Rindera graeca (A. DC.) Boiss. & Heldr. (Boraginaceae) In Vitro Cultures Targeting Lithospermic Acid B and Rosmarinic Acid Production.

The in vitro cultures of Rindera graeca, a rare endemic plant, were developed as a sustainable source of phenolic acids. Various shoot and root cultures were established and scaled up in a sprinkle bioreactor. A multiplication rate of 7.2 shoots per explant was achieved. HPLC-PDA-ESI-HRMS analysis revealed the presence of rosmarinic acid (RA) and lithospermic acid B (LAB) as the main secondary metabolites in both the shoot and root cultures. The maximum RA (30.0 ± 3.2 mg/g DW) and LAB (49.3 ± 15.5 mg/g DW) yields were determined in root-regenerated shoots. The strongest free radical scavenging activity (87.4 ± 1.1%), according to 2,2-diphenyl-1-picrylhydrazyl-hydrate assay, was noted for roots cultivated in a DCR medium. The highest reducing power (2.3 µM ± 0.4 TE/g DW), determined by the ferric-reducing antioxidant power assay, was noted for shoots cultivated on an SH medium containing 0.5 mg/L 6-benzylaminopurine. A genetic analysis performed using random amplified polymorphic DNA and start codon targeted markers revealed genetic variation of 62.8% to 96.5% among the investigated shoots and roots. This variability reflects the capacity of cultivated shoots and roots to produce phenolic compounds.

HPLC-PDA-ESI-HRMS-Based Profiling of Secondary Metabolites of Rindera graeca Anatomical and Hairy Roots Treated with Drought and Cold Stress.

To cope with environmental harmful conditions, plant cells developed adaptive strategy that involves production of a wide variety of complex secondary metabolites. The spectrum and quantity of biosynthesized compounds in specific plant species is determined by its genotype, tissue, developmental and physiological stage and environmental factors. This phenomenon was used to exploit the potential of anatomical and hairy root cultures of Rindera graeca to produce bioactive compounds. Cultivated in vitro roots were subjected to abiotic stresses i.e., drought or coldness. Next the extract profiling was performed using HPLC-PDA-ESI-HRMS method, as well quantitative determination of caffeic, rosmarinic and lithospermic B acids, that were present in all root extracts. Phenolic acids, flavonoids and iridoids represent the major groups of compounds detected in chemical profiles growing under various conditions roots. The highest number of phytochemicals was determined in roots subjected to coldness. Lithospermic B acid proved to be the most abundant compound in all investigated extracts. Among applied abiotic stress factors it was demonstrated that coldness affected to the most secondary metabolites production. The results of current study suggest that root cultures of R. graeca could serve as a new and abundant source of lithospermic B acid.

Bifunctional Opioid/Melanocortin Peptidomimetics for Use in Neuropathic Pain: Variation in the Type and Length of the Linker Connecting the Two Pharmacophores.

Based on the mechanism of neuropathic pain induction, a new type of bifunctional hybrid peptidomimetics was obtained for potential use in this type of pain. Hybrids consist of two types of pharmacophores that are connected by different types of linkers. The first pharmacophore is an opioid agonist, and the second pharmacophore is an antagonist of the pronociceptive system, i.e., an antagonist of the melanocortin-4 receptor. The results of tests in acute and neuropathic pain models of the obtained compounds have shown that the type of linker used to connect pharmacophores had an effect on antinociceptive activity. Peptidomimetics containing longer flexible linkers were very effective at low doses in the neuropathic pain model. To elucidate the effect of linker lengths, two hybrids showing very high activity and two hybrids with lower activity were further tested for affinity for opioid (mu, delta) and melanocortin-4 receptors. Their complexes with the target receptors were also studied by molecular modelling. Our results do not show a simple relationship between linker length and affinity for particular receptor types but suggest that activity in neuropathic pain is related to a proper balance of receptor affinity rather than maximum binding to any or all of the target receptors.

Novel bifunctional hybrid compounds designed to enhance the effects of opioids and antagonize the pronociceptive effects of nonopioid peptides as potent analgesics in a rat model of neuropathic pain.

