Determination of ginsenoside Rf and Rg2 from Panax ginseng using enzyme immunoassay

We have developed an enzyme immunoassay (EIA) to quantify trace amounts of ginsenoside Rf (Rf), one of the glycosides of protopanaxatriol from Panax ginseng. A carrier protein of bovine serum albumin (BSA) was coupled to the carbohydrate component of Rf using the periodate oxidation method. Antibodies were raised in rabbits using Rf-BSA conjugate as the immunogen and competitive indirect EIA was used for the determination of Rf. The working range was 0.01-10 ng per assay. The anti-Rf antiserum cross-reacted with ginsenoside Rg2 (105%), which is also a component of Panax ginseng and has a very similar chemical structure to Rf. These results suggest that the anti-Rf antiserum could also be used for the quantitation of ginsenoside Rg2 as well as ginsenoside Rf. In a comparison of EIA and HPLC the linear regression equation and correlation coefficient for the two methods were y(EIA) = 1.31x (HPLC)-11.48 and 0.98, respectively.     

The physical map of the chloroplast DNA from Korean ginseng (Panax ginseng C.A. Meyer)

To compare the gene order of the chloroplast genome among dicotyledonous plants, we constructed a physical map of chloroplast DNA (cpDNA) of Korean ginseng (Panax ginseng C.A. Meyer) with four restriction enzymes, BamHI, HindIII, EcoRI, and PstI. The restriction enzyme recognition sites of the physical map were also confirmed by Southern hybridization of total ginseng cpDNA with homologous and heterologous probes. The cpDNA of Korean ginseng was determined as a circular molecule with a total size of about 154 kb, which contain two inverted repeats of 23 kb each that disrupt the rest of the molecule into a large (90 kb) and a small single copy region (18 kb). The genome structure of Korean ginseng cpDNA was similar in size and gene order to that of tobacco cpDNA. The cpDNA of Korean and American ginseng (P. quinquefolius) showed very similar restriction patterns.     

Stimulatory effect of saponin from Panax ginseng on immune function of lymphocytes in the elderly

In order to determine the epitope structure in peptide NP50-63, which has been reported to be the only Kk-restricted T-cell antigen within the influenza virus (A/PR/8/34) nucleoprotein, a series of 13 peptides truncated from C- and N-termini of NP50-63 were synthesized and their sensitizing activities against Kk-restricted nucleoprotein-specific cytotoxic T lymphocytes (CTLs) were examined. One of the 13 peptides, NP50-57, sensitized L929 cells at the nM level, which was 100-1000 times lower in concentration than that at which the other peptides sensitized these cells. The presence of NP50-57 in A/PR/8/34-infected L929 cells was also investigated. Acid extracts of virus-infected cells were separated on a reverse-phase HPLC column and then anion-exchange column. By both separations, only one peak of sensitizing activity against nucleoprotein-specific CTLs was observed. The position of the peak coincided with that of the elution of NP50-57. These results strongly suggest that NP50-57 is the natural epitope in the antigenic structure, NP50-63.     

Inhibition by ethanol of noradrenaline output from peripheral sympathetic nerves: possible interaction of ethanol with neuronal receptors

The sympathetic nerve terminals of the isolated rabbit heart perfused with Tyrode solution were used to study the action of ethanol on the noradrenaline uptake and release. The uptake of exogenous noradrenaline (10 ng/ml) into the sympathetic nerve endings, the noradrenaline output evoked by raising the concentration of potassium ions in the perfusion fluid, and the release in response to electrical stimulation of the nerve axons were inhibited only by lethal concentrations of the alcohol; the concentrations which caused 50% inhibition (IC50) amounted to 760 mM, 830mM, and 1150 mM respectively. However, ethanol at concentrations compatible with moderate intoxication reduced the noradrenaline release in response to activation of the nicotine receptors on the nerve terminals by dimethylphenylpiperazine; the threshold concentration was 36 mM and the IC50 was 129 mM. It is suggested that this effect is due to hydrophobic interaction of the alcohol with receptor proteins, thus inhibiting stimulus formation.     

