A novel analytical ELISA-based methodology for pharmacologically active saikosaponins.

A competitive enzyme-linked immunosorbent assay (ELISA) for saikosaponins was established using monoclonal antibody (MAb) 3G10. Hybridoma 3G10 prepared by fusing splenocytes immunized with saikosaponin a-BSA (SSa-BSA) conjugate and a hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line, P3-X63-Ag8-U1, secreted monoclonal antibodies with wide cross-reactivity to saikosaponins including saikosaponin b(2) (SSb(2)), c (SSc) and d (SSd), which are stereo and/or regio isomers of SSa. The method, at an effective measuring range of 0.6 mug /ml to 2.3 mug/ml of SSa, successfully detected total saikosaponins in Bupleuri radix and Kampo medicines prescribed with Bupleuri radix. Good correlation between ELISA and HPLC analyses of total saikosaponin in a crude extract of Bupleuri radix was obtained after hydrolysis of acyl saikosaponins by treatment with a mild alkaline solution.

Production of monoclonal antibody against ginkgolic acids in Ginkgo biloba Linn.

A competitive enzyme-linked immunosorbent assay (ELISA) for ginkgolic acids (GAs) was developed using monoclonal antibody (MAb) 9F raised against 6-(13-formylheptyl) salicylic acid covalently coupled to bovine serum albumin (BSA). ELISA, at an effective measuring range of 300 ng/ml-1 microgram/ml of GA15:1, was successful in detecting GAs content in ginkgo leaves and standardized extracts due to the lack of cross-reactivity against various related compounds. The sensitive and simple immunoassay developed in this study was validated to be specific for the quantitative determination of total GAs content in ginkgo crude drugs with no interference from the sample matrix. The analytical recovery of spiked GA15:1 was 103% in a concentration range between 10 and 40 mg/g dry weight of ginkgo leaves.

Anti-solasodine glycoside single-chain Fv antibody stimulates biosynthesis of solasodine glycoside in plants.

We constructed a recombinant antibody fragment--single chain fragment-variable (scFv) antibody--derived from hybridoma cell lines to control the concentration of solasodine glycosides in hairy root cultures of Solanum khasianum transformed by the anti-solamargine (As)-scFv gene. The properties of the As-scFv protein expressed in Escherichia coli were almost identical to those of the parent monoclonal antibody (MAb). Up to 220 ng recombinant As-scFv was expressed per milligram of soluble protein in transgenic hairy root cultures of S. khasianum. The concentration of solasodine glycosides was 2.3-fold higher in the transgenic than in the wild-type hairy root, as reflected by the soluble As-scFv level and antigen binding activities. These results suggested that the scFv antibody expressed in transgenic hairy roots controlled the antigen level, thus representing a novel plant breeding methodology that can produce secondary metabolites.

Crocin suppresses tumor necrosis factor-alpha-induced cell death of neuronally differentiated PC-12 cells.

Crocus sativus L. is used in Chinese traditional medicine to treat some disorders of the central nervous system. Crocin is an ethanol-extractable component of Crocus sativus L.; it is reported to prevent ethanol-induced impairment of learning and memory in mice. In this study, we demonstrate that crocin suppresses the effect of tumor necrosis factor (TNF)-alpha on neuronally differentiated PC-12 cells. PC-12 cells dead from exposure to TNF-alpha show apoptotic morphological changes and DNA fragmentation. These hallmark features of cell death did not appear in cells treated in the co-presence of 10 microM crocin. Moreover, crocin suppressed the TNF-alpha-induced expression of Bcl-Xs and LICE mRNAs and simultaneously restored the cytokine-induced reduction of Bcl-X(L) mRNA expression. The modulating effects of crocin on the expression of Bcl-2 family proteins led to a marked reduction of a TNF-alpha-induced release of cytochrome c from the mitochondria. Crocin also blocked the cytochrome c-induced activation of caspase-3. To learn how crocin exhibits these anti-apoptotic actions in PC-12 cells, we tested the effect of crocin on PC-12 cell death induced by daunorubicin. We found that crocin inhibited the effect of daunorubicin as well. Our findings suggest that crocin inhibits neuronal cell death induced by both internal and external apoptotic stimuli.

