Biosystematics and plant proteomics: role of proteomics in plant phylogenetic analysis.

Since time immemorial, systematics has played significant role in every sphere of life. Biosystematics has evolved from folk taxonomy towards natural classification system and then culminated into homology based classification system. A good systematic approach is practical and predictive of phylogenetics of taxa incorporating different data. The morphological, chemical and molecular (genomics and proteomics) informations are used to explore the exact inter-relationship among the organisms. Proteomics is an essential and inevitable aspect in plant biology which can help in deciphering the functions of the genes that are or will be sequenced. Proteomics has proved to be a good tool in characterisation of individual lines and genetic distances among the genera, species, subspecies, verities and populations describing their phylogenetic interrelationships. Two-dimensional electrophoresis (2-DE) is the major technique being applied for polypeptide characterization of each taxon for exploring phylogenetic or physiological relationships among organs, tissues or organisms. Moreover, proteomics can lead to unraveling the natural phenomena of plants development and their response to changing environment. These proteomic derived informations and their application in phylogenetic studies can be useful in agro-biotechnology development for better yield and safe use of food and medicines.

Involvement of NF-kappaB and c-myc signaling pathways in the apoptosis of HL-60 cells induced by alkaloids of Tripterygium hypoglaucum (levl.) Hutch.

Tripterygium hypoglaucum (levl.) Hutch (Celastraceae) (THH) root is a Chinese medicinal herb commonly used for treating autoimmune diseases. In the present study, alkaloids of THH were prepared and their cytotoxicity against the HL-60 cell was investigated. THH-induced apoptosis was observed using flow cytometry, confocal fluorescence microscope, and DNA laddering and caspase assays. The molecular mechanism involved in the induction of HL-60 cell apoptosis by THH alkaloids was examined using cDNA microarrays containing 3000 human genes derived from a leukocyte cDNA library. Sixteen genes were identified to be differentially expressed in HL-60 cells upon THH treatment. Several genes related to the NF-kappaB signaling pathway and cell apoptosis (such as NFKBIB, PRG1 and B2M) were up-regulated. In addition, c-myc binding protein and apoptosis-related cysteine proteases caspase-3 and caspase-8 were also regulated. The changes in c-Myc RNA expression and c-myc protein level were further confirmed by RT-PCR and Western blot analysis. The results demonstrated that THH alkaloids induced apoptosis of HL-60 cells though c-myc and NF-kappaB signaling pathways.

Constituents of Chinese Piper species and their inhibitory activity on prostaglandin and leukotriene biosynthesis in vitro.

The n-hexane extracts of 19 Piper species, predominantly from China, were screened for their 5-lipoxygenase (5-LOX) and cyclooxygenase-1 (COX-1) inhibitory potential. Many of them showed considerable inhibitory activity against at least one of these two key enzymes of the arachidonic acid metabolism, especially against COX-1. The best results in inhibiting the formation of leukotrienes were obtained with the extract of Piper kadsura. In the terms of prostaglandin synthesis inhibition, the extract of Piper boehmeriifolium var. tonkinense was found to have the strongest activity. Furthermore, an analytical investigation by means of TLC, HPLC-DAD and GC-MS resulted in the identification of 20 constituents. Most of them were amides with an interesting variety of amine moieties. Among them were pellitorine, and four higher homologues, piperlonguminine, dihydropiperlonguminine, futoamide, chingchengenamide, the retrofractamides A, B and D, guineensine, brachystamide B, piperanine, piperine, piperdardine, sarmentine, pipataline and benzylbenzoate. In 96 cases, these constituents were new for the particular plant.

Aquiledine and isoaquiledine, novel flavonoid alkaloids from Aquilegia ecalcarata.

Two new flavonoid alkaloids, aquiledine (1) and isoaquiledine (2), were isolated from the whole herb of Aquilegia ecalcarata, and their structures were determined by detailed spectral analysis. Three known flavonoids were also isolated from this extract-apigenin, apigenin-7,4'-dimethyl ether, and luteolin-and have not been reported previously from A. ecalcarata.

