Li-Kang Ho

Analysis and Confirmation of Synthetic Anorexics in Adulterated Traditional Chinese Medicines by high-performance Capillary Electrophoresis

Six synthetic anorexics, clobenzorex, diethylpropion, fenfluramine, methamphetamine, phenylpropanolamine and phentermine, which can be found as adulterants in traditional Chinese medicines were assayed simultaneously by high-performance capillary electrophoresis. The electrolyte was a buffer solution containing 120 mM phosphate buffer (NaH2PO4/H3PO4, pH 2.0) and 15% acetonitrile. Applied voltage was 16 kV and temperature was 30 degrees C. Fluoren-2,7-diammonium chloride was used as an internal standard and detector set at 200 nm. The recoveries of the synthetic anorexic adulterants in traditional Chinese medicinal formula using C8-SCX mixed solid-phase extraction were studied. Several traditional Chinese medicinal powders obtained from clinics were also studied by the above HPCE method and confirmed by GC-MS. Clobenzorex, diethylpropion and fenfluramine were found and determine in these samples.

Analysis of synthetic anti-diabetic drugs in adulterated traditional Chinese medicines by high-performance capillary electrophoresis.

Four synthetic anti-diabetic drugs, acetohexamide (ACE), chlorpropamide (CHL), glibenclamide (GLI) and tolbutamide (TOL), which can be found as adulterants in traditional Chinese medicines (TCMs) were assayed simultaneously using high-performance capillary electrophoresis (HPCE) in 4 min with UV detection at 200 nm. The electrolyte was a buffer solution containing 100 mM phosphate buffer (NaH2PO4/Na2B4O7, pH 7.5). Applied voltage was 15.0 kV and temperature was 30 degrees C. 2-(4-Hydroxyphenyl) ethyl ammonium chloride (HEA) was used as an internal standard. The effects of buffer concentration, pH and supplied voltage on separation were investigated. The relative standard deviations (R.S.D.) of these anti-diabetic drugs for intra-day and inter-day analyses were 0.23-4.27 and 1.23-6.33%, respectively. The recoveries of the synthetic drug adulterants in traditional Chinese medicinal formula ranged from 81.3 to 105.5%. GLI was found and determined in a real sample of TCM.

Determination of matrine and oxymatrine in Sophora subprostata by CE

Shan-dou-gen is the dried roots of Sophora subprostata (Leguminosae) and a commonly used Chinese herbal drug in Taiwan. It possesses antipyretic, anti-inflammatory, analgesic effects and is used to treat sore throat and acute pharyngolaryngeal infections. To evaluate the quality of S. subprostata, a simple, rapid and accurate high-performance capillary electrophoresis (HPCE) method was developed for the assay of two alkaloids: matrine and oxymatrine. The electrolyte was a buffer solution containing 75% 130 mM phosphate buffer (NaH2PO4/H3PO4, pH 3.5) and 25% acetonitrile. Applied voltage was 10 kV and temperature was 30 degrees C. 2-(4-Hydroxyphenyl)ethylammonium chloride was used as an internal standard and detector set at 200 nm. The contents of matrine and oxymatrine of S. subprostata in several different samples of crude drugs and commercial concentrated preparation have also been determined.

OXIDATIVE METABOLISM OF THE ALKALOID RUTAECARPINE BY HUMAN CYTOCHROME P450

Rutaecarpine is the main active alkaloid of the herbal medicine, Evodia rutaecarpa. To identify the major human cytochrome P450 (P450) participating in rutaecarpine oxidative metabolism, human liver microsomes and bacteria-expressed recombinant human P450 were studied. In liver microsomes, rutaecarpine was oxidized to 10-, 11-, 12-, and 3-hydroxyrutaecarpine. Microsomal 10- and 3-hydroxylation activities were strongly inhibited by ketoconazole. The 11- and 12-hydroxylation activities were inhibited by α-naphthoflavone, quinidine, and ketoconazole. These results indicated that multiple hepatic P450s including CYP1A2, CYP2D6, and CYP3A4 participate in rutaecarpine hydroxylations. Among recombinant P450s, CYP1A1 had the highest rutaecarpine hydroxylation activity. Decreased metabolite formation at high substrate concentration indicated that there was substrate inhibition of CYP1A1- and CYP1A2-catalyzed hydroxylations. CYP1A1-catalyzed rutaecarpine hydroxylations had Vmax values of 1388 to ∼1893 pmol/min/nmol P450, Km values of 4.1 to ∼9.5 μM, and Ki values of 45 to ∼103 μM. These results indicated that more than one molecule of rutaecarpine is accessible to the CYP1A active site. The major metabolite 10-hydroxyrutaecarpine decreased CYP1A1, CYP1A2, and CYP1B1 activities with respective IC50 values of 2.56 ± 0.04, 2.57 ± 0.11, and 0.09 ± 0.01 μM, suggesting that product inhibition might occur during rutaecarpine hydroxylation. The metabolite profile and kinetic properties of rutaecarpine hydroxylation by human P450s provide important information relevant to the clinical application of rutaecarpine and E. rutaecarpa.

