2'-(2,3-Dihydroxybenzoyloxy)-7-ketologanin: a novel iridoid glucoside from the leaves of Gentiana kurroo.

A new bitter acylated iridoid glucoside, 2'-(2,3-dihydroxybenzoyloxy)-7-ketologanin (1), has been isolated from the leaves of Gentiana kurroo. The structure of the compound was elucidated conclusively by chemical analysis, and extensive 1D and 2D NMR experiments.

Molecular cloning and characterization of isomultiflorenol synthase, a new triterpene synthase from Luffa cylindrica, involved in biosynthesis of bryonolic acid.

An oxidosqualene cyclase cDNA, LcIMS1, was isolated from cultured cells of Luffa cylindrica Roem. by heterologous hybridization with cDNA of Glycyrrhiza glabra beta-amyrin synthase. Expression of LcIMS1 in yeast lacking endogenous oxidosqualene cyclase activity resulted in the accumulation of isomultiflorenol, a triterpene. This is consistent with LcIMS1 encoding isomultiflorenol synthase, an oxidosqualene cyclase involved in bryonolic acid biosynthesis in cultured Luffa cells. The deduced amino-acid sequence of LcIMS1 shows relatively low identity with other triterpene synthases, suggesting that isomultiflorenol synthase should be classified into a new group of triterpene synthases. The levels of isomultiflorenol synthase and cycloartenol synthase mRNAs, which were measured with gene-specific probes, correlated with the accumulation of bryonolic acid and phytosterols over a growth cycle of the Luffa cell cultures. Isomultiflorenol synthase mRNA was low during the early stages of cell growth and accumulated to relatively high levels in the late stages. Induction of this mRNA preceded accumulation of bryonolic acid. In contrast, cycloartenol synthase mRNA accumulated in the early stages of the culture cycle, whereas phytosterols accumulated at the same relative rate throughout the whole growth cycle. These results suggest independent regulation of these two genes and of the accumulation of bryonolic acid and phytosterols.

Cloning and characterization of a cDNA encoding beta-amyrin synthase involved in glycyrrhizin and soyasaponin biosyntheses in licorice.

An oxidosqualene cyclase cDNA, termed GgbAS1, was isolated from cultured cells of licorice (Glycyrrhiza glabra) by heterologous hybridization with cDNA of Arabidopsis thaliana LUP1 lupeol synthase. The yeast transformed with an expression vector containing the open reading frame of GgbAS1 produced beta-amyrin, indicating that GgbAS1 encodes beta-amyrin synthase involved in the glycyrrhizin and soyasaponin biosyntheses in licorice. Northern blot analysis showed that the level of beta-amyrin synthase mRNA was drastically changed in the cultured licorice cells, whereas the mRNA level of cycloartenol synthase was relatively constant.

Sesquiterpene glucosides from anti-leukotriene B4 release fraction of Taraxacum officinale.

Chemical examination of the MeOH extract of the root of Taraxacum officinale, which exhibited inhibitory activity on the formation of leukotriene B4 from activated human neutrophils, has resulted in the isolation of 14-O-beta-D-glucosyl-11,13-dihydro-taraxinic acid (1) and 14-O-beta-D-glucosyl-taraxinic acid (2). The absolute stereostructure of 1 has been established by X-ray chrystallographic examination.

Ellagitannins and hexahydroxydiphenoyl esters as inhibitors of vertebrate squalene epoxidase.

Ellagitannins isolated from various plant sources as well as newly synthesized n-alkyl (C(1)-C(18)) esters of hexahydroxydiphenyl (HHDP) dicarboxylic acid were evaluated as enzyme inhibitors of recombinant rat squalene epoxidase, a rate-limiting enzyme of cholesterol biosynthesis. Among the ellagitannins tested, pedunculagin (IC(50) = 2.0 microM) and eugeniin (IC(50) = 1.6 microM), both containing (S)-HHDP ester group(s), showed remarkable inhibition, which was more potent than those of previously reported substrate analogue inhibitors. Furthermore, ellagic acid (IC(50) = 2.0 microM), a bislactone formed by hydrolytic release of a HHDP group from ellagitannins, was also a good inhibitor of the enzyme. On the other hand, the synthetic HHDP esters with C(6) (IC(50) = 0.93 microM) and C(8) alkyl side chains (IC(50) = 0.83 microM) showed potent enzyme inhibition at the submicromolar concentration range, while esters with shorter chain lengths (C(1)-C(4)) and a C(18) ester exhibited moderate inhibition (IC(50) = 8-47 microM).

Influence of natural and synthetic compounds on cell surface expression of cell adhesion molecules, ICAM-1 and VCAM-1.

