The protective effects and mechanism of Ruyi Zhenbao Pill, a Tibetan medicinal compound, in a rat model of osteoarthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Ruyi Zhenbao Pill (RZP) is a prescribed Tibetan formulation for the treatment of white-pulse-disease, yellow-water-disease as well as pain-related disease. RZP is composed of 30 medicinal materials including herbal medicine, animal medicine and mineral medicine. They are widely used in the Tibetan area to treat cerebrovascular disease, hemiplegia, rheumatism, and pain diseases for centuries. AIM OF THE STUDY: The aim of the present study was to evaluate the anti-osteoarthritis function of RZP and to clarify the underlying mechanisms. MATERIALS AND METHODS: The active components in RZP were identified using HPLC methods. Osteoarthritis (OA) animal model was established via intra-articular injection of papain in rat knees. After the administration of RZP (0.45, 0.9 g/kg) for 28 days, the clinical observation was conducted, and pathological changes as well as serum biochemical indexes were detected. Moreover, therapeutic targets and pathways of RZP were discussed. RESULTS: The results showed that RZP could suppress knee joint swelling and arthralgia, thus relieving joint pain and inflammation in OA rats. Microcomputed tomography (µCT)-based physiological imaging and staining pictures confirmed the therapeutic effects of RZP on OA symptoms including knee joint swelling and structural changes with progressive inflammation in OA rats. RZP could promote the synthesis or inhibit the degradation of COLⅡ, attenuate OA-induced OPN up-regulation and thus relieve the OA symptom. Furthermore, RZP (0.45-0.9 g/kg) could all ameliorate the imbalance of biomarkers related to OA such as MMP1, TNF-α, COX2, IL-1ß and iNOS in knee joints or serum. CONCLUSION: In conclusion, RZP could effectively relieve inflammatory reaction induced by OA injury and the formulation could be applied to the treatment of OA therapy.

Flavonol glycosides from the leaves of Elaeagnus pungens.

The leaves of Elaeagnus pungens were extracted and successively purified by a set chromatographic techniques. The structures of the obtained constituents were elucidated on the basis of spectroscopic methods including 1D/2D NMR and high resolution-MS. One new flavonoid glycosides, kaempferol 3-O-ß-d-glucopyranosyl-(1 → 3)-α-l-rhamnopyranosyl-(1 → 6)-[α-l-rhamnopyranosyl(1 → 2)]-ß-d-galactopyranoside-7-O-ß-d-glucopyranoside (1), along with five known compounds (2-6) were isolated and identified. The MTT assay in vitro showed that the isolated flavonol glycosides have no proliferating activity, suggesting no aggravating impact on the increased airway smooth muscle cells.

Anti-inflammatory and blood stasis activities of essential oil extracted from Artemisia argyi leaf in animals.

Artemisia argyi leaf is a well-known species in traditional Chinese medicine. However, the anti-inflammatory and activating blood stasis activities of its essential oil (AAEO) have not been explored in vivo. The present study measured the contents of three chemical components by gas chromatography (GC). The anti-acute inflammatory effects of AAEO were investigated in dimethyl benzene, glacial acetic acid and carrageenan-induced animals through skin administration or by oral gavage, respectively. The effects of AAEO on haemorheology were studied in a rat acute blood stasis model. The contents of eucalyptol, camphor and borneol in AAEO were 254.4, 51.6 and 58.7 mg/g, respectively. All dosages of AAEO by skin administration significantly decreased the swelling in dimethyl benzene-induced ear oedema and carrageenan-induced paw oedema, and reduced the permeability in glacial acetic acid-induced abdominal blood capillary (p < 0.01). Meanwhile, haemorheology indexes such as whole blood viscosity and the erythrocyte aggregation index significantly decreased only in the high dosage group. In addition, the effects of AAEO by oral gavage were weaker than skin administration at the medium dose in the experiments. It suggests that AAEO has better absorption bioavailability and pharmacological effects through skin administration due to the better skin permeability of essential oil than gastrointestinal absorption.

Antitoxic effect of Veratrilla baillonii on the acute toxicity in mice induced by Aconitum brachypodum, one of the genus Aconitum.

