Walter K.K. Ho

Characterization of antioxidants present in hawthorn fruits

Hawthorn fruit extract has been shown to have many health benefits including being cardiovascular protective, hypotensive and hypocholesterolemic. The present study was carried out to characterize further the antioxidants of hawthorn fruit and their effect on the oxidation of human low density lipoprotein (LDL) and alpha-tocopherol. The dry hawthorn fruit was extracted successively with ether, ethyl acetate, butanol and water. The ethyl acetate fraction was only effective in inhibition of Cu(+2)-mediated LDL oxidation. The column chromatographic separation led to isolation of eight pure compounds; namely, ursolic acid, hyperoside, isoquercitrin, epicatechin, chlorogenic acid, quercetin, rutin and protocatechuic acid. All of these phenolic compounds, except ursolic acid, were protective to human LDL from Cu(+2)-mediated LDL oxidation. They were also effective in preventing the peroxy free radical-induced oxidation of alpha-tocopherol in human LDL. The inhibitory effect of these compounds on oxidation of LDL and alpha-tocopherol was dose-dependent at concentrations ranging from 5 to 40 µM. In addition, supplementation of 2% hawthorn fruit powder significantly elevated serum alpha-tocopherol by 18-20% in rats fed a 30% polyunsaturated canola oil diet, as compared with the control. The present results suggest that part of the mechanism for cardiovascular protective effects of hawthorn fruit might also involve the direct protection to human LDL from oxidation or indirect protection via maintaining the concentration of alpha-tocopherol in human LDL.

Suppression of withdrawal symptoms by dynorphin in heroin addicts

The effectiveness of dynorphin-(1-13) in suppressing heroin withdrawal was studied. At a dose of 60 micrograms/kg body weight, dynorphin-(1-13) was effective in suppressing withdrawal symptoms. When saline was administered a strong placebo effect was noticed that, however, did not influence the outcome of the results. Only three out of the twelve patients receiving dynorphin-(1-13) experienced some mild side-effects such as feeling warm, dizziness and precordial formication.

Endothelium-dependent relaxation induced by hawthorn extract in rat mesenteric artery

The extract prepared from hawthorn (Crataegus fruit) was examined for its relaxant effect in rat isolated mesenteric arteries. Hawthorn extract induced concentration-dependent relaxation of the U46619-precontracted artery with an IC50 of 0.22 +/- 0.02 mg/ml. Removal of the functional endothelium reduced by approximately 85% the maximum relaxant response to hawthorn extract. Pretreatment of the arterial tissues with N(G)-nitro-L-arginine methyl ester (3-10 microM) or methylene blue (3-10 microM) inhibited the relaxation induced by hawthorn extract, while indomethacin (10 microM) had no effect. L-arginine (3 mM) did not affect the relaxation induced by hawthorn extract but partially reversed the effect of 10 microM N(G)-nitro-L-arginine methyl ester. Iberiotoxin (100 nM) slightly but significantly inhibited the relaxant effect of hawthorn extract whilst glibenclamide (3 microM) was ineffective. Glibenclamide at 3 microM reversed the relaxation induced by pinacidil. N(G)-nitro-L-arginine methyl ester and methylene blue markedly inhibited acetylcholine-induced relaxation in endothelium-intact arteries. Hawthorn extract also reduced the contraction induced by phenylephrine (1 microM) or high Ki (60 mM) with respective IC50 values of 0.13 +/- 0.01 mg/ml and 0.11 +/- 0.01 mg/ml. In high K+-contracted arteries, hawthorn extract induced only 55% of relaxation while it caused a complete inhibition of the U46619- or phenylephrine-induced contraction. These results suggest that hawthorn contains active components which cause vasorelaxation in rat isolated mesenteric arteries. Nitric oxide but not other endothelium-derived vasoactive factors was probably involved in the relaxation induced by hawthorn extract.