Various natural and synthetic compounds including alkaloids, terpenoids and phenolics were tested for inhibition of the cell surface expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), both of which are crucial in the regulation of immune response and inflammation. Of 40 compounds tested, two compounds significantly downregulated the expression of VCAM-1 on murine endothelial cells (F-2) and ten compounds that of ICAM-1 on mouse myeloid leukemia cells (M1). Sanguinarine chloride (5) and isoliquiritigenin (13) were capable of lowering the levels of both ICAM-1 and VCAM-1. The structure-activity relationships study on chalcone and flavone derivatives related to 13 suggested that the inhibitory activity of the chalcone derivatives is attributable to the 4-hydroxy group as well as the possible coplanarity between the phenyl ring and the adjacent conjugated ketone.

3,28-Di-O-(dimethylsuccinyl)-betulin isomers as anti-HIV agents.

Four isomers of 3,28-di-O-(dimethylsuccinyl)-betulin were prepared and evaluated for anti-HIV activity against HIV-1 replication in H9 lymphocyte cells. 3-O-(3',3'-Dimethylsuccinyl)-28-O-(2", 2"-dimethvlsuccinyl)-betulin (11) was the most potent anti-HIV compound with an EC5, value of 0.00087 microM and a TI value of 42,400.

Synthesis and anti-HIV activity of 3-alkylamido-3-deoxy-betulinic acid derivatives.

3-Alkylamido-3-deoxy-betulinic acids were synthesized and evaluated for anti-HIV activity as part of the structure-activity relationship study of the potent anti-HIV agent 3-O-(3',3'-dimethyl)-succinyl-betulinic acid (DSB) (2). 3Alpha-diglycorylamide-3-deoxy-betulinic acid demonstrated relatively potent anti-HIV activity (EC50 0.24 microm, TI 728). However, replacing the ester group at C-3 in 2 and its analogues with an amido group yielded inactive or much less potent compounds against HIV replication, indicating that the ester group at C-3 in 2-4 is essential for potent anti-HIV activity.

Phylogenetic relationship of six Glycyrrhiza species based on rbcL sequences and chemical constituents.

The nucleotide sequences of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene (rbcL) of Glycyrrhiza glabra, G. uralensis, G. inflata, G. echinata, G. macedonica and G. pallidiflora have been determined to construct their phylogenetic tree. Based on these sequences, the six Glycyrrhiza species were divided into two groups: three, G. glabra, G. uralensis, and G. inflata, which produce glycyrrhizin as a major saponin, and the others, G. echinata, G. macedonica and G. pallidiflora, which produce macedonoside C as a major saponin. Among the three glycyrrhizin-producing species, only two nucleotide substitutions were observed between the rbcL sequences of G. glabra and G. uralensis, and the sequence of G. uralensis was identical to that of G. inflata, indicating that G. uralensis and G. inflata are closely related. Among the three macedonoside C-producing species, only one nucleotide substitution was observed between those of G. echinata and G. macedonica, indicating that these two species are also closely related.

Molecular cloning and characterization of a cDNA for Glycyrrhiza glabra cycloartenol synthase.

A cDNA clone (GgCAS1) encoding cycloartenol synthase (CAS) has been isolated from Glycyrrhiza glabra (licorice) by cross-hybridization with that of Pisum sativum CAS as a probe. The deduced amino acid sequence of GgCAS1 exhibits 89%, 83% and 81% identity to those of Pisum sativum, Panax ginseng and Arabidopsis thaliana CASs, respectively. CAS activity has been detected in the homogenate of the yeast transformed with the expression vector containing the open reading frame of GgCAS1. Southern blot analysis suggested that at least two CAS genes exist in the licorice genome. In Northern blot analysis, the strong signal for CAS mRNA is detected in the cultured licorice cells of all growth phases, but no significant increase of CAS mRNA expression was observed in the cells treated with the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, pravastatin.

Anti-AIDS agents 38. Anti-HIV activity of 3-O-acyl ursolic acid derivatives.

Based on our previous finding that 3-O-acyl-betulinic and -oleanolic acids, especially the 3-O-(3',3'-dimethyl)-succinyl derivatives (2 and 4), demonstrated potent anti-HIV activity [EC(50) < 0.00035 and 0.00086 microM; therapeutic index (TI) > 20 000 and 22 326, respectively], several 3-O-acyl-ursolic acids were prepared and evaluated for anti-HIV activity. Ursolic acid (6) was equipotent (EC(50) 4.4 microM) with oleanolic acid (EC(50) 3.7 microM), although it was slightly toxic (IC(50) 14.3 microM, TI 3.3). 3-O-Diglycoryl-ursolic acid (10) demonstrated relatively potent anti-HIV activity with an EC(50) of 0. 31 microM and a TI of 155.5. In contrast, 3-O-(3', 3'-dimethylsuccinyl)-ursolic acid (8), which is analogous to the extremely potent anti-HIV betulinic acid and oleanolic acid derivatives 2 and 4, displayed only weak anti-HIV activity (EC(50) 2.1 microM, TI 23.6).