ETHNOPHARMACOLOGICAL RELEVANCE: Aconitum brachypodum Diels (Family Ranunculaceae) is well known for both its good therapy and high toxicity in Yunnan and Sichuan provinces in China. Noticeably, Veratrilla baillonii Franch (Family Gentianaceae), an ethnodrug used by Naxi and Lisu nationalities in Yunnan Province, has been widely considered to possess antitoxic effects on Aconitum plants in herbal therapy and folklore medicines. MATERIALS AND METHODS: The present study was conducted to determine the detoxic activities of the water decoction of Veratrilla baillonii Franch (WVBF) on the the chloroform fraction of Aconitum brachypodum Diels (CFA) induced acute toxicity in mice. The physiological (symptoms, body weight, etc.) as well as pathological and clinical biochemistry parameters were assessed and used as the markers for the toxicity. (1)H nuclear magnetic resonance (NMR) based metabolic approach was adopted to further discuss the mechanism. RESULTS: The acute poisoning effects of CFA on mice were observed at doses of 20-62.5mgkg(-1), resulting in an oral median lethal dose (LD50) of 41.3mgkg(-1). Histologically, distinct degenerative changes of the heart, liver and kidney were observed. The biochemistry parameters in the serum as well as metabolites in heart and brain were also altered. However, WVBF (25-200mg/kg) attenuated all the acute toxicity and pathological changes, properly regulated the biochemistry parameters, and reversed the concentration alterations for some metabolites in the heart and brain of mice induced by 40mg/kg of CFA to a certain extent. CONCLUSIONS: WVBF significantly reduced the onset of the CFA toxicity. This study may contribute to further understanding of the toxicological and pharmacological profiles of Aconitum brachypodum and the detoxic property of Veratrilla baillonii.

Relaxant effect of 1-butanol fraction from Elaeagnus pungens leaf through inhibiting L-type Ca2+ channel on guinea pig tracheal smooth muscle.

ETHNOPHARMACOLOGICAL RELEVANCE: The leaf of Elaeagnus pungens thunb. (Family Elaeagnaceae) has been documented as an effective herb for the treatment of asthma and chronic bronchitis in traditional Chinese medicine. In the past years, only a few of preliminary studies reported the chemical constituents and pharmacology effects of the herb, but their action on the tracheal relaxation has not been investigated. AIM OF THE STUDY: To investigate the relaxing effect and mechanism of the extracts from Elaeagnus pungens leaves on guinea pig tracheal smooth muscle and bronchi smooth muscle cells. MATERIALS AND METHODS: Four fractions of different polarities from Elaeagnus pungens leaves were tested to the tracheal strips on the resting tension or pre-contracted by histamine (20 µM) and acetylcholine (20 µM). Inhibitory effects of the 1-butanol fraction (400mg/ml) on cumulative histamine and acetylcholine (0.2-20 µM) induced contraction were measured. In order to determine the mediators on the 1-butanol fraction effect, the relaxing effect of the 1-butanol fraction was evaluated in the absence and presence of ß-adrenoceptor antagonists (1 µM propranolol), K(+) channels-blockers (4-aminopyridine (2mM), tetraethylammonium chloride (5mM) or glibenclamide (10 µM)), the cyclooxygenase inhibitor (indomethacin, 10 µM), nitric oxide synthase inhibitor (Nω-nitro-L-arginine methyl ester, 100 µM) or L-type Ca(2+) channel inhibitor (nifedipine, 1 µM). Moreover, [Ca(2+)]i in bronchi smooth muscle cells was analyzed by measuring the fluorescence intensity with confocal system. RESULTS: 1-Butanol fraction induced the highest relaxant effect among four fractions of different polarities from Elaeagnus pungens leaves, and significantly relaxed the tracheal strip in the concentration-dependent manner on the resting tension and pre-contracted by histamine phosphate and acetylcholine. It also produced an unparallel rightward shift of the cumulative concentration-response curve of histamine or acetylcholine. Furthermore, the relaxant effect of 1-butanol fraction was not affected by propranolol, glibenclamide, tetraethylammonium chloride, 4-aminopyridine, indomethacin and Nω-nitro-L-arginine methyl ester. However, 1-butanol fraction-induced relaxation decreased after adding nifedipine. It also concentration-dependently inhibited CaCl2-induced contraction in the Ca(2+)-free, 60mM K(+)-containing solution. Additionally, [Ca(2+)]i in the BSMCs significantly reduced after administration of the 1-butanol fraction. CONCLUSIONS: The 1-butanol fraction from Elaeagnus pungens leaves resulted in a relaxation in the non-precontracted and pre-contracted tracheal strips. The relaxant effect was not related to K(+) channels, NO, cGMP or ß-adrenoceptors, but related to the inhibition of Ca(2+) influx through L-type Ca(2+) channels.