[Studies on polysaccharides in different development stages of Armillaria mellea (Vahl.: Fr.) Quel].

OBJECTIVE: To study the polysaccharides in different development stages of Armillaria mellea. METHOD: Polysaccharides in rhizomorph, fruit-body, mycelia and its fermenting liquor of A. mellea were extracted, isolated and purified. The properties contents, molar ratio and molecular weight of the polysaccharides were determined by IR spectra, HPLC, GPC and gel chromatography. RESULT: The polysaccharides in mycelia and its fermenting liquor contained only glucose, the sugar contents were 9.00% in mycelia and 0.87 g.(100 ml)-1 in fermenting liquor respectively. The polysaccharides in both rhizomorph and fruit-body consisted of glucose and xylose, and the molar ratio was 1:14 in rhizomorph and 1:10 in fruit-body respectively; the polysaccharide contents were 1.12% in rhizomorph and 2.27% in fruit-body. The molecular weight of these polysaccharides was about 10,000-70,000. CONCLUSION: The experimental results supply important scientific data for developing A. mellea as a medicine.

[Research on DNA molecular marker of Magnolia officinalis Rehd. et Wils.--RAPD study on certified species].

AIM: To discuss the intraspecific relationship in Magnolia officinalis and the genuineness of Cortex Magnoliae officinalis, and to find some DNA characters of certified "Houpo". METHODS: Thirty-three samples from eleven locations, which can represent most of the distribution of M. officinalis, were selected. The total DNA was extracted. Severty-four random primers were tried to get good amplification. RESULTS: One hundred and sixteen bands amplified from seventeen primers, were clustered by NTSYS-pc software. Three branches were obtained. Some distinctive primers and bands, which represent certified species or fine breed, were obtained also. CONCLUSION: 1) M. officinalis should be divided into three geographic clans instead of two subspecies or varieties, they are, a) typical officinalis, b) typical biloba and c) Middle type. This conclusion agrees with the leaf form and other characters. 2) The genetic difference between "Chuanpo" and "Wenpo" is evident and the difference is in correspondence with the quantities of their chemical constituents. So, the genetic difference is the main reason of the genuineness of Cortex Magnoliae officinalis. 3) These results may be used to establish DNA database for identification of Cortex Magnoliae officinalis.

[Studies on the chemical constituents from Aquilegia ecalcarata Maxim].

OBJECTIVE: To study the chemical constituents from Aquilegia ecalcarata. METHOD: Compounds were isolated with silica gel and polyamide chromatography and their structures were determined by spectral analysis and chemical evidence. RESULT: Five compounds were obtained and identified as beta-sitosterol, isoorientin-7-O-glucoside, isovitexin-4'-O-glucoside, isovitexin-2'-O-rhamnoside, luteolin-7-O-glucoside. CONCLUSION: All the compounds were isolated from this plant for the first time.

[Comparison of ITS sequences between wild ginseng DNA and garden ginseng DNA].

OBJECTIVE: To compare the genetic differences between wild ginseng and garden ginseng (Panax ginseng). METHOD: The sequences of ITS1 and ITS2 of wild ginsengs were determined on LKB DNA sequencing station through Si-liver Sequence DNA Sequencing System. The sequencies were aligned with DNA SIS software. RESULTS AND CONCLUSION: The ITS1 and ITS2 of Panax were 220-221 and 222-224 bases in length respectively. In Panax ginsehg, the seqences of ITS1 were very stable, but ITS2 were changeable. The ITS2 sequences of No. 87 and No. 110 of the wild ginseng collected from Fusong Heilongjiang (China) were exactly the same as those of No. U41680(Jun Wen) and No. U41682(Jun Wen) of garden ginseng collected from Heilongjiang Province (China) and Korea respectively, but different from those of No. U41681(Jun Wen) from Hubei Province (China) in three bases (447, 449, 450) The result implies that the cultivated ginsengs may have been introduced from two different populations of the wild ginseng.

[A study on AFLP fingerprinting of land races of Panax ginseng L].

