Lycopodiastrum casuarinoides: An overview of their phytochemicals, biological activities, structure-activity relationship, biosynthetic pathway and 13C NMR data.

Huperzine A, a lycodine-type alkaloid, exhibits potent inhibitory activity against acetylcholinesterase (AChE) and has been utilized to treat neurodegenerative diseases' symptoms. Lycopodiastrum casuarinoides, a member of the family Lycopodiaceae, is renowned for its lycodine-type alkaloids. Some of these alkaloids show various pharmacological benefits, such as anti-cholinesterase, neuroprotective, and cytotoxic effects. To date, 113 chemical compounds, including seventy-four lycodine-type alkaloids, ten terpenoids, eleven aliphatics, and eighteen other compounds, have been isolated from this plant. In this review, we have discussed phytochemicals and biological activities of the reported compounds of L. casuarinoides. Moreover, structure-activity relationship (SAR), plausible biosynthetic pathways, and 13C nuclear magnetic resonance spectroscopy (13C NMR) data of the lycodine-type alkaloids are also summarized.

Fractionation of Lycopodiaceae Alkaloids and Evaluation of Their Anticholinesterase and Cytotoxic Activities.

In view of the abundant evidence that Lycopodiaceae alkaloids, including the well-known huperzine A (HupA), are among the potent acetylcholinesterase (AChE) inhibitors, an attempt was made to search for new compounds responsible for this property. For this purpose, three plant species belonging to the Lycopodiaceae family, commonly found in the Euro-Asia region, were subjected to the isolation of bioactive compounds, their identification and subsequent evaluation of their anticholinesterase and cytotoxic activities. Methanolic extracts of two Lycopodium and one Hupezia species were obtained via optimized pressurized liquid extraction (PLE) and then pre-purified using innovative gradient vacuum liquid chromatography (gVLC). For the first time, three sorbents of different porosity packed in polypropylene cartridges and mobile phase systems of different polarity were used to elute the target compounds. This technique proved to be a rapid tool for the obtainment of alkaloid fractions and allowed one to select the appropriate process conditions to yield potent AChE inhibitors in each of the species studied. More than 100 collected fractions were analyzed via HPLC/ESI-QTOF-MS, which enabled one to detect more than 50 compounds, including several new ones previously unreported. Some of them were present in high purity fractions (60-90% of the established purity). TLC bioautography assays proved that the analyzed species are rich sources of AChE inhibitors, but H. selago showed the highest anti-AChE activity. Additionally, the modified silanized silica gel sorbent used allowed one to isolate L. clavatum alkaloids more efficiently using an aqueous reversed-phase solvent system. Furthermore, the tested extracts from the three plant extracts were found to be safe, as they did not exhibit cytotoxicity to skin fibroblasts.

Optimization of Pressurized Liquid Extraction of Lycopodiaceae Alkaloids Obtained from Two Lycopodium Species.

Alkaloids of the Lycopodiaceae family are of great interest to researchers due to their numerous properties and wide applications in medicine. They play a very important role mainly due to their potent antioxidant, antidepressant effects and a reversible ability to inhibit acetylcholinesterase (AChE) enzyme activity. This property is of immense importance due to the growing problem of an increasing number of patients with neurodegenerative diseases in developed countries and a lack of effective and efficient treatment for them. Numerous studies have shown that Lycopodiaceae alkaloids are a rich source of AChE inhibitors. In the obtaining of new therapeutic phytochemicals from plant material, the extraction process and its efficiency is crucial. Therefore, the aim of this work was to optimize the conditions of modern PLE to obtain bioactive alkaloids from two Lycopodium species: L. clavatum L. and L. annotinum L. Five different solvents of different polarity were used for prepared plant extracts in order to compare the alkaloid content in and thereby effectiveness of the entire extraction. PLE parameters were used based on multiple studies conducted that gave the highest alkaloids recovery. Crude extracts were purified using solid-phase extraction (SPE) on Oasis HLB cartridge and examined by HPLC/ESI-QTOF-MS of the highly abundant alkaloids. To the best of our knowledge, this is the first time such high recoveries have been obtained for known Lycopodiaceae alkaloids. The best extraction results of alkaloid-lycopodine were detected in the dichloromethane extract from L. clavatum, where the yield exceeded 45%. The high recovery of annotinine above 40% presented in L. annotinum was noticed in dichloromethane and ethyl acetate extracts. Moreover, chromatograms were obtained with all isolated alkaloids and the best separation and quality of the bands in methanolic extracts. Interestingly, no alkaloid amounts were detected in cyclohexane extracts belonging to the non-polar solvent. These results could be helpful for understanding and optimizing the best conditions for isolating potent AChE inhibitors.