ABSTRACT: The purpose of our work was to determine the role of nonopioid peptides derived from opioid prohormones in sensory hypersensitivity characteristics of neuropathic pain and to propose a pharmacological approach to restore the balance of these endogenous opioid systems. Nonopioid peptides may have a pronociceptive effect and therefore contribute to less effective opioid analgesia in neuropathic pain. In our study, we used unilateral chronic constriction injury (CCI) of the sciatic nerve as a neuropathic pain model in rats. We demonstrated the pronociceptive effects of proopiomelanocortin- and proenkephalin-derived nonopioid peptides assessed by von Frey and cold plate tests, 7 to 14 days after injury. The concentration of proenkephalin-derived pronociceptive peptides was increased more robustly than that of Met-enkephalin in the ipsilateral lumbar spinal cord of CCI-exposed rats, as shown by mass spectrometry, and the pronociceptive effect of one of these peptides was blocked by an antagonist of the melanocortin 4 (MC4) receptor. The above results confirm our hypothesis regarding the possibility of creating an analgesic drug for neuropathic pain based on enhancing opioid activity and blocking the pronociceptive effect of nonopioid peptides. We designed and synthesized bifunctional hybrids composed of opioid (OP) receptor agonist and MC4 receptor antagonist (OP-linker-MC4). Moreover, we demonstrated that they have potent and long-lasting antinociceptive effects after a single administration and a delayed development of tolerance compared with morphine after repeated intrathecal administration to rats subjected to CCI. We conclude that the bifunctional hybrids OP-linker-MC4 we propose are important prototypes of drugs for use in neuropathic pain.

Polyscias filicifolia (Araliaceae) Hairy Roots with Antigenotoxic and Anti-Photogenotoxic Activity.

Hairy root cultures are considered as a valuable source of bioactive phytoconstituents with expanding applicability for their production. In the present study, hairy root cultures of Polyscias filicifolia (Araliaceae), a traditional Southeast Asian medicinal plant, were established. The transformation with Agrobacterium rhizogenes ATCC 15834 allowed to obtain 15 root lines. The K-1 line, demonstrating the highest growth capabilities, was subjected to further investigations. To enhance the biosynthetic potential of hairy roots, methyl jasmonate elicitation approach was applied (MeJA; at different doses and exposure time), with subsequent transfer of elicited roots to control medium. This strategy resulted in chlorogenic acid production up to 1.59 mg/g dry weight. HPLC-PDA-ESI-MS analysis demonstrated variation in extracts composition and allowed to identify different caffeic and ferulic acid derivatives. Next, cytotoxic, antigenotoxic, and anti-photogenotoxic properties of hairy roots extracts were determined. None of the tested extracts were cytotoxic. In addition, they demonstrated significant antigenotoxic activity with the highest protective potential; up to 52% and 49% of inhibition of induction ratio (IR) induced by the 2-aminoanthracene was revealed for extracts derived from hairy roots elicited for 3 days with 50 µM MeJA and roots elicited for 7 days with 100 µM MeJA and then transferred for 30 days to control medium, respectively. These same extracts exhibited the highest anti-photogenotoxic potential, up to 36% of inhibition of chloropromazine-induced genotoxicity.

Huperzine A and Huperzine B Production by Prothallus Cultures of Huperzia selago (L.) Bernh. ex Schrank et Mart.

This is the first report of an efficient and effective procedure to optimize the biosynthesis of huperzine A (HupA) and huperzine B (HupB) in vitro from Huperzia selago gametophytes. Axenic tissue cultures were established using spores collected from the sporophytes growing in the wild. The prothalia were obtained after 7-18 months. Approximately 90 up to 100% of the gametophytes were viable and grew rapidly after each transfer on to a fresh medium every 3 months. The best biomass growth index for prothallus calculated on a fresh (FW) and dry weight (DW) basis, at 24 weeks of culture, was 2500% (FW) and 2200% (DW), respectively. The huperzine A content in the gametophytes was very high and ranged from 0.74 mg/g to 4.73 mg/g DW. The highest yield HupA biosynthesis at >4 mg/g DW was observed on W/S medium without growth regulators at 8 to 24 weeks of culture. The highest HupB content ranged from 0.10 mg/g to 0.52 mg/g DW and was obtained on the same medium. The results demonstrate the superiority of H. selago gametophyte cultures, with the level of HupA biosynthesis approximately 42% higher compared to sporophyte cultures and 35-fold higher than when the alkaloid was isolated from H. serrata, its current source for the pharmaceutical industry. Moreover, the biosynthesis of HupB was several-fold more efficient than in H. selago sporophytes growing in the wild. HPLC-HR-MS analyses of the extracts identified eight new alkaloids previously unreported in H. selago: deacetylfawcettine, fawcettimine, 16-hydroxyhuperzine B, deacetyllycoclavine, annopodine, lycopecurine, des-N-methylfastigiatine and flabelline.