Structural Analysis of an Oligosaccharide and Glycopeptide Mixture from Panax Ginseng Root with Inhibition SHP-1 Function

A mixture of oligosaccharide and glycopeptide was isolated from the aqueous extract of Panax ginseng roots.The mixture inhibits protein tyrosine phosphatase(SHP-I)function,implying it enhances immune activity.The peak molecular mass of the oligosaccharide portion is 1800 calculated via GPC software after separation by HPLC.And the structure of the oligosaccharide portion is the backbone of(1→3)-and(1→4)-linked arabinopyranoside,and(1→4)-and(1→6)-linked glucopyranoside,with non-reducing terminals of arabinopyranoside and glucopyranoside.The peak molecular mass of glycopeptide portion is 1900 calculated via GPC software after separation by HPLC.The structure of glycopeptide portion is the backbone of(1→3)-and(1→4)-linked arabinopyranoside,and(1→3,6)-linked glucopyranoside,with non-reducing terminals of galactopyranose and glucopyranoside.The peptide composition is Glu,Asp,Hyp,Ser,Arg,Gly,Thr,Pro,Ala,Val,Ile,Leu and Lys.The oligosaccharide-peptide linkage is formed by Ara and Hyp.     

Inhibition of melatonin secretion by ethanol in man

To determine whether ethanol inhibits nocturnal melatonin (MT) secretion, three experiments (A, B, and C) were performed in seven normal subjects. In A, ethanol at a dose of 0.34 g/kg was administered orally at 6:00, 8:00, and 10:00 PM. Each dose was increased to 0.52 g/kg in B. In C, water was substituted for ethanol. Blood samples for determination of serum MT levels were drawn every second hour between 6:00 PM and 8:00 AM. Urinary excretion of MT during the night was also determined. In A, serum ethanol reached a maximal level of 13 +/- 1 mmol/L at 12 midnight. In B, the corresponding maximum was 25 +/- 1 mmol/L. The higher alcohol dose inhibited nocturnal MT secretion by 20% +/- 5% (P < .01), whereas the lower dose lacked such effect. Urinary excretion of MT was left unaffected by alcohol at both doses. Five additional normal subjects were given alcohol as described above at a dose of 0.52 g/kg (experiment D). This induced mild nocturnal hypoglycemia as evidenced by a glucose decremental area (5.9 +/- 1.8 mmol/L.h) that differed significantly from zero (P < .05). To determine whether a reduced glucose delivery to pinealocytes might contribute to the decreased MT secretion in alcohol-intoxicated subjects, two experiments (E and F) were performed in eight healthy individuals. In E, ethanol was given orally as in B; three small oral doses of glucose were also given at 8:00 PM, 10:00 PM, and 12 midnight. In F, water was substituted for ethanol and glucose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of adrenaline-induced lipolysis by ginseng polypeptide and its modified peptides

The anti-lipolysis by ginseng polypeptide and its modified peptides was examined using porcine adipose cells. Ginseng polypeptides modified by amino acid substitution or proteolyzation reduced or lost the inhibiting activity of adrenalin-induced lipolysis. Correlation between the anti-lipolytic activity of ginseng polypeptide and its Mg(2+)- and ribose-binding activities is discussed.     

Inhibition of binding of malaria-infected erythrocytes by a tetradecasaccharide fraction from chondroitin sulfate A

Adherence of parasite-infected erythrocytes (IEs) to the microvascular endothelium of various organs, a process known as sequestration, is a feature of Plasmodium falciparum malaria. This event is mediated by specific adhesive interactions between parasite proteins, expressed on the surface of IEs, and host molecules. P. falciparum IEs can bind to purified chondroitin sulfate A (CS-A), to the proteoglycan thrombomodulin through CS-A side chains, and to CS-A present on the surface of brain and lung endothelial cells and placental syncytiotrophoblasts. In order to identify structural characteristics of CS-A important for binding, oligosaccharide fragments ranging in size from 2 to 20 monosaccharide units were isolated from CS-A and CS-C, following controlled chondroitin lyase digestion, and used as competitive inhibitors of IE binding to immobilized ligands. Inhibition of binding to CS-A was highly dependent on molecular size: a CS-A tetradecasaccharide fraction was the minimum length able to almost completely inhibit binding. The effect was dose dependent and similar to that of the parent polysaccharide, and the same degree of inhibition was not found with the CS-C oligosaccharides. There was no effect on binding of IEs to other ligands, e.g., CD36 and intercellular adhesion molecule 1. Hexadeca- and octadecasaccharide fractions of CS-A were required for maximum inhibition of binding to thrombomodulin. Analyses of oligosaccharide fractions and polysaccharides by electrospray mass spectrometry and high-performance liquid chromatography suggest that the differences between the activities of CS-A and CS-C oligosaccharides can be attributed to differences in sulfate content and sulfation pattern and that iduronic acid is not involved in IE binding.     