In Silico Inhibition of BACE-1 by Selective Phytochemicals as Novel Potential Inhibitors: Molecular Docking and DFT Studies.

BACKGROUND: Alzheimer's Disease (AD) has become the most common age-dependent disease of dementia. The trademark pathologies of AD are the presence of amyloid aggregates in neurofibrils. Recently phytochemicals being considered as potential inhibitors against various neurodegenerative, antifungal, antibacterial and antiviral diseases in human beings. OBJECTIVE: This study targets the inhibition of BACE-1 by phytochemicals using in silico drug discovery analysis. METHODS: A total of 3150 phytochemicals were collected from almost 25 different plants through literature assessment. The ADMET studies, molecular docking and density functional theory (DFT) based analysis were performed to analyze the potential inhibitory properties of these phytochemicals. RESULTS: The ADMET and docking results exposed seven compounds that have high potential as an inhibitory agent against BACE-1 and show binding affinity >8.0 kcal/mol against BACE-1. They show binding affinity greater than those of various previously reported inhibitors of BACE-1. Furthermore, DFT based analysis has shown high reactivity for these seven phytochemicals in the binding pocket of BACE- 1, based on ELUMO, EHOMO and Kohn-Sham energy gap. All seven phytochemicals were testified (as compared to experimental ones) as novel inhibitors against BACE-1. CONCLUSION: Out of seven phytochemicals, four were obtained from plant Glycyrrhiza glabra i.e. Shinflavanone, Glabrolide, Glabrol and PrenyllicoflavoneA, one from Huperzia serrate i.e. Macleanine, one from Uncaria rhynchophylla i.e. 3a-dihydro-cadambine and another one was from VolvalerelactoneB from plant Valeriana-officinalis. It is concluded that these phytochemicals are suitable candidates for drug/inhibitor against BACE-1, and can be administered to humans after experimental validation through in vitro and in vivo trials.

Lycodine-type alkaloids from Lycopodiastrum casuarinoides and their acetylcholinesterase inhibitory activity.

Five previously undescribed lycodine-type alkaloids, named huperzine Y (1), 8,15-epoxy-N-demethylhuperzinine (2), 7-hydroxyl-huperzinine (3), huperzine Z (4), and huperzine D N-oxide (5), were isolated from the aerial parts and roots of Lycopodiastrum casuarinoides (Lycopodiaceae), along with ten known analogues. The structures of the new compounds were elucidated by means of spectroscopic technique (IR, UV, MS and NMR). The absolute configurations of the new compounds were established on the basis of comparison of their experimental and TD-DFT (time-dependent density functional theory) calculated ECD spectra. Moreover, all the isolates were evaluated for acetylcholinesterase (AChE) inhibitory activity. Only huperzine C showed moderate activity, with an IC50 value of 0.525 ±â€¯0.140 µM, which was comparable with the positive control, huperzine A (IC50 = 0.143 ±â€¯0.029 µM).

Anti-cholinesterase activities of constituents isolated from Lycopodiastrum casuarinoides.

