Ilex latifolia Thunb is a traditional Chinese tea and herbal medicine. In this study, one new triterpene saponin (1) and six known triterpenoids (2–7) were isolated from the methanol extract of I. latifolia using a PC12 cell bioassay system. The structures and stereochemistry of these compounds were elucidated using spectroscopic methods and chemical derivatization. This new triterpene saponin (1) was characterized as an ursolic type acid with a 19α-hydroxyl and a trisaccharide moiety at C-3. Compound 1 significantly promoted the neurite outgrowth in PC12 cells by 52% at 10 μM, whereas compounds 2–7 showed less neuritogenic activity. Structure activity relationship studies indicated that introducing a trisaccharide moiety at C-3 is important for the neuritogenic activity, but the sugar group at C-28 decreased this activity. In addition, compound 1 increased the neurite outgrowth length in primary cortical neuron cells of mice and also exhibited a neuronal protection effect on H2O2-damaged PC12 cells at optimum concentrations.

•We isolated 5 cerebrosides from Baifuzi and determined their chemical structures.•Four cerebrosides significantly activated the BK channel.•Acyl chain lengths of the cerebrosides potently affect the channel activation.•Double bond configuration of the cerebrosides weakly affected the channel.Our previous study reported that a mixture of cerebrosides from traditional Chinese medicine Baifuzi could activate BKCa channel. It is curious to know the effect of each single cerebroside on the channel. Here we isolated 5 pure cerebrosides from the mixture and determined their chemical structures. The most potent one increased the single channel open probability 6 folds with EC50 value of 1.0 μM. The structure–activity relationship revealed that acyl chain length of cerebrosides has potent effect, while configuration of double bond at C8–C9 on their long chain base has weak effect on the channel activity. Thus, this research provides a guide for design and synthesis of a lead cerebroside with more potent effect on the BKCa channel.The longer the acyl chain length, the more efficient the cerebrosides to activate the BKCa channel.

Two novel ergosterol derivatives, ganodermasides A and B, hydroxylated at C-15 were isolated from the methanol extract of spores of a medicinal mushroom, Ganoderma lucidum, showed to extend the replicative life span of Saccharomyces cerevisiae, a yeast of K6001 strain. The stereostructures of ganodermasides A and B were determined based on the spectroscopic analysis and comparison of spectroscopic data. These new sterols have a 4, 6, 8(14), 22-tetraene-3-one unit with a unique hydroxylation at C-15. The anti-aging activity of these compounds on yeast is comparable to a well-known substance, resveratrol. Based on results of the investigation of the mechanism of biological activity, ganodermasides A and B regulated UTH1 expression in order to extend the replicative life span of yeast.Two novel ergosterol derivatives, ganodermasides A (1) and B (2), were isolated from spores of a medicinal mushroom, Ganoderma lucidum. They exhibited remarkable activity on extending replicative lifespan of a yeast strain via UTH1 gene.

Nine new alkyl 2,3-dihydroxybenzoates, gentisides C–K, were isolated from the traditional Chinese medicine Gentiana rigescens Franch. Their structures and stereochemistry were elucidated by spectroscopic methods, and comparison of the specific rotation with that of the gentiside B. These metabolites are additional members of the gentisides which belong to a novel class of neuritogenic compounds. They are structurally different from one another because they possess varying alkyl chain lengths, with or without an isobutyl or isopropyl group at the end of the alkyl chain. These compounds are potent inducers of neurite outgrowth on PC12 cells. The gentiside C possessing the shortest alkyl chain length exhibited the highest neuritogenic activity among all of the gentisides. Gentiside C showed a significant neuritogenic activity at 1 μM against PC12 cells comparable to that seen for the best nerve growth factor (NGF) concentration of 40 ng/mL. In addition, evident neuritogenic activity was observed in the cells when treated with gentiside C at a concentration as low as 0.03 μM. The structure–activity relationships within the gentisides A–K revealed that alkyl chain length is important for the activity, but structure diversity at the end of the alkyl chain is not.Gentisides C–K are additional members of a novel class of neuritogenic compounds. They exhibited significant neuritogenic activity against PC12 cells. The structure–activity relationships within the gentisides A–K revealed that the alkyl chain length is important for the activity, but structure diversity at the end of alkyl chain is not.

Two new alkyl 2,3-dihydroxybenzoates, gentisides A and B, were isolated from the traditional Chinese medicine Gentiana rigescens Franch. Their structures and stereochemistry were elucidated by spectroscopic methods and chemical derivatization. These compounds showed a significant neuritogenic activity at 30 μM against PC12 cells that was comparable to that seen for the best nerve growth factor (NGF) concentration of 40 ng/mL. Gentisides A and B showed parallel activity, indicating that the observed structural difference at the end of their alkyl chain did not affect neuritogenic activity.Two new alkyl 2,3-dihydroxybenzoates, gentisides A and B, were isolated from the traditional Chinese medicine Gentiana rigescens Franch. They showed a significant neuritogenic activity at 30 μM against PC12 cells that was comparable to that seen for the best nerve growth factor concentration of 40 ng/mL.

