[Diterpenoids from Rabdosia flexicaulis].

The genus Rabdosia is famous for the abundance of diverse and novel ent-kaurane diterpenoids. However, only a few ent-kauranoids have been discovered from R. flexicaulis since the investigation on its chemical constituents is not systematic. To find novel bioactive diterpenoids, the ethyl acetate extract of the above ground part of R. flexicaulis in Daofu County, Sichuan Province was obtained by column chromatography. One new compound and five known ones were identified as flexicaulin E(1), forrestin B(2), inf-lexarabdonin D(3), 7α-hydroxydehydroabietic acid(4), 15-hydroxydehydroabietic acid(5), and pomiferin F(6) by spectral techniques. Compounds 1-3 were the ent-kaurane diterpenoids isolated from this species for the first time. Compounds 4-6, aromatic abie-tanoids, were isolated from the genus Rabdosia for the first time.

[Effect of aluminum phosphate gel and Kangfuxin on esophageal pathology and IL-8 and PGE2 expressions in a rat model of reflux esophagitis].

OBJECTIVE: To explore the effect of aluminum phosphate gel and Kangfuxin on esophageal pathology and expressions of interleukin-8 (IL-8) and prostaglandin E2 (PGE2) in rats with reflux esophagitis and explore the possible mechanisms. METHODS: Sixty SD rats were randomized into aluminum phosphate gel group (n=10), Kangfuxin group (n=10), aluminum phosphate gel+Kangfuxin group (n=10), model group (n=20), and control group (n=10). Except for those in the control group, all the rats were subjected to infusion of diluted lysolecithin with hydrochloric acid in the esophagus for 14 days. Ten rats in the model group and those in the control group were sacrificed to examine the pathological changes and contents of IL-8 and PGE2 in the esophagus using optical and electron microscopes and radioimmunoassay. The next day the rest rats were given corresponding treatments (saline in model group) administered into the esophagus on a daily basis for 14 days, after which esophageal pathologies and IL-8 and PGE2 contents were examined. RESULTS: The model rats showed obvious esophageal pathologies including inflammatory cell infiltration, vacuolar degeneration of the epithelial cells, esophageal erosion and even ulceration, with severe detachment of the epithelial cells. The rats in all the intervention groups showed lessened esophageal pathologies and lowered esophageal IL-8 and PGE2 contents compared with those in the model group. Esophageal mucosal injury index and IL-8 and PGE2 contents were all significantly lower in rats receiving combined treatment with aluminum phosphate and Kangfuxin than in those receiving either of the treatments (P<0.05). CONCLUSIONS: Both Kangfuxin and aluminum phosphate gel are effective in the treatment for reflux esophagitis induced by lysolecithin and hydrochloric acid, and their therapeutic effects are achieved possibly by reducing IL-8 and PGE2 levels in the esophagus.

Diterpene alkaloids with an aza-ent-kaurane skeleton from Isodon rubescens.

Two compounds belonging to a new group of diterpene alkaloids, kaurines A and B (1 and 2), and an alkaloid bearing a succinimide moiety (3) were obtained from Isodon rubescens. Their structures and absolute configurations were determined by spectroscopy and quantum-chemical computational (13)C NMR and ECD data analysis. These alkaloids differ from known diterpene alkaloids and diterpenoids and are presumably biosynthesized from ent-kaurane diterpenoids.

Pharmacokinetics, tissue distribution and excretion of verticinone from F. hupehensis in rats.

Verticinone, the main active component in F. hupehensis, exhibits potent antitussive and expectorant effects. Here, a LC-MS method was developed and applied to study the pharmacokinetics, tissue distribution and excretion of verticinone in rats, and its plasma protein binding in vitro. A significant gender difference in the pharmacokinetics of verticinone in rats was observed, as its absolute oral bioavailability in male and female rats was 45.8% and 2.74%, respectively. The relative bioavailability of verticinone was significantly lower in female rats as compared to male, following intragastrical (i.g.) and intravenous (i.v.) administration. After successive i.g. administration of verticinone, accumulation was observed in female rats but not in the male ones. The tissue distribution study showed that verticinone had a good tissue penetrability and a high tissue affinity in most studied tissues, except brain. After a 2 mg/kg oral dose, less than 4% of the dose was excreted as unchanged parent compound in male rats, and less than 1% in female rats, which indicated that verticinone was metabolized more extensively in female rats than in male rats.

Simultaneous determination of four bioactive flavonoids from Polygonum orientale L. in dog plasma by UPLC-ESI-MS/MS and application of the technique to pharmacokinetic studies.

