NON-POLAR CONSTITUENTS FROM TARAXACUM OFFICINALE WEBER EX WIGG / Монголын эм зүй, эм судлал

The dichloromethane fraction from the ethanol extract of the aerial parts of Taraxacum offi cinale showed a good inhibitory effect on hepatocellular carcinoma cells (HCC). As a result of a series of column chromatographies and usage of nuclear magnetic resonances spectrometric methods and mass spectroscopy 9 known components as taraxasterol (1), taraxasterylacetate (2),pseudotaraxasterol (3), lupeolacetate (4), b-sitosterol (5), b-sitosterylglucopyranose (6), palmitic acid (7), monopalmitin (8) and chrysoerol (9) have been determined. Amongst them palmitic acid, monopalmitin and chrysoeriol have been determined for the fi rst time in the aerial parts of Taraxacum offi cinale. Six compounds as 1, 3, 4, 5, 6 and 7 were tested for their inhibitory activity on HCC and only palmitic acid exhibited more activity against HCC than others, suppressing cell proliferation, migration, adhesion and activated cell apoptosis.Keywords: Triterpenol and sterol derivatives; palmitic acid; hepatocellular carcinoma inhibition activity;IntroductionTaraxacum, commonly called dandelion, is a large genus of fl owering plants in the Asteraceae family. The latin name Taraxacum is from the Greek and means “disease remedy”, while the English name dandelion is originated from the French dent de leon, meaning “lion’s tooth”1. The Mongolian well-recognised name is “baaban beeben”, while in Japan it calls hokouei, respectively. Consequently, Taraxacum is widespread plant throughout the world, in particular, 19 species are found in the Mongolian fl ora2. Generally Taraxacum is considered weedy plant used as a medicinal herb and for food preparation. Traditionally, Taraxacum offi cinale Weber ex Wigg. in Mongolian and Tibetan medicine under the name “khurmong” the root has been used as the composition in a remedy for jaundice and other disorders of the liver and gallbladder, whilethe leaf is used as a diuretic and bitter digestive stimulant. Moreover, fresh dandelion stem latex is used for the warts treatment1,3-6. Taraxacum leaf is included as a medicinal drug in Herbal Pharmacopeia of several European countries. Numerous biological activity tests resulted that Taraxacum possessed an infl ammation modulating activity7-9, diuretic activity comparable to furosemide10, digestive stimulant, appetitive effect and activator for bile fl ow11-12, hypoglycemic activity13 and antitumor activity14. No side effects and carcinogenicity of T. offi cinale extracts and preparations have been noticed. Chemical constituents of T. offi cinale arewell studied. Scientists of different countries reported that whole plant T. offi cinale containedabundance of bitter principles as terpenoids and sterols, bile like terpenes and sterols, various fl avonoids and phenolic acids, large amount of polysaccharides as inulin and fructosans11,15-17.Also, dandelion is a rich in minerals such as iron, potassium and zinc18,19. In this work we are describing activity-guided isolation and the molecular structure elucidation of components from the dichloromethane fraction of the aerial parts of T. offi cinale, from the Mongolian fl ora.

Effect of flavones on rat brain and lung matrix metalloproteinase activity measured by film in-situ zymography.

We have evaluated the inhibitory activity of flavone, nobiletin, and heptamethoxyflavone on matrix metalloproteinase (MMP) activity in the rat. MMP in 9000-g supernatant fraction of lung homogenate was activated by p-aminophenyl mercuric acetate (APMA), and gelatinolytic activity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. This activity should be related to MMP-2 and/or MMP-9 and was confirmed by gelatin zymography. Fluorescent-conjugated collagen used as a substrate for collagenolytic activity wasinvestigated by SDS-PAGE also. The film in-situ zymography method was applied to rat brain and lung tissue in the same manner. Flavone and nobiletin inhibited the APMA-stimulated gelatinolytic activity and also the collagenolytic activity by more than 75%. The film in-situ zymography method indicated that these compounds might be potent inhibitors of MMP, suggesting the specific inhibition of localized MMP in brain hippocampus and/or lung terminal bronchioles, which may contribute to the prevention of some types of brain disease or cancer invasion and metastasis.

Analysis of swertiamarin in Swertia herb and preparations containing this crude drug by capillary electrophoresis.

