Neuroprotective effects of total alkaloids fraction of Huperzia serrata on scopolamine-induced neurodegenerative animals.

Huperzia serrata contains Huperzine A (HupA)-an alkaloid used to treat cognitive dysfunction. In this study, we used the total alkaloids (HsAE) to investigate their potential in managing cognitive impairment in comparison with HupA. The antioxidant activity was measured by DPPH assay. In the cellular study, the cell viability and level of ACh of SH-SY5Y cells were evaluated after pretreated with HsAE and scopolamine. For in vivo assay, mice were pre-treated with HsAE, and HupA and undergone scopolamine injection for cognitive impairment. The behavioral tests including the Y-maze and Morris water maze test and the AChE activity, the SOD, CAT, MDA level in the hippocampus and cortex were evaluated. HsAE showed significant scavenging properties on DPPH radicals. HsAE was not toxic to SH-SY5Y cells, and can rescue these cells upon scopolamine treatment. Intriguingly, HsAE showed the neuroprotection against scopolamine-induced amnesia in mice. Moreover, HsAE decreased AChE activity, MDA level, increased antioxidative enzyme activity in the hippocampus as well as cortex of mice, which was relatively better than that of HupA. These findings suggested that HsAE may significantly protect the neurons of mice with scopolamine-induced memory impairment connected to AChE depletion and oxidative stress.

Polysaccharide extracted from Huperzia serrata using response surface methodology and its biological activity.

In this study, Huperzia serrata polysaccharide (HSP) fraction was isolated using response surface methodology (RSM) and Box-Behnken design (BBD). The extraction time, temperature and ratio of water to raw material were employed effects. And properties of four polysaccharide (60%-HSP, 70%-HSP, 80%-HSP and 90%-HSP) were evaluated. The results indicated that the optimal extraction conditions were the following: 3.07 h, 49.46 °C and a liquid material ratio of 20.73:1. The four HSP presented irregular aggregation of shape. And all HSP exhibited antioxidant and anticancer activities.

Biotransformation of Huperzine A by Irpex lacteus-A fungal endophyte of Huperzia serrata.

The biotransformation of huperzine A (hupA), one of the characteristic bioactive constituents of the medicinal plant Huperzia serrata, by a fungal endophyte of the host plant was studied. Two previously undescribed compounds 1-2, along with a known analog 8α,15α-epoxyhuperzine A (3), were isolated and identified. The structures of all the isolates were established by spectroscopic methods including NMR, MS, IR, and UV spectra. In particular, the absolute configurations of 1 and 2 were elucidated by CD spectra comparison and theoretic NOE strength calculation. In the LPS-induced neuro-inflammation injury assay, 1-3 exhibited moderate neuroprotective activity by increasing the viability of U251 cell lines with EC50 values of 35.3 ±â€¯0.9, 32.1 ±â€¯0.9, and 50.3 ±â€¯0.8 nM, respectively.

Potent effects of alkaloid-rich extract from Huperzia selago against sodium nitroprusside-evoked PC12 cells damage via attenuation of oxidative stress and apoptosis.

Imbalance between production and scavenging of free radicals and other reactive oxygen species (ROS) is a component of many diseases, but it is especially important in aging-related diseases of the central nervous system. Oxidative stress-induced neuronal dysfunction plays an important role in the pathomechanism of neurodegenerative disorders, including Alzheimer's and Parkinson's disease. Experimental data showed that free radical scavengers may protect the brain against oxidative modifications. The need for efficient and safe antioxidants with therapeutic potential stimulated the rise of interest in the medicinal plant products, which are a rich source of phytochemicals possessing biological activity. In our studies we focused on alkaloid fractions (AFs) isolated from club moss, Huperzia selago and Diphasiastrum complanatum, due to their beneficial activity and exclusive chemical structure. Our previous study demonstrated that selected alkaloids from Huperzia selago effectively protect macromolecules from oxidative damage. Therefore, in the present study we investigated the effects and mechanisms of action of AFs isolated from Huperzia selago and Diphasiastrum complanatum against sodium nitroprusside (SNP)-induced oxidative injury in PC12 cells. The results demonstrated that the selected AFs via reduction of nitric oxide (NO) liberation protected cells against oxidative stress, DNA and mitochondrial damage, as well as apoptosis caused by SNP. Selected AF notably decreased SNP-evoked mitochondrial polymerase γ (Polg) up-regulation. Furthermore, AF which contains Lycopodine, Serratidine, Lycoposerramine-G and (probably) Cermizine B completely inhibited the SNP-induced expression of interferon-γ (Ifng) and cyclooxygenase 2 (Ptgs2) as well as significantly down-regulated the expression of 12/15-lipoxygenase (Alox12) and tended to decrease the mRNA level of interleukin-6 gene (Il6). In conclusion, these results suggest that the AFs from Huperzia selago effectively protect PC12 cells against SNP-induced oxidative damage by adjusting the level of reactive nitrogen species, suppression of apoptosis and down-regulation of proinflammatory genes. The compounds present in these AFs could be potential candidates to develop successful drugs preventing oxidative damage and apoptosis in age-related neurodegenerative disorders.

