[Comparison of antioxidative and antitumor activities of six flavonoids from Epimedium koreanum].

OBJECTIVE: To study the antioxidative and antitumor activities of flavonoids isolated from Epimedium koreanum. METHOD: The compounds were separated by column chromatography with silica gel and Sephadex LH-20, and identified by spectral a- nalysis (ESI-MS, 1H-NMR and 13C-NMR) respectively. DPPH radical scavenging assay and MTT assay were used to observe the antioxidative and antitumor abilities. RESULT: Six compounds were isolated from the the ethyl acetate extract of the aerial part. Their structures were identified as icariin (I), luteolin (II), baohuoside II (III), hyperoside (IV), epimedokoreanin B (V) and baohuoside I (VI). The results indicated that at concentrations of 3. 125-200 micromol x L(-1), compound I, III and VI had no ability to scavenge the DPPH radical, but the scavenging ability of compounds II, IV and V were stronger than that of Vit C in dose-dependant manner. Compounds I, II, V and VI could inhibit the proliferation of MCF-7 and HepG2 in dose-dependant manner, but compounds III and IV had no effect on the proliferation. CONCLUSION: The antitumor activity of E. koreanum may be partially related to the antioxidantive activity of flavonoids.

[Correlation between ITS genotype and geographical distribution of Pogostemon cablin].

To investigate the correlation between genotype and distribution of Pogostemon cablin by sequencing ITS1 and ITS2 genes, and provide molecular information for its germplasm evaluation, ITS1 and ITS2 genes of Pogostemon cablin from different localities were identified by PCR direct sequencing. The sequences of ITS1 and ITS2 genes were 424 bp and 380 bp in length, respectively. And nineteen base substitutions were observed in ITS1 gene, and five in ITS2 gene. The results showed a good correlation between genotype and distribution of Pogostemon cablin, and ITS gene sequencing could provide useful molecular information for germplasm evaluation of the plant species verification.

[Study on chemical constituents from rhizomes of Actaea asiatica].

OBJECTIVE: To investigate the chemical constituents of the rhizomes of Actaea asiatica in order to obtain a more comprehensive understanding of its effective components. METHOD: Compounds were separated by silica gel chromatography, RP-C18 chromatography and semi-preparative high performance liquid chromatography, and their structures were established by spectral analysis and chemical evidence. RESULT: Six compounds were isolated from the ethyl acetate extract. Their structures were identified as 25-O-acetylcimigenol (1), 12beta-hydroxycimigenol (2), 23-epi-26-deoxyactein (3), 27-deoxyacetylacteol (4), 26-deoxycimicifugenin (5) and beta-sitosterol (6). CONCLUSION: All these compounds mentioned above were isolated from the plant for the first time.

[Progress in anti-osteoporosis activity of traditional Chinese medicine and medicinal plants].

The anti-osteoporosis activity and mechanism of traditional Chinese medicine and medicinal plants were discussed. It is hoped that we can provide some reference for future drug development and introduction of traditional Chinese medicine to world.

Detection of genetic homogeneity of Panax notoginseng cultivars by sequencing nuclear 18S rRNA and plastid matK genes.

The nuclear 18S rRNA and chloroplast MATK genes of 18 samples of Panax notoginseng and its processed material Sanqi (Radix Notoginseng) were analyzed. The two genes, regardless of cultivar origin, were found to be identical to genotype R1 and M1, respectively, of the published sequences (GenBank accession no. D85171 and AB027526). This phenomenon implies that the species is highly conserved, which is probably caused by the use of the same strain in cultivation and the lack of active mutation in these two genes.

[Review of bioactivities of natural cycloartane triterpenoids].

Cycloartane triterpenoids, which exist widely in nature, are mainly distributed in Astragalus (Leguminosae) species, Trib. Cimicifuga (Ranunculaceae) and Thalictrium (Ranunculaceae) species and possess various bioactivities. Along with the development of isolation techniques of phytochemistry, more and more this kind of compounds are isolated and identified. However, bioactivity researches on the compounds are relatively lagged behind. Most researches are still in screening level, deficient in mechanism elucidation, short of action proven in vivo and SAR analysis. The author summarized the bioactivity of this kind of compounds from all aspects: anti-tumor, anti-virus, antibacterial, anti-inflammation, immune-regulatory, cardiovascular system, hepatic protection and so forth. This will be benefit for the further research and development of the compounds.