Double staining of ginsenosides by Western blotting using anti-ginsenoside Rb1 and Rg1 monoclonal antibodies.

Ginsenosides separated by silica gel TLC blotted to a polyvinylidene difluoride (PVDF) membrane was treated with a NaIO4 solution followed by bovine serum albumin (BSA), resulting in a ginsenoside-BSA conjugate on a PVDF membrane. The blotted bands were stained with anti-ginsenoside Rg1 and Rb1 monoclonal antibodies (MAbs). The newly established double staining with the Western blotting methods was applied for the determination of ginsenosides possessing protopanaxadiol or protopanaxatriol and the number of sugar depending on the stained color and their Rf values in the Panax plants.

Production of monoclonal antibodies against a major purgative component, sennoside B, their characterization and use in ELISA.

For immunization, sennoside B was conjugated with bovine serum albumin. The hapten density in the antigen conjugate was determined to be 3 mol mol(-1) protein by matrix-assisted laser desorption-ionization TOF mass spectrometry. A hybridoma secreting monoclonal antibody against sennoside B was produced by fusing splenocytes from mouse immunized with the sennoside B conjugate and mouse myeloma cells. Weak cross-reactivities occurred with sennoside A which is a stereochemical isomer, and a monomer of sennoside B, rhein, but no cross-reactivity was observed with other related anthraquinones and phenolics. The range of the assay extended from 0.5 ng ml(-1) to 15 ng ml(-1) of sennoside B, and good correlation between ELISA and HPLC methods was obtained when crude extracts of rhubarb were analyzed.

Morphine metabolism in the opium poppy and its possible physiological function. Biochemical characterization of the morphine metabolite, bismorphine.

We identified a novel metabolic system of morphine in the opium poppy (Papaver somniferum L.). In response to stress, morphine is quickly metabolized to bismorphine consisting of two morphine units, followed by accumulation in the cell wall. This bismorphine binds predominantly to pectins, which possess high galacturonic acid residue contents, through ionical bonds. Our newly developed method using artificial polysaccharides demonstrated that bismorphine bridges are formed between the two amino groups of bismorphine and the carboxyl groups of galacturonic acid residues, resulting in cross-linking of galacturonic acid-containing polysaccharides to each other. The ability of bismorphine to cross-link pectins is much higher than that of Ca2+, which also acts as a cross-linker of these polysaccharides. Furthermore, we confirmed that cross-linking of pectins through bismorphine bridges leads to resistance against hydrolysis by pectinases. These results indicated that production of bismorphine is a defense response of the opium poppy. Bismorphine formation is catalyzed by anionic peroxidase that pre-exists in the capsules and leaves of opium poppies. The constitutive presence of morphine, together with bismorphine-forming peroxidase, enables the opium poppy to rapidly induce the defense system.

Inhibition of P-glycoprotein by orange juice components, polymethoxyflavones in adriamycin-resistant human myelogenous leukemia (K562/ADM) cells.

We investigated the effects of the ethyl acetate extract of grapefruit juice (GFJ), that of orange juice (OJ) and their components on the uptake of [(3)H]vincristine into adriamycin-resistant human myelogenous leukemia cells. Its uptake was increased by the extracts of GFJ and OJ up to 7- and 3-fold, respectively, as well as verapamil and cyclosporin A. OJ components, i.e. 3,3',4',5,6,7,8-heptamethoxyflavone, nobiletin and tangeretin, also increased the uptake of [(3)H]vincristine in a concentration-dependent manner. Although GFJ components, dihydroxybergamottin and bergamottin, significantly increased the uptake, their potencies were considerably weaker than those of OJ components. These data suggest that OJ components inhibit P-gp-mediated efflux of [(3)H]vincristine, resulting in the intracellular accumulation of chemotherapeutic drugs. These components may become candidates of multi-drug resistance reversing agents in cancer chemotherapy.

