Bilobalide promotes expression of glial cell line-derived neurotrophic factor and vascular endothelial growth factor in rat astrocytes.

AIM: To study the effects of bilobalide on the expression of glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF) in rat astrocytes in vitro. METHODS: Semiquantification polymerase chain reaction (SQ-PCR) was used to investigate GDNF and VEGF mRNA expression in the astrocytes after bilobalide (5, 15, 50, 100 mumol.L-1) treatment. Immunohistochemistry method was used to detect GDNF and VEGF protein expression in cells treated with bilobalide 50 mumol.L-1 for 24 h. RESULTS: GDNF and VEGF mRNA increased markedly after astrocytes were treated with bilobalide 50 mumol.L-1 for 12 h. GDNF and VEGF protein were detected in the cytoplasm of astrocytes after the cells were treated with bilobalide 50 mumol.L-1 for 24 h. CONCLUSION: Bilobalide induced GDNF and VEGF expression in the cultured astrocytes.

Involvement of monoamine oxidase inhibition in neuroprotective and neurorestorative effects of Ginkgo biloba extract against MPTP-induced nigrostriatal dopaminergic toxicity in C57 mice.

The present study investigated the neuroprotective and neurorestorative effects of Ginkgo biloba extract (EGb 761) and its two components ginkgolides A (BN52020) and B (BN52021) in mice. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (30 mg/kg/d i.p. for six days) significantly reduced striatal dopamine (DA) levels in C57 mice measured by high performance liquid chromatography with electrochemical detection (HPLC-EC). When C57 mice were pretreated with EGb 761 (20, 50, 100 mg/kg/d i.p.) for 7 days and then treated with the same extract 30 min before MPTP injection for 6 days, the neurotoxic effect of MPTP was antagonized in a dose-dependent fashion. Similar treatment with ginkgolides A and B (5, 10, 50 mg/kg/d i.p.) showed no protective effect. When C57 mice were treated with EGb 761 (50 mg/kg/d i.p.) after MPTP-lesion, the recovery of striatal dopamine (DA) levels was accelerated. However, similar treatment with ginkgolides A or B (10 mg/kg/d i.p.) did not show any effect. EGb 761, but not ginkgolides A and B, nonselectively inhibited mouse brain MAO activity in vitro (IC50 = 36.45 +/- 1.56 microg/ml) tested by an improved fluorimetric assay. The results demonstrate that EGb 761 administered before or after MPTP treatment effectively protects against MPTP-induced nigrostriatal dopaminergic neurotoxicity and that the inhibitory effect of EGb 761 on brain MAO may be involved in its neuroprotective effect.

Alliin lyase (Alliinase) from garlic (Allium sativum). Biochemical characterization and cDNA cloning.

The garlic plant (Allium sativum) alliinase (EC 4.4.1.4), which catalyzes the synthesis of allicin, was purified to homogeneity from bulbs using various steps, including hydrophobic chromatography. Molecular and biochemical studies showed that the enzyme is a dimer of two subunits of MW 51.5 kDa each. Its Km using synthetic S-allylcysteine sulfoxide (+ isomer) as substrate was 1.1 mM, its pH optimum 6.5, and its isoelectric point 6.35. The enzyme is a glycoprotein containing 6% carbohydrate. N-terminal sequences of the intact polypeptide chain as well as of a number of peptides obtained after cyanogen bromide cleavage were obtained. Cloning of the cDNAs encoding alliinase was performed by a two-step strategy. In the first, a cDNA fragment (pAli-1-450 bp) was obtained by PCR using a mixed oligonucleotide primer synthesized according to a 6-amino acid segment near the N-terminal of the intact polypeptide. The second step involved screening of garlic lambda gt11 and lambda ZAPII cDNA libraries with pAli-1, which yielded two clones; one was nearly full length and the second was full length. These clones exhibited some degree of DNA sequence divergence, especially in their 3' noncoding regions, suggesting that they were encoded by separate genes. The nearly full length cDNA was fused in frame to a DNA encoding a signal peptide from alpha wheat gliadin, and expressed in Xenopus oocytes. This yielded a 50 kDa protein that interacted with the antibodies against natural bulb alliinase. Northern and Western blot analyses showed that the bulb alliinase was highly expressed in bulbs, whereas a lower expression level was found in leaves, and no expression was detected in roots. Strikingly, the roots exhibited an abundant alliinase activity, suggesting that this tissue expressed a distinct alliinase isozyme with very low homology to the bulb enzyme.

Development of natural products as drugs acting on central nervous system.

We have recently studied several natural product constituents which have effects on the CNS. (1) Tetrahydropalmatine (THP) and its analogues were isolated from Corydalis ambigua and various species of Stephania. (+)-THP and (-)-THP possess not only analgesic activity, but also exert sedative-tranquilizing and hypnotic actions. Results of receptor binding assay and their pre- and post-synaptic effects on dopaminergic system indicate that (-)-THP and (-)-stepholidine are dopamine receptor antagonists while (+)-THP is a selective dopamine depletor. (2) 3-Acetylaconitine (AAC) is an alkaloid isolated from Aconitum flavum. The relative potency of analgesic action of AAC was 5.1-35.6 and 1250-3912 times that of morphine and aspirin, respectively. The analgesic effect of AAC was not antagonized by naloxone, but was eliminated by reserpine. In monkeys, after AAC was injected for 92 days, no abstinence syndrome was seen after sudden AAC withdrawal or when challenged with nalorphine. (3) Huperzine A (Hup-A) is an alkaloid isolated from Huperzia serrata which was found to be a selective ChE inhibitor and could improve learning and retrieval processes. Preliminary clinical studies showed that Hup-A improve short- and long-term memory in patients of cerebral arteriosclerosis with memory impairment. (4) Ranamargarin is a new tetradecapeptide isolated from the skin of the Chinese frog Rana margaratae. This peptide may mainly act on NK-1 receptor.