Analysis of three lupane type triterpenoids in Helicteres angustifolia by high-performance liquid chromatography

Kang-chih-ma is the dried roots and stems of Helicteres angustifolia (Sterculiaceae) and a commonly used folk herbal drug in Taiwan. It possesses antidotal, analgesic, anti-inflammatory and anti-bacterial effects and is also known as a kind of tumor inhibitory plant. To evaluate the quality of H. angustifolia, a simple, rapid and accurate high-performance liquid chromatographic (HPLC) method was developed for the assay of three lupane type triterpenes: 3beta-acetoxy-27-benzoyloxylup-20(29)-en-28-oic acid methyl ester (methyl helicterate), 3beta-acetoxy-27-benzoyloxylup-20(29)-en-28-oic acid, and 3beta-acetoxy-27-(p-hydroxyl) benzoyloxylup-20(29)-en-28-oic acid methyl ester. The present HPLC system used an Inertsil ODS-2 column by gradient elution with acetonitrile and water as the mobile phase and detected at UV 230 nm. Regression equations revealed good linear relationships (correlation coefficients: 0.9922-0.9997). The relative standard deviations of these three constituents ranged between 1.05-3.14% (intraday) and 2.12-4.38% (interday). The contents of these three constituents of the heartwood and the bark of the roots of H. angustifolia in five different samples have also been determined.

Solid-phase extraction for the determination of caffeine in traditional Chinese medicinal prescriptions containing Theae folium by high performance liquid chromatography.

A high performance liquid chromatographic method in combination with C-18 reverse phase solid-phase extraction (SPE) was developed for determination of caffeine (CA) in traditional Chinese medicinal prescriptions which contain Theae folium. The frequently used prescriptions include Shin-Yi-San, Chuan-Chyong-Char-Tyau-San, Tsang-Eel-San, San-Hwang-Shyr-Gau-Tang, Tzy-Shenn-Ming-Mu-Tang and Shiang-Chyong-San. The present HPLC system uses a Merck RP-select B column by isocratic elution with methanol and 1% (v/v) acetic acid (1:4) as the mobile phase and detected at UV 270 nm. 8-Chlorotheophylline was used as an internal standard. The extracts of prescriptions were treated by Supelclean LC-18 SPE tube for eliminating interferences. Blank decoctions of each prescription were also examined as a test for interferences. The recoveries of caffeine from the Chinese medicinal prescriptions ranged from 88.5 to 92.3%. The relative standard deviations of caffeine ranged between 0.86 and 1.97% (intraday) and 1.04 and 3.90% (interday). The contents of caffeine in standard decoctions ranged from 13.98 to 19.62 mg g(-1).

Synthesis and vasorelaxant activity of 4-(cyclic amido)-2H-naphtho[1,2-b]pyrans

A series of 4-(cyclic amido)-2H-naphtho[1,2-b]pyrans related to cromakalim (1) has been prepared and their vasorelaxant activities on isolated rat thoracic aorta precontracted with phenylephrine have been evaluated. The relaxant mechanism of 3a was found not through ATP-sensitive K(+) channels as cromakalim, but through opening voltage-sensitive K(+) channels.

Analysis of N‐cis‐ and N‐trans‐Feruloyl 3‐Methyldopamine in Achyranthes bidentata by HPLC

Abstract Huai‐niu‐xi is the dried root of Achyranthes bidentata (Amaranthaceae) and a commonly used herbal medicine. It possesses analgesic, anti‐inflammatory, diuretic, anti‐bacterial, and anti‐convulsive activity. In clinical use, it was used to strengthen the sinew and bones, supplement the liver and kidney, and cure knee pain. A simple, rapid, and accurate high‐performance liquid chromatographic (HPLC) method was developed for the assay of N‐cis‐feruloyl 3‐methyldopamine (cis‐FMD) and N‐trans‐feruloyl 3‐methyldopamine (trans‐FMD) in Huai‐niu‐xi. The HPLC system uses an Inertsil ODS‐P (5 µm, 250 mm × 4.6 mm I.D.) column with gradient elution, with acetonitrile and water as the mobile phase. A detector was set at 300 nm. Calibration graphs were constructed in the range 8.0–160.0 µg mL−1 for cis‐FMD and 16.4–246.0 µg mL−1 for trans‐FMD. Regression equations revealed good linear relationships (correlation coefficients: 0.9997–0.9998) between the peak‐area to each constituent. The contents of two constituents of A. bidentata in six samples have been determined. This study using HPLC, also compared with those similar herbal medicines, Cyathula officinals (Amaranthaceae) and Strobilanthes forrestii (Acanthaceae), commonly used as an adulterant.

The evaluation of the therapeutic effect of Tao-Shang-Tsao on α-naphthylisothiocyanate and carbon tetrachloride-induced acute liver damage in rats.

The hepatoprotective effects of Tao-shang-tsao, Ixeris laevigata Sch.-Bip. var. oldhami Kitam., were studied on cholestatic hepatitis induced by alpha-naphthylisothiocyanate (ANIT, 100 mg/10 ml/kg, in olive oil, i.p.) and acute hepatitis induced by carbon tetrachloride (20% CCl4/olive oil, 1.5 ml/kg, i.p.) in rats by post-treatment with the crude methanolic extracts of I. laevigata (0.3, 1.0, and 2.0 g/kg) orally in the therapeutic model. Hepatoprotective activity was monitored by estimating the serum transaminases concentrations and histopathological changes in the livers of experimental rats. It was found that the I. laevigata extract significantly decreased the acute elevation of serum transaminases by biochemical examination. According to pathohistological studies in the liver of experimental rats, the crude I. laevigata extract ameliorates the central necrosis, fatty change or proliferation of bile duct epithelium focal necrosis caused by ANIT or CCl4-induced hepatitis rats.