Anti-hepatoma activity and mechanism of ursolic acid and its derivatives isolated from Aralia decaisneana.

AIM: To investigate the anti-tumor activity of ursolic acid (UA) and its derivatives isolated from Aralia decaisneana on hepatocellular carcinoma both in vitro and in vivo. METHODS: In vivo cytotoxicity was first screened by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. Morphological observation, DNA ladder, flow cytometry analysis, Western blot and real time PCR were employed to elucidate the cytotoxic mechanism of UA. Implanted mouse hepatoma H22 was used to evaluate the growth inhibitory effect of UA in vivo. RESULTS: UA could significantly inhibit the proliferation of HepG2 and its drug-resistance strain, R-HepG2 cells, but had no inhibitory effect on primarily cultured normal mouse hepatocytes whereas all the six derivatives of UA could not inhibit the growth of all tested cell lines. Further study on mechanism demonstrated that apoptosis and G0/G1 arrest were involved in the cytotoxicity and cleavage of poly-(ADP-ribose)-polymerase (PARP). Downregulation of cyclooxygenase-2 (COX-2) protein and upregulation of heat shock protein (HSP) 105 mRNA correlated to the apoptosis of HepG2 cells treated with UA. In addition, UA also could inhibit the growth of H22 hepatoma in vivo. CONCLUSION: UA is a promising anti-tumor agent, but further work needs to be done to improve its solubility.

[Cytotoxicity and mechanism of 23-O-acetylcimigenol-3-O-beta-D-xylopyranoside on HepG2 cells].

OBJECTIVE: To elucidate the cytotoxicity and mechanism of 23-O-acetylcimigenol-3-O-beta-D-xylopyranoside isolated from C. dahurica on HepG2 cells and to find the leading compound for new drug development. METHOD: MTT, AO/EB staining observation, flow cytometry and western blot methods were used to study the cytotoxicity, morphological changes, cell cycle distribution and protein expression profile of 23-O-acetylcimigenol-3-O-beta-D-xylopyranoside on HepG2 cells. RESULT: 23-O-acetylcimigenol-3-O-beta-D-xylopyranoside could inhibit the proliferation of HepG2 cells with IC50 at 16 micromol x L(-1), and could also induce apoptosis and G2-M cell cycle arrest. Further study demonstrated that the compound could cleavage PARP, regulate protein expression of bcl-2 family and decrease the expression of cdc 2 and cyclin B. CONCLUSION: 23-O-acetylcimigenol-3-O-beta-D-xylopyranoside exerts its cytotoxicity on HepG2 cells via apoptosis and G2-M arrest. In addition, caspases family activation, regulation of protein expression of bcl-2 family and down regulation of cdc 2 and cyclin B were involved in apoptosis and G2-M arrest induced by it.

Effect of daidzin, genistin, and glycitin on osteogenic and adipogenic differentiation of bone marrow stromal cells and adipocytic transdifferentiation of osteoblasts.

AIM: To examine the effect of daidzin, genistin, and glycitin on the osteogenic and adipogenic differentiation of bone marrow stromal cells (MSC) and the adipogenic transdifferentiation of osteoblasts. METHODS: MTT test, alkaline phosphatase (ALP) activity measurement, Oil Red O stain and measurement were employed. RESULTS: Daidzin, genistin, and glycitin 1*10(-8), 5*10(-7), 1*10(-6), 5*10(-6), and 1*10(-5) mol/L all promoted the proliferation of primary mouse bone MSC and osteoblasts. Daidzin 5*10(-7) mol/L and genistin 1*10(-6) mol/L promoted the osteogenesis of MSC. Genistin 1*10(-8), 5*10(-7), 1*10(-6), 5*10(-6), and 1*10(-5) mol/L and glycitin 1*10(-8), 1*10(-6), and 1*10(-5) mol/L inhibited the adipogenesis of MSC. Daidzin, genistin, and glycitin 1*10(-8), 5*10(-7), 1*10(-6), 5*10(-6), and 1*10(-5) mol/L all inhibited the adipocytic transdifferentiation of osteoblasts. CONCLUSIONS: Daidzin, genistin, and glycitin may modulate differentiation of MSC to cause a lineage shift toward the osteoblast and away from the adipocytes, and could inhibit adipocytic transdifferen-tiation of osteoblasts. They could also be helpful in preventing the development of osteonecrosis.