Triazolopeptides Inhibiting the Interaction between Neuropilin-1 and Vascular Endothelial Growth Factor-165.

Inhibiting the interaction of neuropilin-1 (NRP-1) with vascular endothelial growth factor (VEGF) has become an interesting mechanism for potential anticancer therapies. In our previous works, we have obtained several submicromolar inhibitors of this interaction, including branched pentapeptides of general structure Lys(Har)-Xxx-Xxx-Arg. With the intent to improve the proteolytic stability of our inhibitors, we turned our attention to 1,4-disubstituted 1,2,3-triazoles as peptide bond isosteres. In the present contribution, we report the synthesis of 23 novel triazolopeptides along with their inhibitory activity. The compounds were synthesized using typical peptide chemistry methods, but with a conversion of amine into azide completely on solid support. The inhibitory activity of the synthesized derivatives spans from 9.2% to 58.1% at 10 µM concentration (the best compound Lys(Har)-GlyΨ[Trl]GlyΨ[Trl]Arg, 3, IC50 = 8.39 µM). Synthesized peptidotriazoles were tested for stability in human plasma and showed remarkable resistance toward proteolysis, with half-life times far exceeding 48 h. In vitro cell survival test resulted in no significant impact on bone marrow derived murine cells 32D viability. By means of molecular dynamics, we were able to propose a binding mode for compound 3 and discuss the observed structure-activity relationships.

Enkephalin degradation in serum of patients with inflammatory bowel diseases.

BACKGROUND: Inflammatory bowel diseases (IBD) are a group of chronic and recurrent gastrointestinal disorders that are difficult to control. Recently, a new IBD therapy based on the targeting of the endogenous opioid system has been proposed. Consequently, due to the fact that endogenous enkephalins have an anti-inflammatory effect, we aimed at investigating the degradation of serum enkephalin (Met- and Leu-enkephalin) in patients with IBD. METHODS: Enkephalin degradation in serum of patients with IBD was characterized using mass spectrometry methods. Calculated half-life (T1/2) of enkephalins were compared and correlated with the disease type and gender of the patients. Additionally, statistical analysis was used to examine the dynamics of changes in terms of inhibition of enkephalins degradation within research groups. RESULTS: Our research indicates that the degree of enkephalins degradation depends on the gender of the patients. The difference is most evident for the rate of Met-enkephalin degradation between men (mean T1/2 = 13.61 min) and women (mean T1/2 = 21.84 min) with Crohn's disease (CD). CONCLUSIONS: The most significant alternation of enkephalins degradation in serum samples of IBD patients, compared to control group, were observed in both Crohn's disease and ulcerative colitis (UC) female patients. We suggest that the differences observed between the genders in IBD patients may be explained by regulation of enkephalinases activity by estradiol.

Branched pentapeptides as potent inhibitors of the vascular endothelial growth factor 165 binding to Neuropilin-1: Design, synthesis and biological activity.

The demonstrated involvement of VEGF165/NRP-1 complex in pathological angiogenesis has catalyzed interest in blocking this interaction to combat angiogenesis dependent diseases. It was shown before that Lys-Pro-Pro-Arg is a fairly strong inhibitor of the VEGF165/NRP-1 interaction. Our current findings suggest that the side chain elongation of the Lys1 by branching it with additional homoarginine (Har) residue, to obtain Lys(Har)-Pro-Pro-Arg, allows more effective inhibition. Moreover, increasing the flexibility of the middle part of molecule, in particular with simultaneous introduction of additional interacting elements at the second or third position, produced compounds up to 30-fold more active (IC50 = 0.2 µM) than the heptapeptide ATWLPPR (A7R) which is one of the first peptide known as an effective antagonist of the VEGF165 binding to NRP-1 and in vivo decreases breast cancer angiogenesis and growth. Herein, we present also the structure-activity study of Lys(Har)-Pro-Pro-Arg, discussing the design, synthesis, inhibitory activity, proteolytic stability and molecular modeling of the prepared derivatives. For two of the most active analogs the high proteolytic stability was also observed. These studies provide the next step for elucidating the optimal structure of the small peptidic inhibitors of VEGF165/NRP-1 interaction that could serve as research tools or be prospective drug candidates.

