Polysaccharides of Dendrobium officinale inhibit TNF-α-induced apoptosis in A-253 cell line.

OBJECTIVE: Our previous study demonstrated that polysaccharides of Dendrobium officinale Kimura et Migo (DP) were capable of enhancing immunomodulation in an experimental model of Sjögren's syndrome, a chronic autoimmune disease mainly affecting the salivary glands. In the present study, we further investigated the protective effect of DP on a human salivary gland cell line A-253 against tumor necrosis factor (TNF)-α-induced apoptosis. MATERIALS: TNF-α (100 U/ml) was used as the stimulus for treating the A-253 cells to induce cellular apoptosis. Nuclear factor-kappa B (NF-κB, p65), phosphorylation of mitogen-activated protein kinases (MAPK), reactive oxygen species (ROS) generation, mitochondrial membrane potential and proapoptotic proteins were examined. A-253 cells were pre-treated with DP for 12 h before TNF-α stimulation. RESULTS: We observed translocation of NF-κB into the nuclei, prolonged MAPK, excessive ROS generation and strongly decreased mitochondrial membrane potential, and subsequently cytochrome C release and caspase-3 activation. However, pre-treatment with DP significantly inhibited the TNF-α-induced apoptotic factors. CONCLUSIONS: Our data suggested the inhibitory effect of DP on TNF-α-induced apoptosis in a human salivary gland cell line. This inhibition indicated potential inference of DP in the initial plasma membrane-bound complex of TNF-α and its receptors.

Recent progress in medicinal investigations on trichosanthin and other ribosome inactivating proteins from the plant genus Trichosanthes.

Ribosome inactivating proteins (RIPs) are toxic RNA N-glycosidases that cleave an adenine-ribose glycosidic bond at position adenine(4324) with the conserved ricin/α-sarcin loop in the eukaryotic 28S ribosomal RNA. RIPs have captured the attention of botanists, biochemists, and drug discoverers, due to their diverse potent defensive activities, and inter alia, their antitumor and anti-HIV activities. Out of the 145 families of plants, Trichosanthes ranks among the top 5 genera with a good potential of use for discovery of anticancer drugs. Trichosanthin (TCS) is a famous type I RIP purified from T. kirilowii that has been known for around 30 years. Based on the results of voluminous in vitro and in vivo investigations, TCS is considered a good candidate for the treatment of HIV/AIDS and neoplasms. Here we integrate recent progress of the research on the different medicinal activities of TCS. In addition to TCS, other promising RIPs from the same species (such as TAP29 and trichoanguin), and from the same genus Trichosanthes are included. This review presents a brief panorama of the studies on Trichosanthes RIPs. Regarding the debilitating nature of AIDS and different tumors, further understanding of these multifunctional proteins is worthwhile since it may help to open a novel therapeutic window for these stubborn diseases.

Anti-acetylcholinesterase activities of traditional Chinese medicine for treating Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive memory loss and cognitive impairment. It is the most common type of dementia in the ageing population due to a severe loss of cholinergic neurons in selected brain area. At present, acetylcholinesterase inhibitors (AChEI) are the first group of drugs approved by the FDA to treat mild to moderate Alzheimer's disease. Most of these drugs such as huperzine and galanthamine are originally isolated from plants. In this study, the AChE inhibitory activities from extracts of Chinese medicinal herbs that have traditionally been prescribed to treat insomnia and brain function disorders were examined in a 96-well plate assay based on Ellman's method. Both ethanol and aqueous extracts of 26 traditional Chinese medicinal herbs were tested. Inhibitory effects were expressed as the percentage of inhibition. For the herbal extracts that were shown to exert a significant inhibition, dose-dependent inhibitory assays were also performed. Ethanol and aqueous extracts of six herbs were found to have high AChE inhibitory activities in a dose-dependent manner. The IC(50) of these herbal extracts on inhibition of AChE are at around 5-85 microm/ml. The results of this study indicate that there is a great potential to search for novel usage of these medicinal herbs for the treatment of AD.