Swertia herb (florescent whole plantof Swertia japonica, Gentianaceae) has long been utilized as a folk medicine in Japan. It is often blended in general gastroenteric drugs as a bitter stomachic. Swertiamarin, a bitter secoiridoid glycoside, is the representative constituent of this crude drug and Swertia herb is normally evaluated by the swertiamarin content. To date, papers have described the discrimination of Swertia herbs from other bitter crude drugs, estimation of swertiamarin and seasonal variation in swertiamarin content using thin-layer chromatography, while other papers have reported quantitative determination of swertiamarin using high-performance liquid chromatography (HPLC). In our previous papers, we reported analyses of the constituents of some crude drugs using capillary electrophoresis (CE). To aid in the evaluation of crude drugs, we succeeded in our attempt to separate and determine the quantity of swertiamarin in Swertia herb. Subsequently, we applied the same analytical condition to estimate the swertiamarin contents in Japanese pharmacopoeia stomachic preparations, in OTC gastroenteric drugs and in OTC hair tonics containing Swertia herb.

Augmentation of the pharmacological action of corydalis tuber by saussurea root in isolated mouse ileum.

To understand the meaning of blending crude drugs in Chinese medicinal prescriptions, the influence of Saussurea root on the pharmacological action of Corydalis tuber was examined. Saussurea root increased the depression of acetylcholine-induced contraction caused by the hot water extract solution of Corydalis tuber in mouse ileum at low dosage, which showed no direct influence on acetylcholine. Dehydrocostuslactone in Saussurea root was characterized as the component having increasing activity and the relationship between the concentration of acetylcholine and the variation in the contraction depressed by Corydalis tuber alone or a mixture of the Corydalis tuber and dehydrocostuslactone was investigated for clarification of the mode of action.

Screening of crude drugs for influence on amylase activity and postprandial blood glucose in mouse plasma.

Screening of crude drugs for inhibitory activity on alpha-amylase in mouse plasma was performed. Hot water extracts and ethanolic extracts of Arecae Semen, Ephedrae Herba, Malloti Cortex, Rhei Rhizoma and ethanolic extract of Moutan Cortex inhibited enzyme activity in isolated mouse plasma strongly and dose-dependently. However, the effects of these 9 extracts were not observed in the plasma when they were administered intraperitoneally or orally. Ethanolic extract of Arecae Semen also showed a depressive effect on elevation of postprandial blood glucose.

[The mechanism of alteration of monoamine metabolism in brain regions in marble burying behavior-isolated housing mice and effect of oren-gedoku-to on this alteration].

The mechanism of the alteration in marble burying behavior-isolated housing (MBB-IH) mice was investigated. The determination of hypothalamus monoamine and serum corticosterone contents indicated that MBB-IH mice readily responded to the stress stimuli in conditioned fear stress. Six drugs, such as buspirone (10 mg/kg, p.o.), zimelidine (10 mg/kg, p.o.), clomipramin (10 mg/kg, p.o.), yohimbine (5 mg/kg, p.o.), ethyl beta-carboline-3-carboxylate (beta-CCE, 5 mg/kg, p.o.) and flumazenil (15 mg/kg, p.o.) were singly and/or three times administered to MBB-IH mice. Their inhibitory activity on the MBB-IH mice was considered by the use of activity profiles consisting of spontaneous locomotor activity, marble burying behavior and hypothalamus monoamine content. Using these profiles, we calculated the activities as vector in three-dimensional space, and compared the distance from the control point (DCP). DCPDOPAC and DCP5-HIAA were shortened by single administration of beta-CCE and flumazenil. Oren-gedoku-to (30 and 300 mg/kg, p.o.) shortened the DCPDOPAC and DCP5-HIAA similarly to beta-CCE. The blended crude drugs in Oren-gedoku-to, Coptis rhizome (636.0 mg/kg, p.o.), Scutellaria root (644.4 mg/kg, p.o.) and Gardenia fruit (894.8 mg/kg, p.o.) shortened the DCPDOPAC. Coptis rhizome and Scutellaria root also shortened the DCP5-HIAA. These results suggest that GABA neuron function intensely affects the alteration of MBB-IH and Oren-gedoku-to has the intrinsic benzodiazepine-like activity.

[The anti-Candida activity of shikon].

Antifungal activity of Shikon, roots of Lithospermum erythrorhizon and Arnebia euchroma was investigated in vitro. The extracts containing the pigments of Ko-shikon or Nan-shikon showed the antifungal activities against Candida albicans. Acetylshikonin, one of these pigments, inhibited the fungal growth at MIC 15.6 micrograms/ml (RPMI24 h) or 3.9 micrograms/ml (YNB24 h).