Structure-activity relationship study of tetrapeptide inhibitors of the Vascular Endothelial Growth Factor A binding to Neuropilin-1.

Neuropilin-1 is considered as one of the key receptors responsible for signaling pathways involved in pathological angiogenesis necessary for tumor progression, therefore targeting of VEGF165 binding to NRP-1 could be a relevant strategy for antiangiogenic treatment. It was shown before that the VEGF165/NRP-1 interaction can be inhibited by short tetrapeptides with K/RXXR sequence. Here, we present a structure-activity relationship study of the systematic optimization of amino acid residues in positions 1-3 in the above tetrapeptides. All the 13 synthesized analogs possessed C-terminal arginine that is a necessary element for interaction with NRP-1. The obtained results of the inhibitory activity and modeling by molecular dynamics indicate that simultaneous interactions of the basic amino acid residues in position 1 and 4 (Arg) with Neuropilin-1 are crucial and their cooperation strongly affects the inhibitory activity. In addition, the binding strength is modulated by the flexibility of the peptide backbone (in the central part of the peptide), and the nature of the side chain of the amino acids at the second or third position. A dramatic decrease in the activity to the receptor is observed in flexible derivatives that are missing proline residues. The results described in this paper should prove useful for future studies aimed at establishing the best pharmacophore for inhibitors of VEGF165 binding to NRP-1.

Conformational latitude - activity relationship of KPPR tetrapeptide analogues toward their ability to inhibit binding of vascular endothelial growth factor 165 to neuropilin-1.

Neuropilin-1 has been found to be overexpressed in several kinds of malignant tumors, and it is postulated that its interaction with the vascular endothelial growth factor 165 leads to progression of tumor vascularization and growth. Several analogues (KxxR) with various conformational latitudes have been synthesized and found as inhibitors of NRP-1. Detailed insight provided by molecular dynamics simulation allowed forming a clear relationship between flexibility of xx part of the molecule and its inhibitory activity.

Structure-activity relationship study of a small cyclic peptide H-c[Lys-Pro-Glu]-Arg-OH: a potent inhibitor of Vascular Endothelial Growth Factor interaction with Neuropilin-1.

Inhibition of angiogenesis is one of the most promising approaches in anticancer therapy. It was recently suggested that Neuropilin-1 (NRP-1) in tumour cells may serve as a separate receptor for Vascular Endothelial Growth Factor-165 (VEGF165) which is one of the main pro-angiogenic agents in the organism. Therefore molecules inhibiting VEGF165 binding to NRP-1 could be potential candidates for new antiangiogenic and anticancer drugs. Here we present a structure-activity relationship study of the peptide H-c[Lys-Pro-Glu]-Arg-OH which showed high inhibitory effect on VEGF165/NRP-1 binding (IC50=0.18µM) in our previous study. We report the design, synthesis, in vitro assays and docking analysis of four small cyclic peptides (14-,15-membered ring) and one bigger cyclic compound (30-membered ring). Our study shows that both the ring size and configuration of amino acid residues present in the structure are crucial for high inhibitory effect.

Design, synthesis and in vitro biological evaluation of a small cyclic peptide as inhibitor of vascular endothelial growth factor binding to neuropilin-1.

Neuropilin-1 (NRP-1) is a co-receptor of VEGFR (vascular endothelial growth factor receptor), but it is also suggested that NRP-1 in tumour cells may serve as a separate receptor for VEGF165. Therefore molecules interfering with VEGF165 binding to NRP-1 seem to be promising candidates as new anti-angiogenic and anti-tumour drugs. Here, we report the design, synthesis, biological evaluation and molecular modelling of the small cyclic peptide, which shows a good inhibitory effect on VEGF165/NRP-1 binding (IC50=0.18µM). The reported compound could be considered as one of the smallest cyclic peptides (MW=510) interfering with VEGF165/NRP-1 binding presented up to now.

Imaging and identification of endogenous peptides from rat pituitary embedded in egg yolk.