Cloning of the cDNA of α-momorcharin: a ribosome inactivating protein

Abstract Using a λgt11 cDNA library constructed from the seeds of the bitter melon ( Momordica charantia ), we have obtained a full length cDNA containing the entire sequence of α-momorcharin by immunoscreening. The length of this cDNA is 1044 basepairs long and it consists of an open reading frame coding for a polypeptide of 286 amino acids. The first 23 residues of this polypeptide probably code for a signal sequence. The N-terminal sequence of the deduced protein is exactly identical to that determined by peptide sequencing. The sequence identity between α-momorcharin and other ribosome inactivating proteins, such as trichosanthin and ricin A chain, is high, i.e., 34–63%. Examination of the predicted secondary structure of α-momorcharin and trichosanthin indicates that these proteins have regions of high structural similarities and this may account for the common biological activities that they share, viz., abortificant, immunosuppressive, antitumor and inhibition of HIV-1.

Beta-endorphin-like immunoactivity in the plasma of heroin addicts and normal subjects.

Abstract The plasma beta-endorphin levels of heroin addicted and normal subjects were measured by radioimmunoassay. The mean immunoassayable beta-endorphin activity of the addicted group was 345 pg/ml and that of the normal group was 1024 pg/ml. By the Students T-test, the difference between the two groups was significant.

Tetramethylpyrazine for treatment of experimentally induced stroke in Mongolian gerbils.

Tetramethylpyrazine, a drug originally isolated from the rhizome of Ligusticum walliichi, has been used routinely in China for the treatment of stroke and angina pectoris. We evaluated this drug by testing its effectiveness in increasing the survival rate in a stroke model using Mongolian gerbils. Our results indicate that tetramethylpyrazine can increase survival rate only if it is administered before the induction of cerebral ischemia. Since we administered the drug intraperitoneally, it is possible that pretreatment was necessary to increase its effective concentration in the blood. Receptor binding studies indicated that tetramethylpyrazine was inactive against a variety of pharmacologically active receptors.

High-performance liquid chromatographic method for simultaneous determination of hawthorn active components in rat plasma

A simple HPLC method with photodiode-array (PDA) ultraviolet detection was developed for the simultaneous determination of four active polyphenol components of hawthorn (Crataegus), chlorogenic acid, epicatechin, hyperoside and isoquercitrin, in rat plasma. Following extraction from the plasma samples with ethyl acetate-methanol (2:1, v/v), these four compounds were successfully separated using a C18 column with a gradient elution of 5 and 25% acetonitrile in 25 mM phosphate buffer (pH 2.4). The flow-rate was set at 1 ml/min and the eluent was detected at 325 nm for chlorogenic acid, 278 nm for epicatechin, and 360 nm for both hyperoside and isoquercitrin. Narignin (0.82 microg) was used as the internal standard and was detected at 278 nm. The method is linear over the studied range of 0.16-40, 0.63-160, 0.13-32 and 0.13-30 microg/ml for chlorogenic acid, epicatechin, hyperoside and isoquercitrin, respectively. The correlation coefficient for each analyte was greater than 0.995. The intra-day and inter-day precision of the analysis was better than 4 and 7%, respectively. The extraction recoveries at low to high concentration were greater than 85% for both epicatechin and chlorogenic acid, and greater than 94% for both hyperoside and isoquercitrin. The detection limits were 0.04, 0.20, 0.03 and 0.03 microg/ml for chlorogenic acid, epicatechin, hyperoside and isoquercitrin. The developed method was used to analyze the plasma concentrations of the four analytes after the intravenous administration of hawthorn polyphenol extract to rats.