Accumulation of plastocyanin mRNA lacking 5' region in the green alga Pediastrum boryanum grown under copper-deficient conditions.

In the green alga Pediastrum boryanum NIES-301, plastocyanin accumulates under copper-sufficient conditions and cytochrome c6 accumulates under copper-deficient conditions. We cloned the cDNA which encodes pre-apoplastocyanin from P. boryanum cultured under the copper-sufficient condition. The deduced amino acid sequence of the pre-apoplastocyanin protein consists of 151 amino acid residues including a putative bipartite presequence of 53 amino acid residues. Southern blot analysis of P. boryanum genomic DNA indicated that pre-apoplastocyanin is encoded by a single nuclear gene. Northern blot analysis showed that copper-deficient cells accumulated a shorter form of the mRNA of pre-apoplastocyanin, which did not generate pre-apoplastocyanin in the wheat-germ translation system. The difference in size was ascribed to the absence of the 5' region in the mRNA of pre-apoplastocyanin obtained from the copper-deficient cells, which accounts for the absence of plastocyanin under these conditions. This phenomenon represents a novel regulatory mechanism, although details of the mechanism are not yet known.

Structure comparison between oxidized and reduced plastocyanin from a fern, Dryopteris crassirhizoma.

The X-ray crystal structures of oxidized and reduced plastocyanin obtained from the fern Dryopteris crassirhizoma have been determined at 1.7 and 1.8 A resolution, respectively. The fern plastocyanin is unique in the longer main chain composed of 102 amino acid residues and in the unusual pH dependence due to the pi-pi stacking interaction around the copper site [Kohzuma, T., et al. (1999) J. Biol. Chem. 274, 11817-11823]. Here we report the structural comparison between the fern plastocyanin and other plastocyanins from cyanobacteria, green algae, and other higher plants, together with the structural changes of fern plastocyanin upon reduction. Glu59 hydrogen bonds to the OH of Tyr83, which is thought to be a possible conduit for electrons, in the oxidized state. However, it moves away from Tyr83 upon reduction like poplar plastocyanin.

The structure and unusual pH dependence of plastocyanin from the fern Dryopteris crassirhizoma. The protonation of an active site histidine is hindered by pi-pi interactions.

Spectroscopic properties, amino acid sequence, electron transfer kinetics, and crystal structures of the oxidized (at 1.7 A resolution) and reduced form (at 1.8 A resolution) of a novel plastocyanin from the fern Dryopteris crassirhizoma are presented. Kinetic studies show that the reduced form of Dryopteris plastocyanin remains redox-active at low pH, under conditions where the oxidation of the reduced form of other plastocyanins is inhibited by the protonation of a solvent-exposed active site residue, His87 (equivalent to His90 in Dryopteris plastocyanin). The x-ray crystal structure analysis of Dryopteris plastocyanin reveals pi-pi stacking between Phe12 and His90, suggesting that the active site is uniquely protected against inactivation. Like higher plant plastocyanins, Dryopteris plastocyanin has an acidic patch, but this patch is located closer to the solvent-exposed active site His residue, and the total number of acidic residues is smaller. In the reactions of Dryopteris plastocyanin with inorganic redox reagents, the acidic patch (the "remote" site) and the hydrophobic patch surrounding His90 (the "adjacent" site) are equally efficient for electron transfer. These results indicate the significance of the lack of protonation at the active site of Dryopteris plastocyanin, the equivalence of the two electron transfer sites in this protein, and a possibility of obtaining a novel insight into the photosynthetic electron transfer system of the first vascular plant fern, including its molecular evolutionary aspects. This is the first report on the characterization of plastocyanin and the first three-dimensional protein structure from fern plant.

Novel insight into the copper-ligand geometry in the crystal structure of Ulva pertusa plastocyanin at 1.6-A resolution. Structural basis for regulation of the copper site by residue 88.