Polyacetylene analogs, isolated from hairy roots of Panax ginseng, inhibit Acyl-CoA : cholesterol acyltransferase

In the course of our screening program for acyl-CoA : cholesterol acyltransferase (ACAT) inhibitors from Korean herbal medicines, ACAT inhibitors were isolated from the hairy roots of Panax ginseng (Araliaceae) and identified as panaxynol, panaxydol, panaxydiol, and panaxytriol. These active compounds inhibit rat liver ACAT with IC50 values of 94, 80, 45 and 79 microM, respectively.     

Inhibition of muscarinic receptor-stimulated glial cell proliferation by ethanol

Acetylcholine and other muscarinic agonists stimulate the proliferation of rat cortical astrocytes and 132 1N1 human astrocytoma cells by activating muscarinic m3 cholinergic receptors. Ethanol was a potent inhibitor of carbachol-stimulated proliferation, measured by [3H]thymidine incorporation, with an IC50 of 10 mM. On the other hand, basal and serum-stimulated proliferation of astrocytes and astrocytoma cells was inhibited by ethanol with lower potency (IC50 = 200-250 mM). Concentration-response experiments with carbachol, in the presence of 10 mM ethanol, suggested that inhibition of proliferation by the alcohol was of the noncompetitive type. Experiments with acetaldehyde and with the alcohol dehydrogenase inhibitor 4-methylpyrazole suggested that the inhibitory effect of alcohol was due to ethanol itself and not to its metabolite acetaldehyde. Proliferation of astrocytoma cells induced by carbachol and the inhibitory effects of ethanol were also confirmed by flow cytometry using the 5-bromodeoxyuridine-Hoechst 33258 method. Ethanol (10 mM) had no effect on proliferation induced by 50 micrograms/ml insulin and 100 ng/ml platelet-derived growth factor BB; on the other hand, the mitogenic effect of 1 mM histamine, 100 U/ml interleukin-1, and 100 ng/ml 12-O-tetradecanoylphorbol 13-acetate were inhibited by approximately 50%. These results indicate that proliferation of glial cells induced by muscarinic agonists is especially sensitive to the inhibitory effect of ethanol. This action of ethanol may be relevant to its developmental neurotoxicity, particularly microencephaly, which is one of the common features of the fetal alcohol syndrome.     

Facile method for the preparation of 7-methyl-8-oxoguanosines as an immunomodulator

Reaction of 9-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)-7-methylguaninium iodide (2a) with hydrogen peroxide in acetic acid gave the corresponding 7-methyl-8-oxoguanosine derivative (3a) in good yield. Deprotection of 3a easily gave 7-methyl-8-oxoguanosine (1), which is well-known as an immunomodulator. Substitution of acetyl group at the N2-position of guanine ring accelerated the oxidation reaction of the 7-methylguaninium iodide.     

Anti-tumor-promoting activity of majonoside-R2 from Vietnamese ginseng, Panax vietnamensis Ha et Grushv. (I)

Seven saponins (1-7) isolated from the rhizomes and roots of Panax vietnamensis were tested for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), in Raji cells as a primary screening test for anti-tumor-promoters (cancer chemopreventive agents). The ocotillol-type saponin, majonoside-R2 (2), which is the major and characteristic constituent of this plant, exhibited a significant inhibitory effect on EBV-EA activation. Furthermore, the cell cycle analysis of 2 on Raji cells was also examined and strong inhibition was observed on the effect of the cell cycle induced by TPA. Compound 2 showed potent anti-tumor-promoting activity in two-stage carcinogenesis tests of mouse skin using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and TPA or fumonisin B1 as a promoter. Consequently, these results suggest that majonoside-R2 (2) could be a valuable chemopreventive agent against chemical carcinogenesis.     