Phytochemical investigation of the ethyl acetate extract of Lycopodiastrum casuarinoides (Spring) Holub (Lycopodiaceae) led to the isolation of nine compounds, including two new serratene triterpenoids, serrat-14-en-3α,21α-diol (1), 26-nor-8-oxo-21-one-α-onocerin (6), one new abietane diterpenoid, lycocasuarinone A (7), one new sesquiterpene acid, 7, 9-diene-1,4-epoxy-2-hydroxy-10-carboxylic acid (8) and one new chromone derivative, 5,7-dihydroxy-2-methyl esterchromone (9), together with four known serratene triterpenoids (2-5). Abietane diterpenoid (7) and sesquiterpene acid (8) from Lycopodiastrum casuarinoides are reported for the first time. Their structures and stereochemistry were unambiguously elucidated by spectroscopic analysis and comparison with known ones. All the compounds were tested for acetylcholinesterase (AChE) and butyrocholinesterase (BuChE) inhibitory activities. Bioactivity assays revealed that compound 6 exhibited the most potent AChE inhibitory effect.

Ethnopharmacology of the club moss subfamily Huperzioideae (Lycopodiaceae, Lycopodiophyta): A phylogenetic and chemosystematic perspective.

ETHNOPHARMACOLOGICAL RELEVANCE: The most speciose subfamily Huperzioideae (Lycopodiaceae, Lycopodiophyta) contains about 276 species, and some (ca. 20 species) have traditionally been used for the treatment of e.g., dementia, rheumatism and traumatic injury. Ethnopharmacological studies have also contributed to the development of huperzine A as a drug lead, a compound first isolated from the club moss Huperzia serrata (Thunb. ex Murray) Trevis. AIM OF THE REVIEW: This review, with a phylogenetic and chemosystematic perspective, intends to highlight plant identification challenges in these taxa with examples from club moss phytochemical and ethnopharmacological studies, as these lead to data inconsistency and confusion. We suggest that future studies should include more details on plant identification including for example plant specimen images and DNA barcoding data. An integrative approach combining DNA barcoding and chemical fingerprinting is also introduced. MATERIALS AND METHODS: Literature concerning ethnopharmacology and chemosystematics of Huperzioideae club mosses was searched from databases, e.g. PubMed, Web of Science, SciFinder, etc. Plant names were retrieved from original publications, and compared with up-to-date taxonomic and phylogenetic status. Ethnobotanical uses and herbal preparations were summarized. Production of certain pharmaceutically interesting compounds, such as the alkaloid huperzine A, was explored in a phylogenetic context. RESULTS: Most traditionally used club mosses are associated with psychoactivity, followed by medicinal uses against rheumatism and traumatic injury. Herbs are often prepared as infusions, decoctions or tinctures, and this implies importance of water- or aqueous-alcohol-soluble substances, such as alkaloids. Most ethnopharmacological papers on club mosses need to update or correct plant names according to recent taxonomic nomenclature, and there are still a number of unidentified species with traditional use. Advanced LC-MS chemical profiling techniques, enable distinction of genotypes of the same species as well as annotation of potential chemotaxonomic markers. In combination with DNA barcoding, chemosystematics could also help us select plant taxa with higher pharmaceutical potential. Caution should be taken when interpreting bioassay results, in terms of compounds or extract preparation and bioassay standardization. CONCLUSION: Huperzioideae club mosses have interesting pharmaceutical potential supported by ethnopharmacological investigations. Bioprospecting of these plants should be preceded by careful plant identification to produce consistent and reproducible data. We expect that DNA barcoding and LC-MS-based chemical fingerprinting could facilitate and improve ethnopharmaceutical studies in selection of club moss taxa.

Serratene triterpenoids and their biological activities from Lycopodiaceae plants.

The plants of Lycopodiaceae family, distributed across China, India and also Southeast Asia, have been used as folk medicines. The phytochemical constitutent studies of this family was widely reported. Serratene trierpenoids is one of phytochemical constitutent type, which have been mainly isolated from this plant family. To date, more than 100 serratene-type triterpenoids have been reported and several of them have been shown promising biological activities, especially cytotoxicity and chemopreventive activity. This review covers the structural classification, biological activities and hypotheses about biosynthetic pathways of serratene-type triterpenes.

Lycocasuarines I-Q, new Lycopodium alkaloids isolated from Lycopodiastrum casuarinoides.