The in vivo effects of traditional herbal medicines are generally mediated by multiple bioactive components. The main constituents of Polygonum orientale L. are flavonoids such as orientin, vitexin, cynaroside, and quercitrin. The aim of this study was to develop and validate a method for characterizing these flavonoids, in order to better understand the pharmacokinetics and pharmacodynamics of P. orientale L. We used ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) to analyze the flavonoids. After precipitation of the proteins with methanol, the flavonoids were separated on a BEH C18 column (50mm×2.1mm, i.d., 1.7µm) by using an elution gradient of acetonitrile. Flavonoid content was determined using the multiple reaction monitoring (MRM) mode at m/z 449.2→329.2 for orientin, m/z 433.2→313.0 for vitexin, m/z 449.2→287.1 for cynaroside, m/z 449.2→303.4 for quercitrin, and m/z 417.0→267.0 for the internal standard, puerarin. Pharmacokinetics was assessed after intravenous administration of P. orientale L. extracts (POE) in Beagle dogs at a dose of 22, 44, or 88mg/kg. Analysis of the standard curves by linear regression revealed high linearity over a 243-fold dynamic range for the four flavonoids (the lower limit of quantitation values were 4-21ng/mL). The relative standard deviations of intra- and inter-day measurements were less than 15.1%, and the method was accurate to within -8.7% to 7.2%; the extraction recoveries from dog plasma were 70.6-89.3%, 69.8-88.7%, 72.5-85.7%, and 71.0-79.1% for orientin, vitexin, cynaroside, and quercitrin, respectively. Our results suggest non-linear pharmacokinetic characteristics with rapid clearance of the flavonoids. In conclusion, UPLC-ESI-MS/MS is a rapid and sensitive method for simultaneous quantification of multiple flavonoids from POE in dog plasma and is suitable for pharmacokinetic studies of herbal medicines.

Three new ursane-type triterpenoids from the aerial parts of Isodon excisoides.

Three new ursane-type triterpenoids, 2α,3ß-dihydroxy-11α,12α-epoxy-urs-28,13ß-olide (1), 2α,3ß,24-trihydroxy-11α,12α-epoxy-urs-28,13ß-olide (2), and 2α,3α,24-trihydroxy-11,20(30)-dien-urs-28,13ß-olide (6), together with six known ursane-type triterpenoids (3-5, 7-9), were isolated from the EtOAc extract of the aerial parts of Isodon excisoides. Their structures were elucidated on the basis of 1D NMR and 2D NMR analyses as well as HRMS experiments.

New ent-abietane and ent-kaurane diterpenoids from Isodon rubescens.

Five new ent-abietane diterpenoids, ent-abierubesins A-E (1-5), and two new ent-kauranoids, hubeirubesins A and B (6, 7), together with three known diterpenoids (8-10), were isolated from the aerial parts of Isodon rubescens. Their structures were identified by means of extensive spectroscopic analysis, and the absolute stereochemistry of 1 was determined by single-crystal X-ray diffraction experiment.

Three new 11,20-epoxy-ent-kauranoids from Isodon rubescens.

Three rare and new 11,20-epoxy-ent-kaurane diterpenoids, named jianshirubesins D-F (1-3), along with one known analogue (4), were isolated from the aerial parts of Isodon rubescens. Their structures were established by analysis of spectroscopic data. Found in the MTT assay to evaluate the cytotoxicity of compounds 1, 2, and 4, only 1 could selectively inhibit certain cell lines from proliferating. In addition, a simple structure-activity relationship discussion might suggest a new bioactive moiety, different from the α,ß-unsaturated ketone group.

Enmein-type diterpenoids from the aerial parts of Isodon rubescens and their cytotoxicity.

Three new enmein-type diterpenoids, jianshirubesins A-C (1-3), together with ten known compounds, were isolated from the aerial parts of Isodon rubescens. Their structures were established by using spectroscopic methods, and the absolute configuration of compound 1 was confirmed by a single-crystal X-ray diffraction analysis. All compounds except 3 were evaluated for their in vitro cytotoxicity by MTT assay, and compounds 5 and 10 exhibited significant inhibitory ability on selected cell lines.

New guaianolides from Artemisia anomala.

Two new guaianolides artemanomalides A and B were isolated from the aerial parts of Artemisia anomala S. Moore. Their structures were characterized as 2-oxo-5α, 10α-dihydroxy-guaia-3-en-1α, 6ß, 7α, 11ß H-12, 6-olide (1) and 8α-acetoxy-2-oxo-5α, 10α-dihydroxy-guaia-3, 11(13)-dien-1α, 6ß, 7αH-12, 6-olide (2) on the basis of extensive spectroscopic analyses. Compounds 1 and 2 showed inhibitory activities against COX-2 enzyme with IC(50) values of 8.8 and 3.6 µM.

The interleukin-18 inhibitory activities of echinocystic acid and its saponins from Impatiens pritzellii var. hupehensis.