RATIONALE: Mass spectrometry imaging (MSI) can provide accurate data containing the spatial distribution of endogenous peptides in tissue sections without previous treatment. One of the key issues in analyzing small samples is establishing a proper technique for mounting and manipulating collected tissue in order to avoid contamination of the sample with optimal cutting temperature (OCT) resin. METHODS: We present a method for embedding rat pituitary tissue in a frozen egg yolk block, which enables its further imaging in experiments on a matrix-assisted laser desorption/ionization (MALDI) mass spectrometer with time-of-flight (TOF) analyzer. Embedding the sample in the egg yolk prevents contamination from the OCT resin, which decreases MALDI signal quality. RESULTS: In the present study we detected numerous m/z peaks related to endogenous peptides. We identified fifteen peptides and their post-translational modifications by tandem mass spectrometry (MS/MS) directly on tissue sections of the hypophysis posterior and intermediate lobes; among these peptides were vasopressin, oxytocin, copeptin, melanocyte-stimulating hormones and beta-endorphin. We also showed that egg yolk itself does not affect localization of peptides in the pituitary. CONCLUSIONS: Egg yolk embedding enables preparation of tissue sections from small tissue fragments to organs such as the pituitary gland, which is suitable for localization and identification of endogenous peptides by the MALDI-MSI and MALDI-MS/MS techniques.

Reactions of stabilized aliphatic carbanions with esters of formic acid in the gas phase.

The reactions of nitromethane, acetonitrile, and ethyl acetate carbanions with formic acid esters were investigated in the gas phase by mass spectrometry and quantum chemical calculations. It was found that the carbanion of nitromethane practically does not react with formic acid esters and only traces of the Claisen- type addition-elimination reaction product were observed. In the case of the deprotonated acetonitrile, the Claisen-type condensation that yielded the carbanion of cyanoacetaldehyde proceeded almost quantitatively for all the studied formates. Other products, like the Riveros reaction product, were observed in minor amounts. The ethyl acetate carbanion reacts with formates in a much more complex way because it decomposes in a collision cell, yielding the deprotonated ketene anion, which also can react with formic acid esters. Consequently, the Claisen condensation reaction products of both anions were observed as well as the products of other reactions. All the proposed reaction pathways were confirmed by quantum chemical calculations, including the transition-state energies.

Synthesis of rigid tryptophan mimetics by the diastereoselective Pictet-Spengler reaction of ß³-homo-tryptophan derivatives with chiral α-amino aldehydes.

The Pictet-Spengler (PS) cyclizations of ß(3)-hTrp derivatives as arylethylamine substrates were performed with L-α-amino and D-α-amino aldehydes as carbonyl components. During the PS reaction, a new stereogenic center was created, and the mixture of cis/trans 1,3-disubstituted 1,2,3,4-tetrahydro-ß-carbolines was obtained. The ratio of cis/trans diastereomers depends on the stereogenic centre of used amino aldehyde and the size of substituents. It was confirmed by 1H and 2D NMR (ROESY) spectra. The conformations of cyclic products were studied by 2D NMR ROESY spectra. Products of the PS condensation after removal of protecting group(s) can be incorporated into a peptide chain as tryptophan mimetics with the possibility of the ß-turn induction.

Identification of unknown colorants in pre-Columbian textiles dyed with American cochineal (Dactylopius coccus Costa) using high-performance liquid chromatography and tandem mass spectrometry.

The present study concerns the identification of nine thus-far unknown derivatives of carminic acid extracted from pre-Columbian Peruvian textiles dyed with American cochineal-these derivatives are not found in commercially available preparations of the dye. These compounds probably represent a unique fingerprint of dyed textiles from this region, as they have never been reported to occur in other fabrics of historical value. They were separated by reversed-phase high-performance liquid chromatography (phenyl column) and detected using a UV/vis spectrophotometer and two tandem mass spectrometers. Peaks observed in chromatograms registered at 450 and 500 nm were further identified by ESI QqQ MS (mainly in the negative ion mode), supported by high-resolution ESI QIT/ToF MS data. The characteristic fragmentation pathways of isolated carminic acid and its derivatives provided additional information concerning lost neutrals and thus the functional groups and substituents present in the parent molecules. This information mainly related to multiple cleavages of the hexoside moiety (initially cross-ring cleavage), which are characteristic of C-glucosides (loss of 90, 120, and 148 Da). This is accompanied by the elimination of H2O as well as the further loss of 60 Da from the hexoside moiety. Moreover, other losses from the carbonyl groups (44 Da from CO2 loss, 62 Da from ethylene glycol loss, 32 Da from O2 loss, 138 Da from hydroxybenzoic acid, and 120 Da from oxomethylene cyclohexadienone) provided more specific information about structures of the identified derivatives of carminic acid.