Lycocasuarines I-Q (1-9), nine new lycodine-type Lycopodium alkaloids were isolated from the aerial parts of Lycopodiastrum casuarinoides (Spring) Holub ex R.D.Dixit. Their structures were unambiguously determined via extensive spectroscopic analyses, including HRESIMS, NMR, and electronic circular dichroism (ECD). The new alkaloids were evaluated for their in vitro inhibitory activity against acetylcholinesterase (AChE). Compounds 3, 5-8 exhibited marginal or weak activities with IC50 values ranging from 97.2 to 171.9 µM.

Lycodine type alkaloids from Lycopodiastrum casuarinoides with cytotoxic and cholinesterase inhibitory activities.

A chemical investigation on the 70% EtOH extract of the aerial parts of Lycopodiastrum casuarinoides led to the isolation of six novel lycodine type alkaloids, lycocasuarines A-F (1-6). The structures of the isolated compounds were established based on 1D and 2D (1H1H COSY, HMQC, and HMBC) NMR spectroscopy, in addition to high resolution mass spectrometry. The isolated alkaloids were tested in vitro for cytotoxic potentials against seven malignant melanoma cell lines as well as acetylcholinesterase (AChE) and butyrocholinesterase (BuChE) inhibitory activities. As a result, alkaloids 1 and 3 exhibited significant cytotoxic activities against all the tested tumor cell lines with IC50 values <10 µM and the inhibitory activities for AchE (0.94 ±â€¯0.15 and 0.24 ±â€¯0.03 µM, respectively) and BuchE (1.82 ±â€¯0.12 and 7.31 ±â€¯0.42 µM, respectively).

Lycodine-type alkaloids from Lycopodiastrum casuarinoides and their cholinesterase inhibitory activities.

Four new trace alkaloids with lycodine-related structures, Lycocasuarinines A-D (1-4), together with seven known analogues (5-11), were isolated from the chloroform extract of Lycopodiastrum casuarinoides. The structures and stereochemistry of 1-4 were elucidated by spectroscopic analysis (IR, UV, MS, NMR, HRESIMS and CD) and comparison with known ones. The acetylcholinesterase (AChE) and butyrocholinesterase (BuChE) inhibitory activities of nine isolates were evaluated. Lycocasuarinine D (4) showed the most potent AChE inhibitory effect. In addition, a plausible biogenetic pathway of compound 4 was proposed.

Serratene-type triterpenoids from Palhinhaea cernua.

Phytochemical investigation of the 95% ethanol extract of the whole plant of Palhinhaea cernua afforded five new serratene-type triterpenoids (1-5) together with four known analogues (6-9). The structures of the new compounds were elucidated by extensive spectroscopic analysis and X-ray crystallographic data. Compounds 1-3 triggered apoptosis of Hela-/- cell with EC50 values of 3.49 ±â€¯0.05, 0.28 ±â€¯0.13 and 7.98 ±â€¯0.03 µM, respectively.

An alkaloid extract obtained from Phlegmariurus Saururus induces neuroprotection after status epilepticus.

BACKGROUND: The brain is exposed to many excitotoxic insults that can lead to neuronal damage. Among these, Epilepsy is a neurological disease that affects a large percentage of world population and is commonly associated with cognitive disorders and excitotoxic neuronal death. Most experimental strategies are focused on preventing Status Epilepticus (SE), but once it has already occurred, the key question is whether it is possible to save neurons. PURPOSE: The aim of this study was to determine if a purified alkaloid extract (AE) obtained from Phlegmariurus saururus, a genus of Lycophyte plants (sometimes known as firmossesor fir club mosses) could induce neuroprotection following SE. METHODS: In vitro and in vivo techniques were applied for this purpose. Protein levels were measured by western blotting procedures. Neuronal death analysis was performed by calcein-ethidium staining and the presence of the NeuN protein as a marker for presence or absence of cells (in vitro experiments) and by Fluoro Jade B staining for the in vivo experiments. RESULTS: The effect of AE in the hippocampal neurons culture was the first determination, where we found an increase in neuronal survival and in the level of pErk and TrkB activation, 24 h after the addition of AE. In a well-established in vitro model of SE, we found that 24 h after being added to the hippocampal neuron-astrocyte co-culture, the AE induces a significant increase in neuronal survival. In addition to this, in the in vivo Li-pilocarpine model of SE, the AE induced a remarkable neuroprotection in areas such as the entorhinal cortex and hippocampal CA1 area. CONCLUSION: These results make the AE an excellent candidate for potential clinical use in neurological disorders where memory impairment and neuronal death occurs.