Ethnopharmacological relevanceG. rigescens Franch (Long Dan Cao in Chinese) is a well-known TCM herb. It is clinically used with other drugs for the treatment of brain diseases such as epilepsy, postherpetic neuralgia in China.Aim of studyIn our previous study, the 11 dihydroxybenzoates compounds with NGF mimicking activity from G. rigescens Franch were found. In the present study, the neurogenesis and neuroprotection of a mixture of benzoates ( n-GS) were investigated in animal level.Materials and methodsThe NGF mimicking activity of n-GS from G. rigescens Franch was examined in PC12 cells. The neurogenesis effects of n-GS were investigated in ICR mice with 5-bromo-2-deoxyuridine (BrdU) and neuronal neclei (NeuN) double immunostaining. Furthermore, the neuroprotection effects of n-GS on the memory in a scopolamine (SCO)-induced mouse model were evaluated with animal behavior tests.ResultsThe NGF-mimicking function and neurogenesis of n-GS were observed in PC12 cells and in normal mice. Subsequently, we investigated the effects of n-GS on the memory in a SCO-induced mouse model. In Y-maze test, SCO significantly lowered the alternation. This finding was reversed by n-GS and donepezil (DONE). SCO significantly impaired the mice's performance in novel object recognition (NOR) and Morris water maze (MWM) tests. The time spent to explore the novel object was longer in the n-GS- and DONE-treated groups than in the SCO control group. In the MWM test, the escape latency of n-GS- and DONE-treated groups was shorter than that of the SCO control group. Mechanism study showed that SCO significantly reduced superoxide dismutase (SOD) but increased the activities of acetylcholinesterase (AChE) and the levels of malondialdehyde (MDA) in the hippocampus and cerebral cortex, which all can be improved by n-GS and DONE. Additionally, the phosphorylation of type 1 insulin-like growth factor (IGF-1) receptor, extracellular signal-regulated kinase (ERK), and cAMP responsive element-binding (CREB) protein in the hippocampus was significantly up-regulated in the treatment group compared with that in the SCO group.Conclusionsn-GS could alleviate impaired memory of the SCO-induced mice model by inhibiting AChE activity and oxidative stress, and regulating the IGF-1R/ERK signaling pathway.Download high-res image (199KB)Download full-size image

BackgroundNeurotrophic factors such as nerve growth factor (NGF) play important roles in nervous system. NGF is a potential therapeutic drug for treatment of neurodegenerative diseases. However, because of physicochemical property, NGF cannot pass through the blood–brain barrier (BBB). Hence, small molecules which exhibit NGF-mimic activity and can pass through the BBB are considered to be promising drug candidates for treatment of such diseases.PurposeThe present study was designed to isolate NGF-mimic substance from extract of natural products, determine their structures and investigate mechanism of action of the active substance.MethodsExtract of Lindernia crustacean was partitioned between water and ethyl acetate to obtain water layer and ethyl acetate layer samples, respectively, and then evaluated their neuritogenic activity in PC12 cells. The active sample was separated by open columns, followed by HPLC purification to obtain active compound. Then, specific inhibitors were used to investigate signaling pathway of neurite outgrowth induced by the active compound. Finally, western blot analysis was performed to confirm the pathway proposed by inhibitor experiments.ResultsThe ethyl acetate layer sample of extract of Lindernia crustacea exhibited significant neuritogenic activity. Two new compounds, named as linderside A and lindersin B, were isolated; their structures were elucidated by spectroscopic and chemical derivatization methods. Linderside A is a cucurbitane glycoside, whereas lindersin B is a cucurbitane triterpenoid. Each compound has an unusual isopentene unit, namely, a double bond bound to an unmodified isopropyl group at the end of cucurbitane triterpenoid side chain. Among them, lindersin B induced significant neurite outgrowth in PC12 cells, while linderside A was inactive against PC12 cells. Western blotting analysis results showed that lindersin B-induced neuritogenic activity depended on the activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK). Moreover, tyrosine kinase A (TrKA) and phosphatidylinositol 3 kinase (PI3K) were also involved in the signaling pathway.ConclusionsTwo new cucurbitane triterpenoids, linderside A and lindersin B, were isolated from Lindernia crustacean. Neurite outgrowth induced by lindersin B in PC12 cells depends on activation of TrkA/PI3K/ERK signaling pathway.Download high-res image (105KB)Download full-size imageTwo new cucurbitane triterpenoids, linderside A and lindersin B, with unusual side chain were isolated. Neurite outgrowth induced by lindersin B in PC12 cells depends on activation of TrkA/PI3K/ERK signaling pathway.

•Three new (1–3) and a known (4) steroidal saponins were isolated from O. japonicus.•All of the isolated saponins exhibited neuritogenic activity.•Compound 1-induced neurite outgrowth depends on the activation of MEK/ERK signaling pathway.Three new steroidal saponins (1–3) and a known saponin (4) were isolated from Ophiopogon japonicus (Thunb.) Ker-Gawl. Their structures were determined by spectroscopic analyses and chemical derivatization. The isolated compounds (1–4) were potent inducers of neuritogenesis on PC12 cells. Compound 1 showed the highest neuritogenic activity of 46% at 1 μM. The study of structure–activity relationships suggests that aglycone is important for the neuritogenic activity of the compounds. Specific inhibitor experiments and Western blot analysis suggest that 1-induced neuritogenic activity depends on the activation of mitogen-activated protein kinase kinase (MEK)/extracellular signal regulated kinase (ERK) signaling pathway on PC12 cells.Download full-size image