New neuroprotective derivatives of cinnamic acid by biotransformation.

Microbial transformation is extensively utilized to generate new metabolites in bulk amounts with more specificity and improved activity. As cinnamic acid was reported to exhibit several important pharmacological properties, microbial transformation was used to obtain its new derivatives with enhanced biological activities. By manipulating the 2-stage fermentation protocol of biotransformation, five metabolites were produced from cinnamic acid. Two of them were new derivatives; N-propyl cinnamamide 2̲ and 2-methyl heptyl benzoate 3̲ produced by Alternaria alternata. The other 3 metabolites, p-hydroxy benzoic acid 4̲, cinnamyl alcohol 5̲ and methyl cinnamate 6̲, were produced by Rhodotorula rubra, Rhizopus species and Penicillium chrysogeneum, respectively. Cinnamic acid and its metabolites were evaluated for their cyclooxygenase (COX) and acetylcholinesterase (AChE) inhibitory activities. Protection against H2O2 and Aß1-42 induced-neurotoxicity in human neuroblastoma (SH-SY5Y) cells was also monitored. Metabolite 4̲ was more potent as a COX-2 inhibitor than the parent compound with an IC50 value of 1.85 ± 0.07 µM. Out of the tested compounds, only metabolite 2̲ showed AChE inhibitory activity with an IC50 value of 8.27 µM. These results were further correlated with an in silico study of the binding interactions of the active metabolites with the active sites of the studied enzymes. Metabolite 3̲ was more potent as a neuroprotective agent against H2O2 and Aß1-42 induced-neurotoxicity than catechin and epigallocatechin-3-gallate as positive controls. This study suggested the two new metabolites 2̲ and 3̲ along with metabolite 4̲ as potential leads for neurodegenerative diseases associated with cholinergic deficiency, neurotoxicity or neuroinflammation.

In Vitro and In Silico Investigation of Polyacetylenes from Launaea capitata (Spreng.) Dandy as Potential COX-2, 5-LOX, and BchE Inhibitors.

Diverse secondary metabolites are biosynthesized by plants via various enzymatic cascades. These have the capacity to interact with various human receptors, particularly enzymes implicated in the etiology of several diseases. The n-hexane fraction of the whole plant extract of the wild edible plant, Launaea capitata (Spreng.) Dandy was purified by column chromatography. Five polyacetylene derivatives were identified, including (3S,8E)-deca-8-en-4,6-diyne-1,3-diol (1A), (3S)-deca-4,6,8-triyne-1,3-diol (1B), (3S)-(6E,12E)-tetradecadiene-8,10-diyne-1,3-diol (2), bidensyneoside (3), and (3S)-(6E,12E)-tetradecadiene-8,10-diyne-1-ol-3-O-ß-D-glucopyranoside (4). These compounds were investigated for their in vitro inhibitory activity against enzymes involved in neuroinflammatory disorders, including cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), and butyrylcholinesterase (BchE) enzymes. All isolates recorded weak-moderate activities against COX-2. However, the polyacetylene glycoside (4) showed dual inhibition against BchE (IC50 14.77 ± 1.55 µM) and 5-LOX (IC50 34.59 ± 4.26 µM). Molecular docking experiments were conducted to explain these results, which showed that compound 4 exhibited greater binding affinity to 5-LOX (-8.132 kcal/mol) compared to the cocrystallized ligand (-6.218 kcal/mol). Similarly, 4 showed a good binding affinity to BchE (-7.305 kcal/mol), which was comparable to the cocrystallized ligand (-8.049 kcal/mol). Simultaneous docking was used to study the combinatorial affinity of the unresolved mixture 1A/1B to the active sites of the tested enzymes. Generally, the individual molecules showed lower docking scores against all the investigated targets compared to their combination, which was consistent with the in vitro results. This study demonstrated that the presence of a sugar moiety (in 3 and 4) resulted in dual inhibition of 5-LOX and BchE enzymes compared to their free polyacetylenes analogs. Thus, polyacetylene glycosides could be suggested as potential leads for developing new inhibitors against the enzymes involved in neuroinflammation.

