Bioassay-guided isolation of neuroprotective compounds from Loranthus parasiticus against H₂O₂-induced oxidative damage in NG108-15 cells.
A parasite plant, Loranthus parasiticus (Loranthaceae), which is generally known as benalu teh (in Malay), Sang Ji Sheng (in Chinese), and baso-kisei (in Japan) distributed in south and southwest part of China, has been used as a folk medicine for the treatment of schizophrenia in southwest China. Loranthus parasiticus has various uses in folk and traditional medicines for bone, brain, kidney, liver, expels wind-damp, and prevents miscarriage.
The current study was designed to evaluate the neuroprotective activity of Loranthus parasiticus against hydrogen peroxide (H(2)O(2))-induced oxidative damage in NG108-15 hybridoma cells, so as to present evidence for the traditional use of this parasite plant in the treatment of oxidative stress related neurodegenerative diseases.
Dried and ground leaves of Loranthus parasiticus were extracted and fractionated into Loranthus parasiticus ethanol extract (LPEE), Loranthus parasiticus ethyl acetate fraction (LPEAF), and Loranthus parasiticus aqueous fraction (LPAF), which were subjected to neuroprotective activity assessment by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay. Bioassay-guided fractionation and isolation was performed subsequently to identify the most neuroactive compound. Mechanism studies underlying the present neuroprotection model of the isolated neuroactive compound was evaluated by employing 2,7-dichlorofluorescein diacetate (DCFH-DA) for intracellular reactive oxygen species (ROS) measurement, annexin V/propidium iodide (PI) for translocation of membrane phosphatidyleserine to identify cells undergo apoptosis, 5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide (JC-1) for mitochondrial membrane potential (MMP) detection, and PI for cell cycle events analysis.
Among those tested extract and fractions (LPEE, LPEAF, and LPAF) in our previous screening, LPAF significantly (P<0.01) yielded the highest neuroprotective activity with 78.00±1.85% cell viability in a dose dependent manner, supporting a neuroprotective role for LPAF in vitro. AC trimer and (+)-catechin have been isolated from LPAF successfully through bioassay-guided fractionation and isolation approach and (+)-catechin exhibited stronger neuroprotective activity as compared with AC trimer. (+)-Catechin increased cell viability and decreased the level of intracellular reactive oxygen species (ROS) in a dose-dependent manner against H(2)O(2)-induced oxidative stress in NG108-15 cells. Moreover, (+)-catechin reduced total annexin V positive cells (early and late apoptotic cells), attenuated the collapsed of MMP, and inhibited cell cycle arrested at sub-G(1) population following H(2)O(2) insult.
Collectively, the use of Loranthus parasiticus as folk medicine in the treatment of mental disorder was attributed to the presence of proanthocyanidins in this parasite plant. These findings support the traditional use of Loranthus parasiticus which is capable in managing neurological disorder where oxidative stress is implicated.
Wong DZ
，Kadir HA
，Ling SK
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Neuroprotective effects of allocryptopine-rich alkaloid extracts against oxidative stress-induced neuronal damage.
Oxidative stress is a significant feature in the pathomechanism of neurodegenerative diseases. Thus, the search for an effective and safe novel antioxidant agent with neuroprotective properties has increased the interest in medicinal plant products as a bioactive phytochemical source. However, little is known about the potential effects of the medically important Glaucium corniculatum as a natural antioxidant.
In the present study, it was aimed to investigate the anti-oxidative, anti-apoptotic, and cell cycle regulatory mechanisms underlying the neuroprotective effects of alkaloid extracts (chloroform, methanol, and water) from G. corniculatum, which was profiled for major alkaloid/alkaloids, against H2O2-induced neuronal damage in differentiated PC12 cells.
The profiles of the alkaloid extracts were analyzed by GC-MS. The effects of the alkaloid extracts on intracellular ROS production, level of apoptotic cells, and cell cycle dysregulation were analyzed by flow cytometry; the effects on mRNA expression of apoptosis-related genes were also analyzed by qRT-PCR.