Authentication of Panax ginseng and Panax quinquefolius using amplified fragment length polymorphism (AFLP) and directed amplification of minisatellite region DNA (DAMD).

AFLP profiles characteristic to Panax ginseng and Panax quinquefolius were generated using primers E-AGG/M-CAA. P. ginseng samples from different farms in China and Korea are homogeneous genetically [similarity index (SI) = 0.88-0.99], whereas samples of P. quinquefolius from different sources are much more heterogeneous (SI = 0.64-0.96). Detailed analysis of one of the polymorphic bands in P. ginseng led to the identification of a minisatellite Pg2, which contains eight repeats of 5'-AGGACTCATCACATTGTTACTC. The minisatellite DNA was consequently used in directed amplification minisatellite region DNA analysis to authenticate the two ginsengs.

Application of sequence characterized amplified region (SCAR) analysis to authenticate Panax species and their adulterants.

A 420-bp RAPD fragment from Panax quinquefolius was converted to a sequence characterized amplified region (SCAR) marker. The main difference between the SCAR of P. quinquefolius and its homolog in P. ginseng is the presence of a 25 bp insertion in the latter. Primers derived from this sequence were successfully used to authenticate six Panax species and two common adulterants.

Direct amplification of length polymorphism analysis differentiates Panax ginseng from P. quinquefolius.

The method of direct amplification of length polymorphism (DALP) was applied to authenticate Panax ginseng and P. quinquefolius. A 636-bp DALP fragment was present in all P. ginseng but absent in all the P. quinquefolius cultivars examined. We have shown that the use of DALP and conversion of specific polymorphic band to sequence-tagged site (STS) for quick authentication may be applied to authenticate related medicinal materials.

Authentication of medicinal Dendrobium species by the internal transcribed spacer of ribosomal DNA.

Herba Dendrobii (Shihu) is a commonly used Chinese medicine derived from the stem of several orchid species belonging to the genus Dendrobium. It is rather expensive and adulteration is frequent. Proper authentication of the medicinal species is necessary to protect consumers and support conservation measures. DNA sequences of the internal transcribed spacer 2 (ITS 2) of 16 Dendrobium species were shown to be significantly different from one another by an average of 12.4% and from non-orchids and Pholidota (an adulterant of Shihu) by 29.8% and 18.8%, respectively. The intra-specific variation among the Dendrobium species studied was only about 1%. Therefore, ITS 2 regions could be adopted as a molecular marker for differentiating medicinal Dendrobium species from one another and also from non-orchids and adulterants.

Differentiation of medicinal Codonopsis species from adulterants by polymerase chain reaction-restriction fragment length polymorphism.

DNA sequence analysis of rDNA internal transcribed spacer (ITS) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were exploited for their applications in differentiating medicinal species Codonopsis pilosula, C. tangshen, C. modesta, and C. nervosa var. macrantha, from two related adulterants Campanumoea javania and Platycodon grandiflorus. The data demonstrated that the rDNA ITSI and ITSII sequences of the four Codonopsis are highly homologous but not identical, and are significantly different from those of the two adulterants. The sequence difference allows effective and reliable differentiation of Codonopsis from the adulterants by PCR-RFLP.

Molecular cloning and characterization of the human glycogen synthase kinase-3beta promoter.