Glycosidic constituents from in vitro Anoectochilus formosanus.

The glycosidic constituents of whole plants of Anoectochilus formosanus propagated by tissue culture were investigated. A new compound, 2-(beta-D-glucopyranosyloxymethyl)-5-hydroxymethylfuran, along with the known compounds, 3-(R)-3-beta-D-glucopyranosyloxybutanolide (kinsenoside), 3-(R)-3-beta-D-glucopyranosyloxy-4-hydroxybutanoic acid, 1-O-isopropyl-beta-D-glucopyranoside, (R)-(+)-3,4-dihydroxy-butanoic acid y-lactone, 4-(beta-D-glucopyranosyloxy)benzyl alcohol, (6R,9S)-9-hydroxy-megastigma-4,7-dien-3-one-9-O-beta-glucopy ranoside, and corchoionoside C were isolated.

[Pharmacognosical study on secondary metabolites].

Clonal micropropagation on various medicinal plants was set up resulting in the regenerated plants which possessed a homogeneous quality. The ratio of hapten to bovine serum albumin (BSA) in an antigen conjugate was determined by matrix-assisted laser desorption/ionization of mass spectrometry. A hybridoma secreting monoclonal antibody (MAb) was produced by fusing splenocytes immunized with an antigene-BSA conjugate with mouse myeloma cells. Competitive enzyme-linked immunosorbent assay (ELISA) using MAb was set up as a high sensitive, specific and reproducible qualitative method. A method of determination for ginsenosides by using a unique western blotting was established. Immunoaffinity column chromatography using an anti-ginsenoside Rb1MAb has made possible a single-step separation of ginsenoside Rb1 from a crude ginseng extract. Single chain Fv gene of anti-forskolin MAb was prepared from mRNA of hybridoma secreting anti-forskolin MAb and cloned. Gene was constructed into a pET-28a(+) vector producing a scFv protein. Modeling of forskolin and scFV was investigated. THCA synthase was purified from the homogenate of Cannabis sativa leaves on successive column chromatographies. THCA synthase was confirmed to be homogeneity having 75 kDa. To obtain the corresponding cDNA clone of THCA synthase, a set of degenerate promers was constructed based on N-terinal and internal amino acid sequences of THCA synthase. The 5' and 3' ends of cDNA were amplified by RACE. A full sequencing has been determined to be corded a polypeptide having 545 amino acid residues. The cDNA clone was expressed in yeast system via PUC19 vector resulting in THCA synthase activity.

Applications of ELISA, western blotting and immunoaffinity concentration for survey of ginsenosides in crude drugs of Panax species and traditional Chinese herbal medicines.

A combination of ELISA, Western blotting and immunoaffinity concentration using an anti-ginsenoside Rb1 monoclonal antibody was applied for qualitative and quantitative surveys of ginsenoside Rb1 and related ginsenosides in roots and traditional Chinese herbal medicines. To improve the low correlation between ELISA and HPLC analysis, the crude extract of roots was immunoaffinity concentrated to make evident the effect of malonyl ginsenoside Rb1. Immunoaffinity column chromatography also concentrated an unknown ginsenoside which had the same cross-reaction with ginsenoside Rb1.

Effect of furanocoumarin derivatives in grapefruit juice on the uptake of vinblastine by Caco-2 cells and on the activity of cytochrome P450 3A4.