The same alkaloid components, allocryptopine, tetrahydropalmatine, and tetrahydroberberine N-oxide were obtained in all three solvents, but the ratios of the components differed according to the solvents. Allocryptopine was determined to be the major alkaloid ingredient in the alkaloid extracts, with the highest amount of allocryptopine (497 μg/mg) being found in the chloroform alkaloid extract (CAE) (*p < 0.05). The best results were obtained from CAE, which has the highest amount of allocryptopine among alkaloid extracts in all studies. CAE suppressed intracellular ROS production (5.7-fold), percentage of apoptotic cells (3.0-fold), and cells in the sub G1 phase (6.8-fold); additionally, it increased cells in the G1 phase (1.5-fold) (**p < 0.01). CAE remarkably reduced the expressions of Bax, Caspase-9/-3 mRNA (2.4-3.5-fold) while increasing the expression of Bcl-2 mRNA (3.0-fold) (*p < 0.05).
Our results demonstrated that alkaloid extracts from G. corniculatum, which contain allocryptopine, tetrahydropalmatine, and tetrahydroberberine N-oxide suppressed oxidative stress-induced neuronal apoptosis, possibly by suppressing the mitochondrial apoptotic pathway and regulating the cell cycle. These results are the first report that related alkaloids have played a neuroprotective role by regulating multiple mechanisms. Thus, our study indicated that these alkaloids especially allocryptopine could offer an efficient and novel strategy to explore novel drugs for neuroprotection and cognitive improvement.
Nigdelioglu Dolanbay S
，Kocanci FG
，Aslim B
《-》
New insights into the antioxidant and apoptotic potential of Glycyrrhiza glabra L. during hydrogen peroxide mediated oxidative stress: An in vitro and in silico evaluation.
Plant-derived substances (phytochemicals) are well recognized as sources of pharmacologically potent drugs in the treatment of several oxidative stress related disorders. Our study aims to evaluate the antioxidant and apoptotic effects of Glycyrrhiza glabra L. in both cell free and cell culture system. Plant fractions have been prepared with hexane, chloroform, ethyl acetate, methanol and water and their antioxidant properties are reviewed. Potent antioxidant activity has been well established in both in vitro and in silico studies which is believed to be responsible for the anticancerous nature of the plant. Results obtained indicate that methanol fraction of G. glabra L. exhibited maximum scavenging activity against DPPH and nitric oxide free radicals comparable to standard antioxidant L-AA. Administration of methanol fraction also considerably reduced the malondialdehyde produced due to lipid peroxidation in mammalian liver tissues. Moreover, the levels of antioxidant enzymes SOD, CAT, GST, GPx and GR in the oxidative stress induced tissues were refurbished significantly after treatment with plant's methanol fraction. Moreover, methanol fraction was found to be nontoxic to normal human cell line whereas it inhibited cancer cells HeLa and HepG2 considerably. Apoptosis was established by DAPI fluorescent staining and western blot analysis of pro apoptotic protein caspase-8, caspase-3 and anti-apoptotic protein Bcl-2.There is an up regulation in the levels of pro apoptotic caspase-8 and caspase-3 and down regulation of anti-apoptotic Bcl-2. Furthermore, GC-MS analysis of the methanol fraction revealed the presence of many compounds. In silico experiments using Autodock 4.2 tools showed strong affinity of plant compounds towards antioxidant enzymes (proteins) thus validating with the conclusions of antioxidant enzyme assays and establishing a role in cancer pathogenesis.
Hejazi II
，Khanam R
，Mehdi SH
，Bhat AR
，Moshahid Alam Rizvi M
，Islam A
，Thakur SC
，Athar F
... -
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Neuroprotective effect of trans-N-caffeoyltyramine from Lycium chinense against H(2)O(2) induced cytotoxicity in PC12 cells by attenuating oxidative stress.
Natural products play a critical role in the promotion of good health as regards the prevention and management of oxidative stress related and neurodegenerative disorders. Oxidative stress has been implicated in several apoptotic pathways associated with cell damages in neuronal disorders. The aim of this study was to investigate the antioxidant effect of trans-N-caffeoyltyramine (TNC) isolated from Cortex lycii against hydrogen peroxide (H2O2) induced cell damages in PC12 cells as well as its mechanism of action. The results obtained indicated that pretreatment with TNC before the exposure of cells to H2O2 toxicity lead to a significant increase in the cell viability and the antioxidant enzyme activities catalase (CAT), superoxide dismutase (SOD), glutathione (GSH) and reduced the level of malondialdehyde (MDA). Furthermore, TNC attenuated the influx of Ca2+, ROS formation and restored the impaired mitochondria membrane potential (MMP). Thus TNC may be used as an alternative therapy for the prevention and treatment of neuronal disorders elicited by oxidative stress.
Olatunji OJ
，Chen H
，Zhou Y
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