Inhibitory effect of jasmine green tea epicatechin isomers on LDL-oxidation

Oxidative modification of low-density lipoproteins (LDL) is thought to play an important role in the development of atherosclerosis. The present study examined inhibitory effect of jasmine green tea polyphenol (GTP) extracts and its individual epicatechin isomers on Cu+2-mediated oxidation of human LDL. The jasmine tea GTP extracts consisted mainly of four epicatechin isomers with (−) epigallocatechin gallate (EGCG) being most abundant (51.2%) followed by (−) epigallocatechin (EGC, 18.7%), (−) epicatechin (EC, 12.3%), and (−) epicatechin gallate (ECG, 11.8%). Jasmine tea GTP as a mixture or its four epicatechin isomers demonstrated strong antioxidant activity in Cu+2-mediated oxidation of human LDL. The inhibitory effect of these epicatechin isomers on LDL oxidation was dose-dependent at the concentrations ranging from 5 to 40 μM. EC and EGC were less protective against LDL oxidation than their gallate derivatives, ECG and EGCG, respectively. ECG and EGCG seemed to have a similar antioxidant activity. In contrast, EGC was less effective than EC as an antioxidant against LDL oxidation. The inhibitory effect of jasmine green tea epicatechin isomers on Cu+2-mediated oxidation was also characterized by protecting docosahexaenoic, arachidonic, α-linolenic, and linoleic acid in LDL against oxidative degradation. We conclude that, in addition to their hypocholesterolemic effect, jasmine tea epicatechin isomers may serve as a source of natural antioxidative agents in human diet.

Hypocholesterolemic activity of hawthorn fruit is mediated by regulation of cholesterol-7α-hydroxylase and acyl CoA: cholesterol acyltransferase

The present study was to investigate the mechanisms by which hawthorn fruit lowers serum cholesterol in hamsters. The control group was fed a semisynthetic diet containing 0.1% cholesterol while the tested group was maintained on the same diet but supplemented with 0.5% hawthorn fruit aqueous ethanolic extract for 4 weeks. Serum total cholesterol (TC) and triacylglycerols (TG) were decreased by 10 and 13%, respectively, in hawthorn fruit group as compared with the control (P<0.05). Supplementation of hawthorn fruit aqueous ethanolic extract led to greater excretion of both neutral and acidic sterols. Further enzymatic assays suggest the mechanisms by which hawthorn fruit decreases serum cholesterol involve a greater excretion of bile acids mediated by up-regulation of hepatic cholesterol 7α-hydroxylase (CH) activity, and an inhibition of cholesterol absorption mediated by down-regulation of intestinal acyl CoA:cholesterol acyltransferase (ACAT) activity.

Comparison of the Pharmacokinetics of Hawthorn Phenolics in Extract Versus Individual Pure Compound

The pharmacokinetics of an active herbal substance may be different when administered in an extract form as compared to that when administered as a pure compound. This study investigated the pharmacokinetics of 4 active compounds of hawthorn fruits—namely, (−)‐epicatechin, chlorogenic acid, hyperoside, and isoquercitrin—following administration of an extract formulation (as hawthorn phenolic extract, which contained the active compounds) or equivalent doses of individual pure compound in male Sprague‐Dawley rats (n = 5 per group). The hawthron phenolic extract or pure compounds were administered both orally and intravenously. Following administration, multiple plasma samples were obtained, and the plasma concentrations were determined by high‐performance liquid chromatography. After the intravenous injection of hawthorn phenolic extract, higher plasma drug concentration, larger area under the plasma concentration‐time curve from 0 to infinity, longer terminal elimination half‐life, smaller apparent volume of distribution, lower total body clearance, and higher urinary excretion of each compound were obtained when compared to that after the pure compound. Following the oral administration of either hawthorn phenolic extract or pure compound, only epicatechin was absorbed, and their pharmacokinetics were generally not significantly different between these 2 formulations. The differences in the pharmacokinetics of the 2 formulations following intravenous but not oral administration may be attributable to the existence of other co‐occurring components in the hawthorn phonolic extract (which may be present in the body after intravenous but not oral administration). The results showed that an herbal extract formulation, when administered intravenously, could potentially alter the pharmacokinetics of its active ingredients.