Molecular cloning and characterization of two cDNAs for Glycyrrhiza glabra squalene synthase.

Two cDNAs (GgSQS1 and GgSQS2) encoding squalene synthase have been isolated from licorice, Glycyrrhiza glabra L., and characterized. The deduced amino acid sequence of GgSQS1 was 88%, 81%, 78%, 45-44%, and 45-41% identical to those of GgSQS2, Nicotiana, Arabidopsis, mammal and yeast squalene synthases, respectively. Squalene synthase activity was found in the cell-free extracts of Escherichia coli transformed with the recombinant plasmids for GgSQS1 and GgSQS2, respectively. Genomic Southern blot hybridization indicated that there are three squalene synthase genes in the licorice genome. Northern blot analysis showed that GgSQS2 mRNA is mainly expressed during the exponential growth phase of the cultured licorice cells.

Seasonal variation of glycyrrhizin and isoliquiritigenin glycosides in the root of Glycyrrhiza glabra L.

The time courses of the glycyrrhizin and isoliquiritigenin glycoside contents in the thickening roots of licorice, Glycyrrhiza glabra L., have been determined. The glycyrrhizin content in 1-year-old roots rapidly increased from October to November, whereas the isoliquiritigenin glycoside content increased up to October. In 3-year-old plants, although the isoliquiritigenin glycoside content rapidly increased from June to July, the glycyrrhizin content did not show any significant increase from May to August. The glycyrrhizin content increased during the senescence of the aerial parts as well as during the early stage of shoot elongation. The incorporation of [14C]mevalonic acid into the glycyrrhizin fraction by the root segments was high in May, June and September, and low in August and winter. These results indicated that the biosynthesis of glycyrrhizin is differently regulated from that of isoliquiritigenin glycoside in the thickening root of G. glabra.

Anti-AIDS agents. 30. Anti-HIV activity of oleanolic acid, pomolic acid, and structurally related triterpenoids.

Oleanolic acid (1) was identified as an anti-HIV principle from several plants, including Rosa woodsii (leaves), Prosopis glandulosa (leaves and twigs), Phoradendron juniperinum (whole plant), Syzygium claviflorum (leaves), Hyptis capitata (whole plant), and Ternstromia gymnanthera (aerial part). It inhibited HIV-1 replication in acutely infected H9 cells with an EC50 value of 1.7 microg/mL, and inhibited H9 cell growth with an IC50 value of 21.8 microg/mL [therapeutic index (T. I.) 12.8]. Pomolic acid, isolated from R. woodsii and H. capitata, was also identified as an anti-HIV agent (EC50 1.4 microg/mL, T. I. 16.6). Although ursolic acid did show anti-HIV activity (EC50 2.0 microg/mL), it was slightly toxic (IC50 6.5 microg/mL, T. I. 3.3). A new triterpene (11) was also isolated from the CHCl3-soluble fraction of R. woodsii, though it showed no anti-HIV activity. The structure of 11 was determined to be 1beta-hydroxy-2-oxopomolic acid by spectral examination. Based on these results, we examined the anti-HIV activity of oleanolic acid- or pomolic acid-related triterpenes isolated from several plants. In addition, we previously demonstrated that derivatives of betulinic acid, isolated from the leaves of S. claviflorum as an anti-HIV principle, exhibited extremely potent anti-HIV activity. Accordingly, we prepared derivatives of oleanolic acid and evaluated their anti-HIV activity. Among the oleanolic acid derivatives, 18 demonstrated most potent anti-HIV activity, with an EC50 value of 0. 0005 microg/mL and a T. I. value of 22 400.

Phylogenetic relationship of Glycyrrhiza plants based on rbcL sequences.

The nucleotide sequences of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene (rbcL) of Glycyrrhiza glabra, G. uralensis, G. inflata, G. echinata, and G. pallidiflora have been determined to construct the phylogenetic tree. In the phylogenetic tree based on the rbcL sequences, the five Glycyrrhiza species were divided into two groups: the three glycyrrhizin-producing species G. glabra, G. uralensis, and G. inflata; and the two glycyrrhizin-nonproducing species G. echinata and G. pallidiflora. Among the three glycyrrhizin-producing species, only two nucleotide substitutions were observed between the rbcL sequence of G. glabra and G. uralensis, and the sequence of G. uralensis was identical to that of G. inflata, indicating that the three glycyrrhizin-producing species are closely related.