Addition-elimination versus Tishchenko reaction in the gas phase.

Gas phase reactions of the substituted phenide ions with methyl formate have been studied. It was found that the results of these reactions depend mainly on the basicity of the phenide ion, which is related to the presence of the electron-accepting or electron-donating substituents in the benzene ring. It was shown that the phenide ions substituted with electron-withdrawing groups react with methyl formate in the gas phase in a two-step reaction. The first step that proceeds according to the typical addition-elimination mechanism results in the formation of the anion of the respective benzaldehyde derivative with the negative charge located either in the aldehyde group (acyl anion) or in the benzene ring (phenide anion) in position ortho to an aldehyde moiety. In the second step, the preliminary-formed anion reacts with the second molecule of methyl formate yielding formally product of the second addition-elimination reaction. Theoretical calculations as well as collision induced dissociation spectra of the model compounds suggest that this reaction proceeds according to the Tishchenko reaction mechanism yielding the respective phthalide anion. According to our knowledge, this is the first example of the Tishchenko-type reaction in the gas phase.

Enantioseparation of ß(2)-amino acids on cinchona alkaloid-based zwitterionic chiral stationary phases. Structural and temperature effects.

The enantiomers of sixteen unusual ß(2)-amino acids were directly separated on chiral stationary phases containing quinine- or quinidine-based zwitterionic selectors. The effects of the mobile phase composition, the structure of the analyte and temperature on the separations were investigated. Experiments were performed at constant mobile phase compositions in the temperature range -5 to 55°C in order to study the effects of temperature, and thermodynamic parameters were estimated from plots of lnk or lnα vs. 1/T. Some mechanistic aspects of the chiral recognition process are discussed with respect to the structures of the analytes. It was found that the enantiomeric separations were in most cases enthalpically driven, but entropically driven separation was also observed. The sequence of elution of the enantiomers was determined in some cases.

Assessment of antioxidative activity of alkaloids from Huperzia selago and Diphasiastrum complanatum using in vitro systems.

Free radical-induced oxidative damage is implicated in the pathogenesis of neurodegenerative disorders, and antioxidants are presumably of therapeutic value in such diseases. Our previous data indicated that free radicals are strongly associated with brain aging and also play an important role in cytotoxicity of amyloidogenic proteins including -synuclein and amyloid , which accumulate in brains during Parkinson's and Alzheimer's diseases. Disruption of the equilibrium of pro-oxidants and antioxidants results in oxidative stress that leads to the modification of DNA, proteins, carbohydrates, and lipids. It is widely accepted that antioxidants acting as radical scavengers protect the brain against oxidative damage in neurodegenerative diseases. Plant products are rich sources of phytochemicals and have been found to possess a variety of biological activities, including antioxidative potential. The aim of this study was to analyse the antioxidative potential of alkaloid fractions from Huperzia selago and Diphasiastrum complanatum to protect macromolecules against oxidative damage. Thin layer chromatography (TLC) and high-performance liquid chromatography with diode array (HPLC-DAD) and electrospray ionisation mass spectrometric detection (ESI-MS/MS) were used to carry out a comprehensive characterization of alkaloids isolated from the plant material. The effect of the tested compounds on iron/ascorbate-induced lipid peroxidation and carbonyl group formation was analysed in the rat brain homogenate. Direct free radical scavenging (DPPH assay) and the effect on dityrosine formation were measured in cell-free systems. Our results indicated that a number of alkaloid extracts at concentration of 25 µg/ml exhibited antioxidant activity as indicated by DPPH radical scavenging potential (up to 59% inhibition) and inhibition of dityrosine formation. Selected alkaloid fractions provided significant protection against lipid peroxidation and protein oxidation in rat brain tissue homogenate, reducing iron/ascorbate-induced damage by about 20% and 76%, respectively. Overall, the results indicated that selected alkaloids isolated from Huperzia selago effectively protect macromolecules from oxidative stress injury, which will give us an insight into the potential of alkaloids in terms of opening up a new therapeutic approach for oxidative stress-dependent disorders.