Anticholinesterase activity and identification of huperzine A in three Mexican lycopods: Huperzia cuernavacensis, Huperzia dichotoma and Huperzia linifolia (Lycopodiaceae).

Huperzine A (Hup A), the alkaloid produced by the Chinese medicinal plant Huperzia serrata, has been documented to be a promising agent for the treatment of Alzheimer's disease due to its potent acetylcholinesterase inhibitory (AChEI) activity. The search for anticholinesterase natural products, as well as for alternative sources of Hup A in Mexican lycopods, prompted us to investigate these plants. The action of methanolic and alkaloidal extracts of three Huperzia species (H. cuernavacensis, H. dichotoma, and H. linifolia) was evaluated using an in vitro anticholinesterase activity assay. Also, chromatographic and spectroscopic analyses were employed to detect the presence of Hup A. Methanolic and alkaloidal extracts of H. cuernavacensis showed IC50 =5.32±0.8µg/mL and 0.74±0.05µg/mL; H. dichotoma displayed AChEI with IC50 values =14.11±2.1µg/mL and 0.64±0.09µg/mL; and H. linifolia presented IC50 =158.37±8.7µg/mL and 4.2±1.24µg/mL, respectively, compared to the control Hup A (IC50= 0.16±0.03µg/mL). Hup A was identified in the extracts of H. dichotoma, but it was not detected in the extracts of H. cuernavacensis and H. linifolia by 1H NMR techniques. This study reveals H. dichotoma as a new source of Hup A, and presents H. linifolia and H. cuernavacensis as potential candidates to obtain other anticholinesterase compounds useful in the Alzheimer's disease treatment.

Neolignans and serratane triterpenoids with inhibitory effects on xanthine oxidase from Palhinhaea cernua.

Three new neolignans, lycocernuasides B-D (1-3), three new serratane triterpenoids, lycernuic ketones D (8) and E (9), and lycernuic A (10), together with six known compounds, were isolated from the 75% aqueous EtOH extract of Palhinhaea cernua. Their structures and absolute configurations were established primarily by NMR, HRESIMS and circular dichroism (CD). All compounds were evaluated the inhibitory activities of xanthine oxidase. Compounds 1-3 displayed moderate inhibitory effects on xanthine oxidase with IC50 values of 30.36µM, 42.65µM and 35.33µM, respectively.

Five new Lycopodium alkaloids from the aerial parts of Phlegmariurus henryi.

A series of new Lycopodium alkaloids, namely 1-epi-malycorin A (1), 1-epi-17S-hydroxymalycorin A (2), 6α-hydroxyphlegmariurine A (3), 2S,4R-dihydroxyfawcettimine (4), and 16-hydroxylycodine (5), together with 24 known ones, have been isolated from the club moss Phlegmariurus henryi. The structures of the new compounds were determined by extensive spectroscopic analysis, including 1D and 2D NMR, as well as X-ray crystallographic analysis. Among them, the absolute configurations of 1, 2, and 4 and the structure of 3 were confirmed on the basis of the single-crystal X-ray diffraction analysis. 1-Epi-17S-hydroxymalycorin A (2) was a unique C19N-type Lycopodium alkaloid consisting of a serratinine skeleton with 1,2-propanediol unit. 2S,4R-dihydroxyfawcettimine (4) was a 2,4-dihydroxy derivative of fawcettimine. 16-Hydroxylycodine (5) was the oxidative product of lycodine with an unusual hydroxymethyl group at C-15. All new compounds were evaluated for in vitro acetylcholinesterase (AChE) inhibitory activity and cytotoxicity against four human cancer cell lines.