Construction of Spirooxindole Analogues Engrafted with Indole and Pyrazole Scaffolds as Acetylcholinesterase Inhibitors.

Twenty-five new hits of spirooxindole analogs 8a-y engrafted with indole and pyrazole scaffolds were designed and constructed via a [3+2]cycloaddition (32CA) reaction starting from three components: new chalcone-based indole and pyrazole scaffolds 5a-d, substituted isatins 6a-c, and secondary amines 7a-d. The potency of the compounds were assessed in modulating cholinesterase (AChE) activity using Ellman's method. Compounds 8i and 8y showed the strongest acetylcholine esterase inhibition (AChEI) with IC50 values of 24.1 and 27.8 µM, respectively. Molecular docking was used to study their interaction with the active site of hAChE.

Acetylcholine esterase inhibitory activity of green synthesized nanosilver by naphthopyrones isolated from marine-derived Aspergillus niger.

Aspergillus niger metabolites exhibited a wide range of biological properties including antioxidant and neuro-protective effects and some physical properties as green synthesis of silver nanoparticles AgNP. The present study presents a novel evidence for the various biological activities of green synthesized AgNPs. For the first time, some isolated naphtho-γ-pyrones from marine-derived Aspergillus niger, flavasperone (1), rubrofusarin B (2), aurasperone A (3), fonsecinone A (4) in addition to one alkaloid aspernigrin A (7) were invistigated for their inhibitory activity of acetylcholine esterase AChE, a hallmark of Alzheimer's disease (AD). The ability to synthesize AgNPs by compounds 3, 4 and 7 has been also tested for the first time. Green synthesized AgNPs were well-dispersed, and their size was ranging from 8-30 nm in diameter, their morphology was obviously spherical capped with the organic compounds. Further biological evaluation of their AChE inhibitory activity was compared to the parent compounds. AgNps dramatically increased the inhibitory activity of Compounds 4, 3 and 7 by 84, 16 and 13 fold, respectively to be more potent than galanthamine as a positive control with IC50 value of 1.43 compared to 0.089, 0.311 and 1.53 of AgNPs of Compounds 4, 3 and 7, respectively. Also compound 2 showed moderate inhibitory activity. This is could be probably explained by closer fitting to the active sites or the synergistic effect of the stabilized AgNPs by the organic compouds. These results, in addition to other intrinsic chemical and biological properties of naphtho-γ-pyrones, suggest that the latter could be further explored with a view towards other neuroprotective studies for alleviating AD.

New prenylated flavonoid and neuroprotective compounds from Tephrosia purpurea subsp. dunensis.

Continuation of the phytochemical investigation of the aerial parts of Tephrosia purpurea subsp. dunensis resulted in the isolation and structural elucidation of a new prenylated flavonoid demeapollinin (1), glabratephrinol (2) and a mixture (3) of tephroapollin G (3a) and epi-tephroapollin G (3b). The neuroprotective activity of compounds (1-3) besides the previously isolated compounds; dunensin (4), pseudosemiglabrin (6), glabratephrin (7), apollinin (5), kampferol 3, 7-O-α-L-dirhamnoside (8) and quercetin 3, 7-O-α-L-dirhamnoside (9) was examined. Molecular docking, acetylcholine esterase inhibitory assay and protection against both H2O2 and induced neurotoxicity were used to evaluate their neuroprotective effect. Compound 2 showed the highest acetylcholine esterase inhibitory activity (IC50 4.31 ± 0.75 µM) compared to galantamine (IC50 1.64 ± 0.32 µM), compounds 4 and 3 exhibited potent protective effect against induced neurotoxicity (IC50 7.70 ± 5.23 and 10.91 ± 6.27 µM, respectively) compared to standard epigallocatechin gallate (IC50 18.36 ± 6.22 µM).