Echinocystic acid (1), an echinocystic acid saponin, 2, and four of its ester saponins, 3-6, obtained from the active fraction of Impatiens pritzellii var. hupehensis, an traditional Chinese medicine for rheumatoid arthritis, were investigated for their effects on lipopolysaccharide (LPS)-induced interleukin (IL)-18 in human peripheral blood mononuclear cells. Three of them, 1, 2 and 6, showed obvious activity to inhibit the production of IL-18, especially the ester saponins with a sugar chain at C-28, 6. Structure-activity relationships are discussed in brief.

Addictive evaluation of cholic acid-verticinone ester, a potential cough therapeutic agent with agonist action of opioid receptor.

AIM: The purpose of this work was to search for potential drugs with potent antitussive and expectorant activities as well as a low toxicity, but without addictive properties. Cholic acid-verticinone ester (CA-Ver) was synthesized based on the clearly elucidated antitussive and expectorant activities of verticinone in bulbs of Fritillaria and different bile acids in Snake Bile. In our previous study, CA-Ver showed a much more potent activity than codeine phosphate. This study was carried out to investigate the central antitussive mechanism and the addictive evaluation of CA-Ver. METHODS: Testing on a capsaicin-induced cough model of mice pretreated with naloxone, a non-selective opioid receptor antagonist, was performed for the observation of CA-Ver's central antitussive mechanism. We then took naloxone-induced withdrawal tests of mice for the judgment of CA-Ver's addiction. Lastly, we determined the opioid dependence of CA-Ver in the guinea pig ileum. RESULTS: The test on the capsaicin-induced cough model showed that naloxone could block the antitussive effect of CA-Ver, suggesting the antitussive mechanism of CA-Ver was related to the central opioid receptors. The naloxone-urged withdrawal tests of the mice showed that CA-Ver was not addictive, and the test of the opioid dependence in the guinea pig ileum showed that CA-Ver had no withdrawal response. CONCLUSION: These findings suggested that CA-Ver deserved attention for its potent antitussive effects related to the central opioid receptors, but without addiction, and had a good development perspective.

Structural analysis and antitussive evaluation of five novel esters of verticinone and bile acids.

Shedan-Chuanbei powder, a complex of traditional Chinese medicine preparation, which consists of Snake Bile (Chinese name "Shedan") and Fritillariae Cirrhosae (Chinese name "Chuanbei"), is the most popular antitussive and expectorant formulation in Chinese communities. However, the clinical application of Shedan-Chuanbei powder is now stringently limited because of the shortage of the two crude medicinal materials, especially for the sake of animal protection. In addition, the inherent defects of the most of the complex of traditional Chinese medicine such as the indistinct basal pharmacodynamic materials and the difficulties in quality control had blocked them heading into the international medicinal market. So we attempted to seek new substitute for Shedan-Chuanbei powder for antitussive drugs. In order to gain some new compounds with better bioactivity and attenuated toxicity, we tried to combine two kinds of drugs through ester bond. Enlightened with "combination principle" in drug discovery, we synthesized five novel esters of verticinone and bile acids, both of which are the major bioactive components in Shedan-Chuanbei powder. We then evaluated the antitussive activity and the acute toxicity of the five ester-linked compounds. The five ester-linked compounds had much more potent antitussive activity and expectorant activity than single bile acids at the same doses, and had equivalent antitussive activity and expectorant activity in comparison with about double moles dose of the monomer verticinone. Especially, cholic acid-verticinone ester had much more potent antitussive effects than the monomer verticinone or cholic acid at the same dose. A further acute toxicity study showed that the LD(50) values of the five ester-linked compounds exceeded 3.5g/kg by intraperitoneal injection in mice. Based on the studies of pharmacology and acute toxicity, the five ester-linked compounds have synergic pharmacodynamic action and attenuated toxicity compared with single verticinone and single bile acids.

Two new triterpenoids from the leaves and stems of Fritillaria hupehensis.

Two new cycloartane-type triterpenoids 25-hydroxyl-9,19-cycloart-22-ene-3-one (1) and (23Z)-9,19-cycloart-23-ene-3alpha,25-diol (2) along with 9,19-cycloart-25-ene-3beta, 24xi-diol (3) and cycloeucalenol (4) have been isolated from the leaves and stems of Fritillaria hupehensis Hsiao et K.C. Hsia. Their structures were elucidated on the basis of spectroscopic analysis.

[Studies on chemical constituents from Anoectochilus roxburghii].