1. The presence of inhibitors of drug efflux transporters, such as P-glycoprotein (P-gp), in grapefruit juice (GFJ) was confirmed based on the uptake of [(3)H]-vinblastine (VBL) by Caco-2 cells. 2. The uptake of [(3)H]-VBL by Caco-2 cells was significantly increased by the ethyl acetate extract of GFJ as well as by cyclosporin A. The extract was separated on a Cosmosil column and the eluate with 60% methanol increased [(3)H]-VBL uptake, while the activity to inhibit CYP3A4 was greatest in the 70 and 80% eluates. 3. These results show that the major inhibitor of efflux transport of VBL is different from that of CYP3A4. 4. Further separation of the 60% methanol eluate afforded dihydroxybergamottin (DHBG). Both ethyl acetate extract of GFJ and DHBG increased steady-state [(3)H]-VBL uptake by LLC-GA5-COL300 cells. Besides DHBG, other furanocoumarins contained in GFJ, such as bergamottin, FC726, bergaptol and bergapten, increased the steady-state uptake of [(3)H]-VBL by Caco-2 cells. 5. The order of inhibitory potency of these compounds was FC726>DHBG>bergamottin>bergapten>bergaptol . While, the IC(50) values for inhibition of CYP3A4 were 0.075, 0.45, 1.0, 1.0 and >20 microM, respectively. Bergaptol specifically inhibited VBL efflux. 6. DHBG was thus identified as a candidate for inhibitors of VBL transport, together with other furanocoumarins. Moreover, partly involvement of the P-gp inhibition was suggested. 7. Therefore, the inhibition of efflux transport of drugs as well as of drug metabolism by CYP3A4 could be an important cause of drug-GFJ interaction.

Steroid 5alpha-reductase inhibitory activity and hair regrowth effects of an extract from Boehmeria nipononivea.

The acetone extract of Boehmeria nipononivea showed both potent 5alpha-reductase inhibitory activity and hair regrowth promotion effects on mice. 5alpha-Reductase inhibitory activity-guided fractionation led to six active fatty acids: alpha-linolenic, linoleic, palmitic, elaidic, oleic and stearic acids. The extract of B. nipononivea, and alphalinolenic, elaidic and stearic acids exhibited a hair regrowth effect.

Hepatoprotective aliphatic glycosides from three Goodyera species.

Hepatoprotective aliphatic glycosides 3-(S)-3-beta-D-glucopyranosyloxybutanolide (1) and its congener, 3-(S)-3-beta-D-glucopyranosyloxy-4-hydroxybutanoic acid (2) were isolated as major constituents from the whole plants of three Goodyera species, G. schlechtendaliana Reichb. fil., G. matsumurana Schltr. and G. discolor Ker-Gawl. The structures of 1 and 2 have been determined by NMR, MS spectroscopic and chemical means. Compound 1 was converted into its methyl ester form (5) during the purification step, when the lactone ring was cleaved by catalysis of silica gel with the CHCl3-MeOH-H2O as a solvent. On the other hand, 1 was obtained in a high yield by the same purification procedure without MeOH. Based on this fact, a simple and economic method for the purification of 1 was confirmed. Compounds 1 and 2 were found to have a hepatoprotective effect on liver injury induced by carbon tetrachloride in primary cultured rat hepatocytes.

Membrane associated antitumor effects of crocine-, ginsenoside- and cannabinoid derivates.

In the present work a systematic study was initiated with crocine, ginsenoside and cannabinoid derivatives on multidrug resistant mouse lymphoma cells, viral tumor antigen expression and some human leukocyte functions. Among saffron derivatives, crocin and picrocrocin, triglucosyl and diglucosyl crocetin were ineffective on the reversal of multidrug resistance of lymphoma cells. Ginsenoside increased drug accumulation and tumor antigen expression at 2.0-20.0 micrograms/mL. Some cannabinoid derivatives such as cannabinol, cannabispirol and cannabidiol increased drug accumulation, while cannabidiolic acid, delta-9-THC and tetrahydro-cannabidiolic acid reduced drug accumulation of the human mdr1-gene transfected mouse lymphoma cells. The reversal of multidrug resistance is the result of the inhibition of the efflux pump function in the tumor cells. Crocetin esters were less potent than crocin itself in the inhibition of EBV early antigen expression. However crocin and diglucosylcrocetin inhibited early tumor antigen expression of adenovirus infected cells, but triglucosylcrocetin was less effective at 0.01-1.0 microgram/mL. The crocin had no antiviral effect [on HSV-2 infected vero cells] up to 25 micrograms/mL concentration. Ginsenosides had a moderate inhibitory effect except ginsenoside Rb1 (was the less effective) on the drug efflux pump. Among the cannabinoid derivatives the cannabinol and cannabispirol increased drug accumulation, while cannabidiolic acid and delta-8-THC, delta-9-THC and tetrahydro-cannabinol reduced drug accumulation in multidrug resistant mouse lymphoma cells. It is interesting that ginsenosides had a chemical structure-dependent immunomodulating effect by enhancing the activity of NK-cells and ADCC activities.