Pyranocoumarin(+/-)-4'-O-acetyl-3'-O-angeloyl-cis-khellactone induces mitochondrial-dependent apoptosis in HL-60 cells.

The pyranocoumarin (+)-4'-O-acetyl-3 'O-angeloyl-cis-khellactone (PC) isolated from Radix Peucedani (root of Peucedanum praeruptorum Dunn) showed a dose-dependent effect at 10 -30 pg/mL on causing apoptotic DNA and nuclear fragmentations in HL-60 cells. After 24 h of PC treatment there were losses of mitochondrial membrane potential and cytochrome c. PC also increased total cellular and mitochondrial Bax protein, stimulated an increase in caspase-dependent Bcl-2 cleavage but showed no effect on Bcl-Xv. These observations strongly suggest activation of the mitochondria apoptotic pathway. The pan-specific caspase inhibitor, ZVAD-fmk, abolished the PC-induced apoptosis,whereas the caspase-8 inhibitor IETD-fmk showed no effect, implying the involvement of the caspase 9 pathway. PC caused a 2 to 12 hour transient increase in phospho-ERK, and a 72 h-long activation of JNK. Pre-treatment with the MEK inhibitor PD98059, which suppresses ERK activation, paradoxically promoted PC-induced mitochondrial cytochrome c release, procaspase-3 and -8 cleavage, and enhanced apoptosis. Our results show that PC triggers mitochondria-mediated apoptosis in HL-60 cells, and the involvement of ERK and JNK signal pathways in the process.

Genotyping and species identification of Fritillaria by DNA chips.

AIM: To investigate the genetic polymorphism of several species of Fritillaria and to develop a DNA chip for the genotyping and identification of the origin of various species of Fritillaria at molecular level. METHODS: Genomic DNA from bulbs of several Fritillaria species was extracted and the polymorphisms of the D2 and D3 regions inside the 26S rDNA gene were identified by direct sequencing. Oligonucleotide probes specific for these polymorphisms were designed and printed on the poly-lysine coated slides to prepare the DNA chip. PCR products from the Fritillaria species were labeled with fluorescence by incorporation of dye-labeled dideoxyribonucleotides and hybridized to the immobilized probes on the chip. RESULTS: The polymorphisms were used as markers for discrimination among various species. Specific oligonucleotide probes were designed and immobilized on a DNA chip. Differentiation of the various Fritillaria species was accomplished based on hybridization of fluorescent labeled PCR products with the DNA chip. CONCLUSION: The results demonstrated the reliability of using DNA chips to identify different species of Fritillaria, and the DNA chip technology can provide a rapid, high throughput tool for genotyping and quality assurance of the plant species verification.

Reversal of multidrug resistance in cancer cells by pyranocoumarins isolated from Radix Peucedani.

The pyranocoumarins, (+/-)-3'-angeloyl-4'-acetoxy-cis-khellactone, were isolated from Radix Peucedani, the dry root of Peucedanum praeruptorum Dunn, through bioassay-guided fractionation. The chemical structure of pyranocoumarins was determined by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. X-ray crystallography showed that there are eight molecules (i.e. two each of four conformers) in each unit cell with their optical activities equally cancelled out. The four conformers are 3'(R)-angeloyl-4'(R)-acetoxy-khellactone in two conformational forms, and 3'(S)-angeloyl-4'(S)-acetoxy-khellactone in two conformational forms. Pyranocoumarins caused apoptotic cell death with IC50 of 41.9+/-2.8 and 17.3+/-8.2 microM for drug-sensitive KB-3-1 and multidrug resistant (MDR) KB-V1, respectively. The two- to threefold sensitivity difference between the two cell lines is interesting considering that the same ratio for doxorubicin is 50-300. Strong synergistic interactions were demonstrated when pyranocoumarins were combined with common anti-tumor drugs including doxorubicin, paclitaxel, puromycin or vincristine in MDR KB-V1 cell line, but not in drug-sensitive KB-3-1 cells. Pyranocoumarins increased doxorubicin accumulation in KB-V1 cells by about 25% after 6 h of incubation. Pyranocoumarins treatment for 24 h down-regulated the expression of P-glycoprotein in KB-V1 cells at both protein and mRNA levels. Pyranocoumarins also transiently reduced the cellular ATP contents in KB-V1 cells in a dose-dependent manner. Our results suggest that pyranocoumarins could be a potential MDR reversing agent.