Glycogen synthase kinase 3beta (GSK-3beta) is a proline-directed kinase that forms part of the wingless signaling pathway. Recent studies have shown that GSK-3beta phosphorylates the microtubule-associated protein tau in vitro and in cell culture. Tau is the principal component of the paired helical filaments (PHFs) found in the brains of patients with Alzheimer disease, and PHF-tau is hyperphosphorylated. GSK-3beta is therefore one of the candidate kinases for phosphorylating tau in Alzheimer disease. GSK-3beta activity is negatively regulated by phosphorylation on serine 9 and positively regulated by phosphorylation on tyrosine 216. However, since overexpression of GSK-3beta by transfection leads to increased activity in the absence of any stimuli, GSK-3beta activity may also be regulated at the transcriptional level. Indeed, increased GSK-3beta protein levels are found in Alzheimer disease brains, and GSK-3beta is found associated with PHFs in Alzheimer disease. To understand how GSK-3beta activity may be regulated at the transcriptional level, we have isolated the human GSK-3beta promoter. The GSK-3beta promoter does not contain a conventional TATA box although several other transcription factor binding sites were identified. A putative transcription start site was mapped by 5' RACE. Transfection of various GSK-3beta promoter CAT reporter genes into both COS-7 cells and SHSY5Y neuronal cells revealed that the GSK-3beta promoter is more active in neuronal cells. Such transfection studies involving promoter deletion mutants revealed that a negative transcriptional response element may be present at position -1421 to -1363 and an activator sequence at position -427 to -384. CP2 binding sites were also present within the promoter. CP2 has recently been shown to interact with the Alzheimer disease amyloid precursor protein binding protein Fe65. The significance of these results with respect to Alzheimer disease pathogenesis are discussed.

Random primed polymerase chain reaction differentiates Codonopsis pilosula from different localities.

DNA fingerprints distinctive among the samples from different localities in China were successfully reproduced for the Chinese herb Dangshen, the roots of CODONOPSIS PILOSULA, (Campanulaceae). Similarity index (S.I.) analysis revealed that C. PILOSULA samples from the same province generated similar DNA fingerprints, while samples of different provinces displayed different DNA fingerprints. This method may be a general and valuable tool for locality authentication of other Chinese herbal medicinal materials.

Authentication of the Chinese drug "ku-di-dan" (herba elephantopi) and its substitutes using random-primed polymerase chain reaction (PCR).

DNA fingerprinting and polymorphism among the Chinese drug "Ku-Di-Dan" and its substitutes were demonstrated with arbitrarily primed polymerase chain reaction (AP-PCR) and random amplified polymorphic DNA (RAPD). Distinctive, reproducible genomic fingerprints from DNA of the Chinese drug Ku-Di-Dan and 9 species of Compositae were generated with six long (18-24 mer) and one short (10 mer) random-chosen primers with PCR. Ku-Di-Dan samples can be distinguished according to the banding patterns of their amplified DNA on agarose gels. Results showed that AP-PCR and RAPD fingerprints of the commercial samples of Ku-Di-Dan retailed in Fujian, Taiwan, Hong Kong and Macau markets are identical with that of Elephantopus scaber L. The relatedness of Ku-Di-Dan among 9 plants may also be estimated by the Similarity Indexes values of the genomic DNA fingerprints.

Authentication of Panax species and their adulterants by random-primed polymerase chain reaction.

DNA from dried or fresh roots of three medicinal Panax species and their adulterants were amplified by AP-PCR or RAPD. Fingerprints for P. ginseng or P. quinquefolius were found to be consistent irrespective of plant source or age. On the other hand, very different fingerprints were obtained from adulterants. Hence the PCR approach may be used as a means to authenticate the concerned Panax species. Analysis of the degree of similarity of the fingerprints confirmed that P. ginseng is more closely related to P. quinquefolius than to P. notoginseng.

Minireview: trichosanthin--a protein with multiple pharmacological properties.

Trichosanthin (TCS), a protein isolated from the root tuber of the Chinese medicinal herb Trichosanthes kirilowii Maxim., is used to induce abortion in China. It also possesses immunomodulatory, anti-tumor and anti-human immunodeficiency virus properties. TCS is a member of the family of ribosome-inactivating proteins and inactivates eukaryotic ribosomes via its N-glycosidase activity. The gene encoding TCS has been cloned and over-expressed and the crystal structure of this protein resolved to 1.73A. In this review, the various pharmacological properties of TCS are discussed and assessed.