reduced in presence of aminogluthetimide, a P450scc inhibitor. These first neuroanatomical and neurochemical results demonstrate the occurrence of neurosteroidogenesis in nociceptive pathways and strongly suggest that neurosteroids may control pain mechanisms.

Neurosteroid quantification in human brain regions: comparison between Alzheimer's and nondemented patients.

Some neurosteroids have been shown to display beneficial effects on neuroprotection in rodents. To investigate the physiopathological significance of neurosteroids in Alzheimer's disease (AD), we compared the concentrations of pregnenolone, pregnenolone sulfate (PREGS), dehydroepiandrosterone, dehydroepiandrosterone sulfate (DHEAS), progesterone, and allopregnanolone, measured by gas chromatography-mass spectrometry, in individual brain regions of AD patients and aged nondemented controls, including hippocampus, amygdala, frontal cortex, striatum, hypothalamus, and cerebellum. A general trend toward decreased levels of all steroids was observed in all AD patients' brain regions compared with controls: PREGS and DHEAS were significantly lower in the striatum and cerebellum, and DHEAS was also significantly reduced in the hypothalamus. A significant negative correlation was found between the levels of cortical beta-amyloid peptides and those of PREGS in the striatum and cerebellum and between the levels of phosphorylated tau proteins and DHEAS in the hypothalamus. This study provides reference values for steroid concentrations determined by gas chromatography-mass spectrometry in various regions of the aged human brain. High levels of key proteins implicated in the formation of plaques and neurofibrillary tangles were correlated with decreased brain levels of PREGS and DHEAS, suggesting a possible neuroprotective role of these neurosteroids in AD.

The octadecaneuropeptide ODN stimulates neurosteroid biosynthesis through activation of central-type benzodiazepine receptors.

Neurosteroids may play a major role in the regulation of various neurophysiological and behavioural processes. However, while the biochemical pathways involved in the synthesis of neuroactive steroids in the central nervous system are now elucidated, the mechanisms controlling the activity of neurosteroid-producing cells remain almost completely unknown. In the present study, we have investigated the effect of the octadecaneuropeptide (ODN), an endogenous ligand of benzodiazepine receptors, in the control of steroid biosynthesis in the frog hypothalamus. Glial cells containing ODN-like immunoreactivity were found to send their thick processes in the close vicinity of neurones expressing the steroidogenic enzyme 3 beta-hydroxysteroid dehydrogenase. Exposure of frog hypothalamic explants to graded concentrations of ODN (10(-10)-10(-5) M) produced a dose-dependent increase in the conversion of tritiated pregnenolone into various radioactive steroids, including 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone and dihydrotestosterone. The ODN-induced stimulation of neurosteroid biosynthesis was mimicked by the central-type benzodiazepine receptor (CBR) inverse agonists methyl beta-carboline-3-carboxylate (beta-CCM) and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). The stimulatory effects of ODN, beta-CCM and DMCM on steroid formation was markedly reduced by the CBR antagonist flumazenil. The ODN-evoked stimulation of neurosteroid production was also significantly attenuated by GABA. Collectively, these data indicate that the endozepine ODN, released by glial cell processes in the vicinity of 3 beta-hydroxysteroid dehydrogenase-containing neurones, stimulates the biosynthesis of neurosteroids through activation of central-type benzodiazepines receptors.

Pregnenolone, dehydroepiandrosterone, and their sulfate and fatty acid esters in the rat brain.

The rat brain contains large amounts of pregnenolone (P) and dehydroepiandrosterone (D) arising from local biosynthetic pathways. We have devised a procedure for the measurement of both "neurosteroids" either unconjugated or released from their sulfate (S) or fatty acid (L) esters. The measurements were performed at the acrophase of the circadian variation of neurosteroids, and confirmed the large accumulation of P (25 +/- 8 ng/g, mean +/- SD) and of PS (19 +/- 6 ng/g) and DS (2.1 +/- 0.5 ng/g) in the brain of adult male rats. We found that fatty acid esters constitute the major species of neurosteroids in brain (PL 46 +/- 14, and DL 36 +/- 7 ng/g, in adult males). The levels of P and DS were increased by daily injection of vehicle to intact males, whereas castration, without or with testosterone or estradiol supplementation (2 mg daily for 7 days), did not produce a significant change of neurosteroids concentrations. Measurements of neurosteroids had not been previously reported in cyclic females. The levels of P, PL, and DS were identical in proestrous females and in intact males, whereas PS (26 +/- 6 ng/g) and DL (50 +/- 16 ng/g) were increased in females. Compared to proestrous females, diestrous females had lower levels of PS (19 +/- 6 ng/g), DS (1.7 +/- 0.4 ng/g), and PL (43 +/- 19 ng/g). These differences suggested a modulatory role of ovarian secretions on the metabolism of neurosteroids.