Flavonoids from Goodyera schlechtendaliana.

A flavonol glycoside, 3-[[6-O-(6-deoxy-alpha-L-mannopyranosyl)-beta-D- glucopyranosyl]oxi]-5,7-dihydroxy-8-[(4-hydroxy-3,5-dimethoxyphenyl)meth yl]- 2-(3,4-dihydroxypheny)-4H-1-benzopyran-4-one, trivially named goodyerin, was isolated from the whole plant of Goodyera schlechtendaliana, along with three known flavonoids, rutin, kaempferol-3-O-rutinoside and isorhamnetin-3-O-rutinoside. The structures were established by spectroscopic analysis.

Isolation of the pharmacologically active saponin ginsenoside Rb1 from ginseng by immunoaffinity column chromatography.

Immunoaffinity column chromatography using an anti-ginsenoside Rb1 monoclonal antibody has made possible a single-step separation of ginsenoside Rb1 from a crude extract of ginseng roots (Panax ginseng). The combination of immunoaffinity column chromatography and an enzyme-linked immunosorbent assay (ELISA) was also investigated.

Biologically active triterpene saponins from callus tissue of Polygala amarella.

A new bioactive saponin (1), together with a known saponin (polygalasaponin XXVIII) has been isolated from the callus tissue culture of Polygala amarella. Based on spectroscopic data, especially direct and long-range heteronuclear 2D NMR analysis and on chemical transformations, the structure of 1 was elucidated as 3-O-beta-D-glucopyranosyl presenegenin-28-O-beta-D-galactopyranosyl-(1 --> 3)-beta-D-xylopyranosyl-(1 --> 4)-alpha-L-rhamnopyranosyl-(1 --> 2)-[beta-D-glucopyranosyl-(1 --> 3)]-beta-D-fucopyranoside. Both saponins showed significant immunological properties based on the enhancement of granulocyte phagocytosis in vitro.

Western blotting for ginseng saponins, ginsenosides using anti-ginsenoside Rb1 monoclonal antibody.

Ginsenosides separated by silica gel TLC were blotted to a polyvinylidene difluoride (PVDF) membrane which was treated with a NaIO4 solution followed by bovine serum albumine (BSA), resulting in a ginsenoside-BSA conjugate on a PVDF membrane. The blotted bands were stained with monoclonal antibody (MAb). The newly established Western blotting method was used for the determination of ginsenosides and their distribution in various Panax species.

Genetic and alkaloid analysis of Papaver species and their F1 hybrid by RAPD, HPLC and ELISA.

Total DNA was extracted from the leaves of a F1 hybrid and its parents, Papaver bracteatum Lindle and P. pseudo-orientale Medw. Analysis of random-amplified polymorphic DNA (RAPD) using ten arbitrary oligonucleotide 10-mers, showed that F1 hybrid was confirmed to be genetically intermediate of both parental plants compared with the genetic distance between F1 hybrid and individual parents. Furthermore, the comparison of the band patterns between a F1 hybrid, P. bracteatum and P. pseudo-orientale clearly showed that part of the bands of both parents were induced into a F1 hybrid. The content of thebaine was determined by HPLC and ELISA used anti-thebaine monoclonal antibody.