Two new flavonol glycosides from the leaves of Elaeagnus pungens.

The leaves of Elaeagnus pungens were extracted with 70% ethanol and successively purified by column chromatography. Seven constituents were obtained and characterized, all of which belong to the class of flavonol glycosides. Their structures were elucidated on the basis of spectroscopic methods including 1D/2D NMR and MS analysis techniques. The seven flavonol glycosides were determined as kaempferol 3-O-ß-d-glucopyranosyl-(1 â†’ 3)-α-l-rhamnopyranosyl-(1 â†’ 6)-[α-l-rhamnopyranosyl(1 â†’ 2)]-ß-d-galactopyranoside (1), isorhamnetin 3-O-ß-d-glucopyranosyl-(1 â†’ 3)-α-l-rhamnopyranosyl-(1 â†’ 6)-ß-d-galactopyranoside (2), together with five known compounds, respectively. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliu-m bromide assay in vitro showed that the isolated flavonol glycosides showed no proliferation activity in the asthma airway smooth muscle cells, comparing with solvent as the control group.

[Transport and metabolism of daidzein in Caco-2 cell model].

OBJECTIVE: To investigate the intestinal absorption and metabolism of daidzein in Caco-2 cell model. METHODS: The damage of daidzein to Caco-2 cell was evaluated by MTT value and alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) activity. With the reference to propranolol, the apparent permeability coefficient (Papp) of daidzein was measured through the monolayer under the different concentrations and pH media. The metabolites were also measured by enzyme hydrolysis. RESULTS: Daidzein had no damage to the growth of the Caco-2 cells in the concentration of 1-50 microg/mL. The apparent permeability coefficient (Papp) of daidzein across the monolayer showed concentration- and pH- independent manner, which was similar to that of transcellcular marker-propranolol, suggesting the good absorption of daidzein in vivo. By hydrolysis with beta-glucuronidase, low metabolites were detected in monolayer and transport medium, verifying the existence of glucuronides and sulfates. CONCLUSION: The daidzein absorption in Caco-2 cell model is a passive and transcellular transport and quite good in the intestines.

Optimized preparation of daidzein-loaded chitosan microspheres and in vivo evaluation after intramuscular injection in rats.

A spherical symmetric design-response surface methodology was applied to optimize the preparation of daidzein-loaded chitosan microspheres by the emulsification/chemical cross-linking technique. The influence of polymer concentration, ratio of drug to polymer, and the stirring speed on the encapsulation efficiency, particle size, particle size distribution, and accumulative drug release percent in microspheres were evaluated. Scan electron microscopy of the optimized microspheres showed spherical particles, loading with drug microcrystal uniformly on the surface of and inside the microspheres. In vivo pharmacokinetic characteristics were evaluated after intramuscular injection of the microspheres in rats. The time-resolved fluoroimmunoassay method was used to determine plasma concentrations of daidzein. The data showed that the release of daidzein in the microspheres in vitro and in vivo almost lasted for 35 days. The bioavailability of daidzein in the microspheres by intramuscular injection increased up to 39% in rats, suggesting that the cross-linked chitosan microspheres are a valuable system for the long-term delivery of isoflavones.

[Preparation of inclusion complex of daidzein and hydropropyl-beta-cyclodextrin].

OBJECTIVE: To prepare an inclusion complex of daidzein and hydropropyl-beta-cyclodextrin to enhance the solubility of daidzein. METHOD: The inclusion complex of daidzein was prepared by the solution stirring method. The binary system of daidzein and HP-beta-CD was confirmed by differential thermal, thermogravimetry analysis, infrared spectroscopy and X-ray diffractometry. RESULT: The drug content in the inclusion complex was 6. 76% and the solubility was 13.68 mg x mL(-1). The identification results showed that the inclusion complex was formed. CONCLUSION: The preparation method of the inclusion complex of daidzein and hydropropyl-beta-cyclodextrin is simple and available, with a increased solubility of daidzein.

[Study on the preparation of rapid-dissoluted EGb droppills].

OBJECTIVE: To study the formulation and technique of preparation of rapid-dissoluted EGb (Extract of Ginkgo biloba) droppills. METHOD: Taking the dissolution percentage of total flavonoids in EGb and weight variation as index, the formulation and technique of EGb droppills were optimized by the orthogonal experiment. RESULT: T50 was 3.62 min and mean weight variation was 2.80%. CONCLUSION: Rapid-dissoluted EGb droppills can increase the dissoluting rate distinctly and reach the purpose of preparation.