Novel cytotoxic diterpenes from the stem of Dysoxylum kuskusense.

Three novel diterpenes, dysokusones A (1), B (2), and C (3), were isolated from the stem of Dysoxylum kuskusense as cytotoxic substances. The structures were established by spectroscopic examinations. Compounds 1, 2, and 3 were cytotoxic toward HL-60(TB) cells with EC50 values of 2.25, 6.35, and 2.37 microM, respectively. Compound 1 also displayed cytotoxicity against K-562 and NCI-H522 cells with EC50 values of 5.04 and 4.80 microM, respectively.

Antitumor agents. 180. Chemical Studies and cytotoxic evaluation of cumingianosides and cumindysoside A, antileukemic triterpene glucosides with a 14,18-cycloapotirucallane skeleton.

Treatment of cumingianosides and cumindysoside A, which possess a 14,18-cycloapotirucallane skeleton, with p-toluenesulfonic acid in CH2Cl2 yielded new triterpene glucosides. Cumingianoside A (1) gave 10 and 11, along with cumingianoside Q (5). The structures of 10 and 11 were determined on the basis of spectral examination and contained a dammar-13(17)-ene and a 17(R),23(R)-epoxydammarane skeleton, respectively. Cumingianoside C (2) afforded, together with cumingianoside P (6), products 12 and 13, which were similar to 10 and 11, respectively. With a short reaction time at room temperature, cumingianoside E (3) yielded cumingianoside D (4). In contrast, when 3 was treated with p-toluenesulfonic acid in CH2Cl2 overnight at 5 degrees C, it gave two products, 9 and 14. Extensive spectroscopic examination revealed that 9 possessed a dammar-12-ene skeleton, while 14 was a pentacyclic tetranortriterpene glucoside with a novel skeleton. Cumindysoside A (8) gave a product (15) similar to 14. The cytotoxicities of 9-15 were evaluated against a panel of 58 human tumor cell lines. Compounds 11-15 exhibited potent cytotoxicity with log GI50 values ranging from -7.11 to -4.94, especially against leukemia and colon-tumor cell lines.

Inhibitory activity of soyasaponin II on virus replication in vitro.

The antiviral activities of two saponins, soyasaponin I and II, isolated from soybean (Glycine max Merrill) were studied in vitro against herpes simplex virus type 1 (HSV-1). Soyasaponin II was more potent than soyasaponin I as shown by reduction of HSV-1 production. Soyasaponin II was also found to inhibit the replication of human cytomegalovirus, influenza virus, and human immunodeficiency virus type 1. The action was not due to inhibition of virus penetration and protein synthesis, but might involve a virucidal effect. When acyclovir and soyasaponin II were evaluated in combination for anti-HSV-1 activity, additive antiviral effects were observed for this virus.

Molecular cloning and functional expression of cDNAs for Glycyrrhiza glabra squalene synthase.

We have isolated two cDNAs (GgSQS1 and GgSQS2) encoding squalene synthase of Glycyrrhiza glabra L. by cross-hybridization with that of Arabidopsis thaliana squalene synthase under conditions of low stringency. Their nucleotide sequences contained an open reading frame for a polypeptide of 413 amino acids (GgSQS1) and 412 amino acids (GgSQS2), respectively. The deduced amino acid sequence of GgSQS1 exhibits 88%, 81%, 78%, 45-44%, and 45-41% identity to those of the GgSQS2, Nicotiana benthamiana. Arabidopsis thaliana, mammal, and yeast squalene synthases, respectively. To express the G. glabra enzymes in Escherichia coli, the entire coding region was subcloned into an expression vector. The cell-free extracts of E.coli transformed with the respective plasmid converted 3H-farnesyl diphosphate into squalene.

Gastric cytoprotection of the non-steroidal anti-inflammatory sesquiterpene, beta-caryophyllene.

The gastric cytoprotective effect of beta-caryophyllene, an anti-inflammatory sesquiterpene, was investigated in rats. The oral administration of beta-caryophyllene to rats significantly inhibited gastric mucosal injuries induced by necrotizing agents such as absolute ethanol and 0.6 N HCl, although it failed to prevent water immersion stress- and indomethacin-induced gastric lesions. In addition, this compound hardly affected the secretion of gastric acid and pepsin. Thus, beta-caryophyllene elicited anti-inflammatory effects without any indication of gastric mucosal damage typical of non-steroidal anti-inflammatory agents. Furthermore, this compound manifested cytoprotective effects, rendering the two-dimensional efficacious beta-caryophyllene to be a clinically safe and potentially useful agent.