OBJECTIVE: To obtain more information on DNA fingerprintings of five land races of Chinese ginseng, namely, Damaya (DMY), Changbo (CB), Yuanbangyuanlu (YBYL) and Huangguo (HG). METHODS: The five land races were detected by amplified restriction fragment polymorphism (AFLP) markers with 11 combined primers (M2, M3, M16, M20, M53, M56, M57, M68, M69, M72, M84 in Mse I). RESULT AND CONCLUSION: Only 4.6% polymorphic sites was found. It was further verified that only a little diversity existed among the land races. The polymorphic sites of CB were much more than those of the others, which suggests that there are more heterozygotes in CB populations, and it is closer to wild ginseng than the others.

Arsenic trioxide induces apoptosis of HPV16 DNA-immortalized human cervical epithelial cells and selectively inhibits viral gene expression.

Arsenic trioxide (As2O3), a major ingredient of arsenic compounds in traditional Chinese medicine, exhibits anti-acute promyelocytic leukemic activity. Considering that over 80% of human malignant tumors derive from epithelial cells, we studied the effect of As2O3 on HPV 16 DNA-immortalized human cervical epithelial cells (HCE16/3 cells) in vitro. As2O3 reduced HCE16/3 cell survival, induced apoptosis at a low concentration and selectively inhibited expression of viral early genes. This effect was evidenced by a reduction of cell viability in the MTT assay, G1 arrest and significant apoptosis upon flow-cytometric analysis, presence of apoptotic bodies, formation of DNA ladders upon gel electrophoresis and inhibition of viral early gene expression by RT-PCR and Western blot. There was a good correlation between cell apoptosis and viral early gene inhibition after As2O3 treatment, suggesting that induction of apoptosis of HCE16/3 cells by As2O3 treatment might be associated with down-regulation of viral oncogene expression. In conclusion, our findings indicate that As2O3 induces apoptosis of HCE16/3 cells, which may provide a new approach for treating HPV-associated tumors.

Isobutylamides and a new methylbutylamide from Piper sarmentosum.

A homologous series of six 2 E,4 E-diene-isobutylamides and a new N-2'-methylbutyl-2 E,4 E-decadieneamide were isolated from the N-hexane extract of the aerial parts of PIPER SARMENTOSUM. While the crude extract was found to possess cyclooxygenase-1 (COX-1) and 5-lipoxygenase (5-LO) inhibitory activity (COX-1 IC (50) = 19 microg/ml; 5-LO IC (50) = 10 microg/ml), the isolated compounds showed only weak effects in both test systems.

Xanthones from Hypericum japonicum and H. henryi.

From the aerial part of Hypericum japonicum, one new xanthone glycoside, 1,5-dihydroxyxanthone-6-O-beta-D-glucoside, one novel dimer xanthone, bijaponicaxanthone, and the first natural prenylated xanthone, 1,3,5,6-tetrahydroxy-4-prenylxanthone, were isolated together with the four known xanthones, 1,5,6-trihydroxyxanthone, isojacereubin, 6-deoxyisojacareubin and 4',5'-dihydro-1,5,6-trihydroxy-4',4',5'-trimethylfurano (2',3':4,5) xanthone. five previously known xanthones, kielcorin, cadensin, 1,7,-dihydroxyxanthone, 1,5-dihydroxy-4-methoxyxanthone and 1,2,5-trihydroxyxanthone were also found in the dichoromethane extract of the stems and leaves of H. henryi. Their structures were elucidated by spectroscopic and chemical methods. Some of the compounds from H. japonicum were found to exert an interesting coagulant activity in an in vitro test. The chemotaxonomic value of xanthones is discussed briefly.

Effect of berbamine on T-cell mediated immunity and the prevention of rejection on skin transplants in mice.