The crystal structure of plastocyanin from a green alga, Ulva pertusa, has been determined at 1.6-A resolution. At its copper site, U. pertusa plastocyanin has a distorted tetrahedral coordination geometry similar to other plastocyanins. In comparison with structures of plastocyanins reported formerly, a Cu(II)-Sdelta(Met92) bond distance (2.69 A) is shorter by about 0.2 A and a Cu(II)-Sgamma(Cys84) distance is longer by less than 0.1 A in U. pertusa plastocyanin. These subtle but significant differences are caused by the structural change at a His-Met loop (His87-Met92) due to an absence of a O(Asp85)-Ogamma(Ser88) hydrogen bond which is found in Enteromorpha prolifera plastocyanin. In addition, poplar and Chlamydomonas reinhardtii plastocyanins with a glutamine at residue 88 have a weak cation-pi interaction with Tyr83. This interaction lengthens the Cu(II)-Sdelta(Met92) bond of poplar and C. reinhardtii plastocyanins by 0.14 and 0.20 A, respectively. As a result of structural differences, U. pertusa plastocyanin has a less distorted geometry than the other plastocyanins. Thus, the cupric geometry is finely tuned by the interactions between residues 85 and 88 and between residues 83 and 88. This result implies that the copper site is more flexible than reported formerly and that the rack mechanism would be preferable to the entatic theory. The His-Met loop may regulate the electron transfer rate within the complex between plastocyanin and cytochrome f.

Increase in monoamine levels caused by emotional stress in mice brain regions is attenuated by Saiko-ka-ryukotsu-borei-to.

The effect of Saiko-ka-ryukotsu-borei-to (SRBT) on the stress-induced increase of monoamines in brain regions was investigated in three mouse emotional stress models. Dopamine (DA) and 3,4-dihydroxy-phenyl acetic acid (DOPAC) contents were elevated significantly by electric shock stress, psychological stress and conditioned fear stress in thalamus, hypothalamus and amygdala. The DA and DOPAC levels were decreased by preadministration of SRBT (600 mg/kg, p.o.) in the last two models, but were not altered in electric shock stress. Therefore, this compound seems to be effective in stress involving emotional factors. These results indicate that SRBT affects the brain monoamine neurons leading to psychological change in mice.

[Evaluation of marble burying behavior: induced alteration of monoamine metabolism in mouse brain].

Evaluation of marble burying behavior (MBB) housing-induced alteration of monoamine metabolism in mouse brain was performed by measuring metabolite levels with HPLC-ECD. Isolated housing of mice, each in a cage (22 x 32 x 14 cm; sawdust, 1-mm diameter; 5 cm in thickness) with 15 evenly spaced glass marbles on the floor (2.5-cm diameter; control, without marbles) for 24-168 hr, 5-HIAA contents were decreased in three regions: the midbrain, thalamus and hypothalamus. 5-HT turnover was not inhibited except for in the hypothalamus due to the decreases of 5-HT in the other two regions. On the other hand, DOPAC content and DA turnover were decreased in four regions: striatum, midbrain, thalamus, hypothalamus. The decrease in hypothalamic monoamine neurons was observed notably after 72 hr of MBB housing. No alterations were observed in feeding, water-drinking, spontaneous locomotor activity, number of buried marbles, serum corticosterone and serum glucose concentrations during MBB keeping compared with the control mice. These results suggested that the isolated mouse housed in a cage with evenly spaced glass marbles for a long period may be a model animal for alteration of monoamine metabolism in brain regions without physical infringement.

Effect of saiko-ka-ryukotsu-borei-to on the stress-induced increase of serum corticosterone in mice.

The effect of Saiko-ka-ryukotsu-borei-to (SRBT) on the stress-induced enhancement of serum corticosterone in various stress models was investigated in mice. The serum corticosterone was elevated significantly by immobilized stress, forced-swim stress, electric-shock stress, psychological stress and conditioned-fear stress, respectively. The concentration in the last two models was decreased in a dose-dependent manner by pre-administration of SRBT (10, 60, 100, 600 and 1000 mg/kg, p.o.). Therefore, this action seems to be effective in stress involving emotional factors but ineffective in physical stress models. These results indicate that the anti-stress effect of SRBT is dependent strongly on the degree of psychological change compared with physical change in mice.

[Effect of Oren-gedoku-to on changes in hexobarbital-induced sleeping time in chlorpromazine-treated mice].

The effects of Oren-gedoku-to on the hypnotic duration induced by hexobarbital, and on the change of this duration in chlorpromazine-treated mice were investigated. Single dose of Oren-gedoku-to (1.0 or 2.0 g/kg, p.o.) or chlorpromazine (10 mg/kg, p.o.) prolonged the hexobarbital hypnosis time. And the simultaneous dose of chlorpromazine (6.0 mg/kg, p.o.) and Orengedoku-to (1.0 g/kg, p.o.) indicated the equal effect for the administration of chlorpromazine (10.0 mg/kg, p.o.) alone. Although the mechanism of the action remains to be unknown, administration of Oren-gedoku-to in combination with chlorpromazine seems to be useful from the standpoint of alleviation of the side-effects caused by less dose.