Inhibition of ethanol production by Saccharomyces cerevisiae in human blood by sodium fluoride

Production of ethanol in antemortem blood samples inoculated with an efficient ethanol-producing microorganism and incubated at various temperatures is discussed. Whole blood samples inoculated with Saccharomyces cerevisiae were incubated in gray stoppered Venoject tubes (approximate draw volume 7 mL) containing sodium fluoride (17.5 mg) and potassium oxalate (14.0 mg) at 4 degrees C, 25 degrees C, and 37 degrees C for 0, 24, 96, 192, and 408 h. No volatile substances (such as ethanol, methanol, isopropanol, acetone, or acetaldehyde) (< 0.010 g/dL) were produced in any of the samples at 4 or 25 degrees C. At 24 h incubation a trace amount (< 0.018 g/dL) of ethanol was detected at 37 degrees C.     

Estimation of S-phase fraction in tumor tissue sections by immunohistochemical staining of PCNA

We describe a method for measuring the size of the S-phase fraction in human tumor tissue sections using an antibody to PCNA (PC10). Although treatment with Triton X-100 before fixation extracted a large amount of PCNA from the cells even in frozen tissue sections, PCNA remained exclusively in S-phase cells. Immunohistochemical staining of PCNA after the detergent treatment allowed estimation of the S-phase fraction in solid tumors. The validity of the method was directly proven by double staining of bromodeoxyuridine (BrdU) and PCNA in HeLa cells. The PCNA-positive cells were identical with BrdUrd-positive cells after the detergent treatment. In contrast, almost all HeLa cells in the exponentially growing phase were positive for PC10 without treatment with Triton X-100.     

Inhibition of gastric acid secretion by blood drawing from an indwelling venous needle

To investigate a possible inhibitory effect of blood drawing through an indwelling forearm vein needle on gastric acid secretion, 11 subjects were studied on four occasions each. The first session was for adapting the subject to the 3-hr collection of gastric juice. In 7 subjects the second through fourth sessions gave three conditions in balanced order: (1) an indwelling forearm vein needle and the withdrawal of 5 or 10 cc of blood every 20 min, (2) only a nonfunctional "dummy" needle implanted subcutaneously in the forearm skin, and (3) the control condition with no needle. In four additional subjects the sessions were identical except that condition (1) involved an indwelling forearm vein needle kept open by a slow infusion of saline solution and no blood was drawn. Phenol red recovery from an initial intragastric injection was measured in all. Results showed that blood drawing, but not saline infusion or venipuncture per se, inhibited gastric acid output.     

Inhibition by ginseng total saponin of the development of morphine reverse tolerance and dopamine receptor supersensitivity in mice

1. Ginseng total saponin (GTS), 200 mg/kg i.p. 3 hr prior to morphine, inhibited the development of reverse tolerance to the ambulatory-accelerating effect of morphine. 2. GTS, 200 mg/kg, also prevented the development of dopamine receptor supersensitivity induced by the chronic administration of morphine, 10 mg/kg a day for 7 days. 3. These results suggest that GTS may be useful for the prevention and therapy of the adverse action of morphine.     

Inhibition of tumor necrosis factor and subsequent endotoxin shock by pirfenidone

Tumor necrosis factor-alpha (TNF) is an extremely potent cytokine which is involved in the pathogenesis of a number of diseases. Interruption of its synthesis can result in a reduction of inflammation and subsequent pathology. A new experimental drug pirfenidone (5-methyl-L-phenyl-2-(1H)-pyridone, trade name: Deskar) has been reported to have beneficial effects for the treatment of certain fibrotic diseases. The present study describes the inhibition of TNF in vitro as well as the inhibition of circulating TNF in vivo by pirfenidone. Isolated, thioglycollate-induced peritoneal macrophages (Mphi) from C57BL/6 mice were exposed to either lipopolysaccharide (LPS) or mannosylated bovine serum albumin then incubated with 0.1-0.9 mg/ml of pirfenidone. This substance inhibited the production of TNF in a dose-dependent manner as measured by ELISA. One i.p. injection of either 100 or 200 mg/kg pirfenidone inhibited the induction of circulating TNF following a single i.v. injection of LPS. Endotoxin shock was induced in mice using an i.p. injection of galactosamine and LPS. The higher dose of pirfenidone (200 mg/kg) completely inhibited shock and subsequent mortality. Lower doses of pirfenidone or administration either prior to or post challenge only partially inhibited symptoms. These results indicate that pirfenidone is able to inhibit both TNF induction and subsequent endotoxin shock. Additional studies are warranted to establish this drug as a potential treatment for diseases where TNF plays a major role.