OBJECTIVE: To investigate the chemical constituents from Anoectochilus roxburghii. METHODS: The compounds were isolated and purified by repeated column chromatographies with silica gel, Macroporous resin and Sephadex LH-20, and their structures were identified by their physical and spectral datas. RESULTS: Ten compounds were isolated and elucidated as: beta-D-glucopyranosyl-(3R)-hydroxybutanolide (I), stearic acid (II), palmitic acid (III), beta-sitosterol (IV) and succinic acid (V), p-hydroxy benzaldehyde (VI), daucosterol (VII), and methyl 4-beta-D-glucopyranosyl-hutanoate (VIII); as well as p-hydroxy cinnamic acid (IX) and o-hydroxy phenol (X) were identified. CONCLUSION: Compound I ,VIII, X are firstly isolated from this species.

Synthesis and antitussive evaluation of verticinone-cholic acid salt, a novel and potential cough therapeutic agent.

AIM: To seek a novel and potent antitussive drug based on Shedan-Chuanbei powder, a complex of traditional Chinese medicine preparation for cough therapy. METHODS: Verticinone-cholic acid (Ver-CA) salt, a novel, salifying derivative of verticinone and cholic acid, both of which are the major bioactive components in Shedan-Chuanbei powder, was synthesized. We then evaluated the antitussive activity and the acute toxicity of the salt. RESULTS: The new compound, with good solubility in water, has much more potent antitussive activity in comparison with the same dose of single verticinone and single cholic acid. The administration 3 mg/kg of Ver-CA could result in over 50% reduction of a citric acid-induced cough. Pretreatment with naloxone (0.8 mg/kg, ip) can only partially antagonize its antitussive effect. On the other hand, glybenclamide (3 mg/kg, ip), an ATP-sensitive K+ channel blocker, can also significantly reduce the antitussive effect of Ver-CA. A further acute toxicity study showed that the LD(50) values of Ver-CA were 3 times that of verticinone. CONCLUSION: Based on the studies of pharmacology and acute toxicity, the salt has a synergic and attenuated toxicity compared with single verticinone and cholic acid. Moreover, the present study also suggests that Ver-CA, a potential novel antitussive agent, may exert its antitussive effect via both the peripheral (modulated by ATP-sensitive K+ channels) and central mechanisms (modulated by the opioid receptor).

[Preparation and antitussive, expectorant and antiasthmatic activities of verticinone-bile acids salts].

To search for potential drugs with potent antitussive, expectorant, antiasthmatic activities and low toxicity, a series of verticinone-bile acids salts were prepared based on the clearly elucidated antitussive, expectorant and antiasthmatic activities of verticinone in bulbs of Fritillaria and different bile acids in Snake Bile. The antitussive, expectorant and antiasthmatic activities of these verticinone-bile acid salts were then screened with different animal models. Ver-CA (verticinone-cholic acid salt) and Ver-CDCA (verticinone-chenodeoxycholic acid salt) showed much more potent activities than other compounds. The bioactivities of Ver-CA and Ver-CDCA are worthy to be intensively studied, and it is also deserved to pay much attention to their much more potent antitussive effects than codeine phosphate. In order to elucidate whether they have synergistic effect and attenuated toxicity, their activities will be continuously compared with single verticinone, cholic acid and chenodeoxycholic acid at the same doses on different animal models. The application of "combination principles" in traditional Chinese medicinal formulations may be a novel way in triditional Chinese medicine research and discovery.

Ferulic acid esters from Euphorbia hylonoma.

Octacosyl cis-ferulate (1), along with the trans isomer (2), cholest-5-en-3beta-ylhexadecanoate, chrysophanol and octadecanoic acid was isolated from the roots of Euphorbia hylonoma.

[Studies on constituents from roots of Euphorbia hylonoma].

OBJECTIVE: To study the constituents from roots of Euphorbia hylonoma. METHOD: Column chromatographic techniques were used for isolation and purification of the chemical constituents and their structures were identified by spectral analysis (IR, 1H-NMR, 13C-NMR, 2D-NMR and MS). RESULT: Six compounds were isolated and elucidated as nonane (1), bis (2-ethylhexyl) phthalate (2), euphol (3), beta-sitosterol (4), acalyphol (5) and daucosterol (6) respectively. CONCLUSION: Compounds 1, 2, 3, 5 and 6 were isolated from the plant for the first time.

[HPLC fingerprint of Fritillaria hupehensis].

AIM: To establish a fingerprint analysis method of Fritillaria hupehensis. METHODS: fingerprint was performed by HPLC-ELSD. Hypersil ODS column was used; the mobile phase was composed of methanol (with 0.05% triethylamine) and water with gradient elution; flow rate was 1.0 mL x min(-1); recording time was 60 min; drift tube temperature was 75 degrees C; gas flow rate was 1.9 L x min(-1). RESULTS: HPLC fingerprint of Fritillaria hupehensis was obtained. CONCLUSION: A reliable method was provided for controlling the quality of Fritillaria hupehensis.