[The medicine chest handed down in the Takaya family, doctors of Sendai-han].

There is a medicine chest with design of Chrysanthemums in Makie made for Edo-era and handed down in the Takaya family, doctors of Sendai-han. Many kinds of crude drugs, named by one Chinese character, are kept in this chest. They were classified into three types, namely, [Japanese characters]. Identification of these crude drugs was carried out and the meaning of the classification was brought up as a question.

The syntheses of plastocyanin and cytochrome c-553 are regulated by copper at the pre-translational level in a green alga, Pediastrum boryanum.

The green alga Pediastrum boryanum synthesizes alternatively two photosynthetic electron carrier proteins, plastocyanin and cytochrome c-553, depending on the copper concentration of the medium. We studied the levels at which the syntheses of the two proteins are regulated. Plastocyanin and cytochrome c-553 were purified from P. boryanum NIES-301 cells, having apparent molecular weights of 14,600 and about 12,000, respectively. Western blotting with antisera raised against these proteins showed accumulation of (apo)plastocyanin and (apo)cytochrome c-553 in the cells grown with (2 microM) and without added CuSO4, respectively, but no accumulation of the precursor proteins in both cultures. The translatable mRNAs for the two proteins were examined by in vitro translation with total RNA and wheat germ extract followed by immunoprecipitation and SDS-PAGE. The 21-kDa polypeptide (preapoplastocyanin) was detected with anti-plastocyanin serum in copper-sufficient cells; the 23-kDa polypeptide (preapocytochrome c-553) with anti-cytochrome c-553 serum in copper-deficient cells. The translatable mRNA for preapoplastocyanin appeared in 1 h and (apo)plastocyanin in 2-3 h after the addition of 2 microM CuSO4 to the copper-deficient culture. The translatable mRNA for preapocytochrome c-553 disappeared within 4-5 h, while (apo)cytochrome c-553 disappeared more slowly. It is concluded that the syntheses of plastocyanin and cytochrome c-553 are regulated by copper at the pre-translational (i.e., transcriptional or post-transcriptional) level in P. boryanum NIES-301.

Anti-ulcer effects of Trichosanthes fruits.

Anti-ulcer activities of fruits of Trichosanthes kirilowii Maximowicz var. japonica Kitamura (50% ethanolic extract, TKE) were investigated in rats. TKE, at doses of 100-1000 mg/kg, showed potent protection against experimental gastric lesions, namely, those induced by water-immersion, histamine, serotonin, HCl.ethanol, 0.6 N HCl, 0.2 N NaOH, 35% NaCl, and Shay' ulcer and acetic acid-induced gastric ulcer. At doses of 500-1000 mg/kg, TKE decreased the gastric secretion and acid output in pylorus-ligation (for 7 h), but 100 mg/kg of TKE had no influence on the gastric secretion. On the other hand, TKE exerted inhibition on the contractile responses of the isolated ileum of mouse to acetylcholine. These results suggested that TKE has an anti-ulcer effect.

Crystal structure of plastocyanin from a green alga, Enteromorpha prolifera.

The crystal structure of the Cu-containing protein plastocyanin (Mr 10,500) from the green alga Enteromorpha prolifera has been solved by molecular replacement. The structure was refined by constrained-restrained and restrained reciprocal space least-squares techniques. The refined model includes 111 solvent sites. There is evidence for alternate conformers at eight residues. The residual is 0.12 for a data set comprising 74% of all observations accessible at 1.85 A resolution. The beta-sandwich structure of the algal plastocyanin is effectively the same as that of poplar leaf (Populus nigra var. italica) plastocyanin determined at 1.6 A resolution. The sequence homology between the two proteins is 56%. Differences between the contacts in the hydrophobic core create some significant (0.5 to 1.2 A) movements of the polypeptide backbone, resulting in small differences between the orientations and separations of corresponding beta-strands. These differences are most pronounced at the end of the molecule remote from the Cu site. The largest structural differences occur in the single non-beta strand, which includes the sole turn of helix in the molecule: two of the residues in a prominent kink of the poplar plastocyanin backbone are missing from the algal plastocyanin sequence, and there is a significant change in the position of the helical segment in relation to the beta-sandwich. Several other small but significant structural differences can be correlated with intermolecular contacts in the crystals. An intramolecular carboxyl-carboxylate hydrogen bond in the algal plastocyanin may be associated with an unusually high pKa. The dimensions of the Cu site in the two plastocyanins are, within the limits of precision, identical.