Molecular Evolution and Functional Characterization of a Bifunctional Decarboxylase Involved in Lycopodium Alkaloid Biosynthesis.

Lycopodium alkaloids (LAs) are derived from lysine (Lys) and are found mainly in Huperziaceae and Lycopodiaceae. LAs are potentially useful against Alzheimer's disease, schizophrenia, and myasthenia gravis. Here, we cloned the bifunctional lysine/ornithine decarboxylase (L/ODC), the first gene involved in LA biosynthesis, from the LA-producing plants Lycopodium clavatum and Huperzia serrata We describe the in vitro and in vivo functional characterization of the L. clavatum L/ODC (LcL/ODC). The recombinant LcL/ODC preferentially catalyzed the decarboxylation of l-Lys over l-ornithine (l-Orn) by about 5 times. Transient expression of LcL/ODC fused with the amino or carboxyl terminus of green fluorescent protein, in onion (Allium cepa) epidermal cells and Nicotiana benthamiana leaves, showed LcL/ODC localization in the cytosol. Transgenic tobacco (Nicotiana tabacum) hairy roots and Arabidopsis (Arabidopsis thaliana) plants expressing LcL/ODC enhanced the production of a Lys-derived alkaloid, anabasine, and cadaverine, respectively, thus, confirming the function of LcL/ODC in plants. In addition, we present an example of the convergent evolution of plant Lys decarboxylase that resulted in the production of Lys-derived alkaloids in Leguminosae (legumes) and Lycopodiaceae (clubmosses). This convergent evolution event probably occurred via the promiscuous functions of the ancestral Orn decarboxylase, which is an enzyme involved in the primary metabolism of polyamine. The positive selection sites were detected by statistical analyses using phylogenetic trees and were confirmed by site-directed mutagenesis, suggesting the importance of those sites in granting the promiscuous function to Lys decarboxylase while retaining the ancestral Orn decarboxylase function. This study contributes to a better understanding of LA biosynthesis and the molecular evolution of plant Lys decarboxylase.

An Unprecedented High Content of the Bioactive Flavone Tricin in Huperzia Medicinal Species Used by the Saraguro in Ecuador.

The flavone tricin (5,7,4'-trihydroxy-3',5'-dimethoxyflavone) is considered to be a selective potent inhibitor of different cancer cell lines and a potential colorectal cancer chemopreventive agent. In this paper we describe a reliable UHPLC-UV-ESIMS method for the determination of tricin in Huperzia plants used in the traditional medicine of the Saraguro community living in Southern Ecuador. An unusually high amount of tricin was found in H. brevifolia and H. compacta, which exceeded the content of this flavone determined so far in other plants.

Effects of huperzine A on secretion of nerve growth factor in cultured rat cortical astrocytes and neurite outgrowth in rat PC12 cells.

AIM: To study the effects of huperzine A (HupA) on neuritogenic activity and the expression of nerve growth factor (NGF). METHODS: After being treated with 10 micromol/L HupA, neurite outgrowth of PC12 cells was observed and counted under phase-contrast microscopy. Mitogenic activity was assayed by [3H]thymidine incorporation. Cell cytotoxicity was evaluated by lactate dehydrogenase (LDH) release. AChE activity, mRNA and protein expression were measured by the Ellman method, RT-PCR, and Western blot, respectively. NGF mRNA and protein levels were determined by RT-PCR and ELISA assays. RESULTS: Treatment of PC12 cells with 10 micromol/L HupA for 48 h markedly increased the number of neurite-bearing cells, but caused no significant alteration in cell viability or other signs of cytotoxicity. In addition to inhibiting AChE activity, 10 micromol/L HupA also increased the mRNA and protein levels of this enzyme. In addition, following 2 h exposure of the astrocytes to 10 micromol/L HupA, there was a significant up-regulation of mRNA for NGF and P75 low-affinity NGF receptor. The protein level of NGF was also increased after 24 h treatment with HupA. CONCLUSION: Our findings demonstrate for the first time that HupA has a direct or indirect neurotrophic activity, which might be beneficial in treatment of neurodegenerative disorders such as Alzheimer disease.

Huperzine B protects rat pheochromocytoma cells against oxygen-glucose deprivation-induced injury.

AIM: To test the ability of huperzine B (HupB) to alleviate injury from oxygen-glucose deprivation (OGD) in the rat pheochromocytoma line PC12 cells. METHODS: After OGD for 3 h and reoxygenation for 24 h, neuronal morphology was observed by phase-contrast microscopy; cell survival was quantified by the reduction of MTT; malondialdehyde (MDA) was determined by the thiobarbituric acid; superoxide dismutase (SOD) was assayed by a modification of the xanthine/xanthine oxidase; and lactate (LA) was measured according to Marbach and Weil. RESULTS: OGD for 3 h and reoxygenation for 24 h triggered death in nearly 70 % of cells, along with major changes in morphology and biochemistry including elevated level of MDA, SOD activity, and LA content. Cells pretreated with HupB 1-100 micromol/L for 2 h showed significantly improved survival and reduced biochemical and morphologic signs of toxicity. CONCLUSION: HupB protected PC12 cells against OGD-induced injury, most likely by alleviating disturbances of oxidative and energy metabolism.