Berbamine, an ingredient of Berberis, which itself is widely utilized in Chinese folk-medicine has been used as a source of leukogenics, anti-arrhythmics and anti-hypertensives. In recent years, the immunosuppressive effects of berbamine has been demonstrated. In order to further investigate the value of berbamine as an immunosuppressive agent, the delayed type hypersensitivity reaction (DTH) response with sheep red blood cells (SRBC), the mixed lymphocyte reaction (MLR) and a skin model of allograft rejection on mice were studied. Berbamine showed suppressive effects on DTH and MLR and significantly prolonged allograft survival compared with untreated transplanted mice. The results indicate that berbamine may be a potential agent in clinical transplantation.

Berbamine inhibits IL-2 receptor expression, but spares IL-2 production and its mRNA expression.

The purpose of this paper was to elucidate the mechanism of action of berbamine on the T-lymphocyte mediated immune responses. Effects of berbamine on IL-2 production, IL-2 receptor expression and the interaction between IL-2 and IL-2 receptor were observed. The results clearly demonstrated that berbamine suppressed the expression of IL-2 receptors on PHA induced T-blast cells, but did not alter the production of IL-2 of mouse splenic cells and human tonsil lymphocytes. Futhermore, the results also showed that berbamine at higher concentrations had a slight inhibitory effect on the interaction between IL-2 and IL-2 receptors, and addition of exogenous IL-2 did not reverse the inhibitory action of berbamine on T-cell proliferation.

Effect of saponins of Panax notoginseng on synaptosomal 45Ca uptake.

AIM: To explore the calcium uptake antagonism of saponins of Panax notoginseng (PNS). METHODS: Synaptosomes were prepared from rat cerebral cortex by using differential Ficoll gradients. The effects of PNS on synaptosomal 45Ca uptake were measured in vitro or after acute treatment. RESULTS: PNS 50-800 mg.L-1 produced a concentration-rated inhibition of Ca2+ uptake [IC50 = 111 (46-176) mg.L-1]. Both initial and maximal uptake were inhibited. Similar effect was obtained after acute PNS treatment with 200 mg.kg-1 i.p. The blocking effect of PNS was reversed by calcium in media. CONCLUSION: PNS is a calcium channel blocker in neurons.

[Determation of strychnine and brucine in Strychnos by HPLC].

This paper describes the determination of strychnine and brucine in the seeds, root, stem and leaves of Strychnos species by HPLC. The analytical column used was ZY110 YNG-C18. The mobile phase was KH2PO4(0.01 mol.L-1)--MeOH(73:27), pH2.5, regulated by 10% H3PO4. Flow rate was 1.0 ml.min-1. The detection wavelength was 264 nm. The linear ranges of strychmine and brucine were 0.18-7.26 micrograms and 0.11-4.32 micrograms, respectively. The recoveries of strychnine and brucine were 98.27% and 98.04%, respectively. The analytical results showed that the contents of strychnine and brucine in samples showed great difference between different species. The contents of strychnine in the seeds of Strychnos wallichiana and S. ignatii were 5.6% and 3.9%, respectively. These results show that the two Strychnos species may be developed as the resources of strychnine.

Flavonoids from Epimedium wanshanense.

A novel flavonol glycoside named wanepimedoside A was isolated from the whole plant of Epimedium wanshanense, along with fifteen known flavonoids, anhydroicaritin, desmethylanhydroicaritin, icarisid I and II, quercetin, ikarisoside A and B, sagittatoside B, 2"-O-rhamnosylicarisid II, icariin, 2"-O-rhamnosylikarisoside A, epimedin B, epimedin C, and diphylloside A and B.

An unusual cycloartane triterpenoid from Cimicifuga foetida.

A new cycloartane triterpenoid glycoside has been isolated from the rhizomes of Cimicifuge foetida L. The spectroscopic characteristics of the new compound are different from previously described cycloartane triterpenoids because of the loss of the 24-isopropyl group as well as the presence of a 11 beta-OH group. Based on spectroscopic evidence, including a series of 2D-NMR analyses, the structure of the new triterpene is assigned as 24-des-isopropyl-7-ene-23-one-9,19; 16,24-dicycloart-3 beta,11 beta,16 alpha,24 alpha-tetraol 3-O-beta-D-xylopryanoside, named here as neocimiside. The structure of the aglycone of neocimiside was confirmed by X-ray analysis.