Rapid screening and identification of lycodine-type alkaloids in Lycopodiaceae and Huperziaceae plants by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Lycodine-type alkaloids have gained significant interest owing to their unique skeletal characteristics and acetylcholinesterase activity. This study established a rapid and reliable method using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q/TOF-MS/MS) for comprehensive characterization of lycodine-type alkaloids for the first time. The lycodine-type alkaloids were detected successfully from Lycopodiastrum casuarinoides, Huperzia serrata and Phlegmarirus carinatus in seven plants of the Lycopodiaceae and Huperziaceae families, based on the established characteristic MS fragmentation of five known alkaloids. Furthermore, a total of 13 lycodine-type alkaloids were identified, of which three pairs of isomers were structurally characterized and differentiated. This study further improves mass analysis of lycodine-type alkaloids and demonstrates the superiority of UPLC with a high-resolution mass spectrometer for the rapid and sensitive structural elucidation of other trace active compounds. Copyright © 2016 John Wiley & Sons, Ltd.

Development and characterization of the α3ß4α5 nicotinic receptor cellular membrane affinity chromatography column and its application for on line screening of plant extracts.

The α3ß4α5 nAChR has been recently shown to be a useful target for smoking cessation pharmacotherapies. Herein, we report on the development and characterization of the α3ß4α5 nicotinic receptor column by frontal displacement chromatography. The binding affinity of the nicotine and minor alkaloids found in tobacco smoke condensates were determined for both the α3ß4 and α3ß4α5 nicotinic receptors. It was demonstrated that while no subtype selectivity was observed for nicotine and nornicotine, anabasine was selective for the α3ß4α5 nicotinic receptor. The non-competitive inhibitor binding site was also studied and it was demonstrated while mecamylamine was not selective between subtypes, buproprion showed subtype selectivity for the α3ß4 nicotinic receptor. The application of this methodology to complex mixtures was then carried out by screening aqueous-alcoholic solutions of targeted plant extracts, including Lycopodium clavatum L. (Lycopodiaceae) and Trigonella foenum graecum L. (Fabaceae) against both the α3ß4 and α3ß4α5 nAChRs.

Kinetics and molecular docking studies of cholinesterase inhibitors derived from water layer of Lycopodiella cernua (L.) Pic. Serm. (II).

Acetylcholinesterase (AChE) inhibitors increase the availability of acetylcholine in central cholinergic synapses and are the most promising drugs currently available for the treatment of Alzheimer's disease (AD). Our screening study indicated that the water fraction of the methanolic extract of Lycopodiella cernua (L.) Pic. Serm. significantly inhibited AChE in vitro. Bioassay-guided fractionation led to the isolation of a new lignan glycoside, lycocernuaside A (12), and fourteen known compounds (1-11 and 13-15). Compound 7 exhibited the most potent AChE inhibitory activity with an IC50 value of 0.23 µM. Compound 15 had the most potent inhibitory activity against BChE and BACE1 with IC50 values of 0.62 and 2.16 µM, respectively. Compounds 4 and 7 showed mixed- and competitive-type AChE inhibition. Compound 7 noncompetitively inhibited BChE whereas 15 showed competitive and 8, 13, and 14 showed mixed-type inhibition. The docking results for complexes with AChE or BChE revealed that inhibitors 4, 7, and 15 stably positioned themselves in several pocket/catalytic domains of the AChE and BChE residues.

The Genus Diphasiastrum and Its Lycopodium Alkaloids.

The genus Diphasiastrum includes at least 23 species distributed primarily across the northern temperate and subarctic areas of the world. These plants produce an array of lycopodium alkaloids, and some species such as Diphasiastrum complanatum have been used in traditional medicine for ages for various conditions. Hybridization is common in this group of plants and they have always been a challenge for taxonomists and other scientists studying them. To date, 11 Diphasiastrum species have been reported to produce lycopodium alkaloids. In this review, reported alkaloids and their distribution patterns across these species along with taxonomical and bioactivity considerations are reviewed and discussed.