A new aliphatic ester of hydroxysalicylic acid from fermented Carica papaya L. preparation with a potential hair growth stimulating activity.

An aliphatic ester of hydroxysalicylic acid (6), reported for the first time from a natural source in addition to five known compounds were isolated from the fermented Carica papaya L. preparation, a commercialized functional food. The known compounds were identified as 5-hydroxymethylfurfuraldehyde (1), trans-caffeic acid (2), butyl 4-hydroxybenzoate (butylparaben) (3), lycopene (4), benzyl isothiocyanate (5). Compounds 1 and 3 were reported for the first time from Papaya fruits through this study. The new compound showed a moderate antioxidant activity and a potent hair growth stimulating activity in vitro.

Derivatization, molecular docking and in vitro acetylcholinesterase inhibitory activity of glycyrrhizin as a selective anti-Alzheimer agent.

Acetylcholinesterase inhibitors (AChE-Is) increase both level and duration of action of acetylcholine (ACh); thus, alleviate symptoms of Alzheimer's disease (AD). Glycyrrhizin, is the main active compound in liquorice root. Its aglycone, glycyrrhetinic acid, has shown several beneficial pharmacological activities. This study reports the synthesis and screening of a series of glycyrrhetinic acid analogs as AChE-Is. Fourteen derivatives were prepared, of which five derivatives are recorded as new viz., 3-phenyl-carbamoyl-18ß-glycyrrhetinic acid (J9), 3-acetyl-18ß-glycyrrhetinic-30-anilinamide (J10), 3-acetyl-18ß-glycyrrhetinic-30-ethanolamide (J11), 3-acetyl-18ß-glycyrrhetinic-30-n-butylamide (J12) and 18ß-glycyrrhetinic acid-30-prenyl ester (J14), in addition to nine known derivatives (J1-J8 & J13). Compounds J12, J11, J0 and J3 showed remarkable AChE-I activity with IC50 values of 3.43, 5.39, 6.27 and 8.68 µM, respectively. These results are in full agreement with the docking study. The active compounds were non-cytotoxic to normal cells (WI-38).

Synthesis, docking, cytotoxicity, and LTA4H inhibitory activity of new gingerol derivatives as potential colorectal cancer therapy.

Leukotriene A4 hydrolase (LTA4H) is a proinflammatory enzyme that generates the inflammatory mediator leukotriene which may play an important role in chronic inflammation associated carcinogenesis. [6]-gingerol, the major bioactive compound of Zingiber officinale, is a potential inhibitor of LTA4H, a highly expressed enzyme in colorectal carcinoma. Eighteen compounds; seven of natural origin (including [4]-, [6]-, [8]-, and [10]-gingerol), five new and six known semi-synthesized [6]-gingerol derivatives were examined using docking, in vitro cytotoxicity against human colon cancer cells (HCT-116) and LTA4H aminopeptidase and epoxide hydrolase inhibitory studies. Methyl shogoal (D8) showed to be the most potent compound against HCT-116 cells (IC50; 1.54µM). Remarkably, D8 proved to be non-cytotoxic to normal cells; (TIG-1) and (HF-19) with high selective index (SI; 52.3). Furthermore [6]-gingerol derivatives showed potent LTA4H inhibitory activities in comparison to the universal positive controls (bestatin and 4BSA). Among the natural gingerols, [10]-gingerol (N3) exhibited the highest LTA4H aminopeptidase and epoxide hydrolase inhibitory activities with IC50; 21.59 and 15.24µM, respectively. Meanwhile, methyl shogoal (D8) and 4'-O-prenyl-[6]-gingerol (D10) retained the highest inhibition with IC50; 4.92 and 3.01µM, for aminopeptidase, and 11.27 and 7.25µM for epoxide hydrolase activities, respectively.

Antiplatelet and Anticoagulant Activities of Angelica shikokiana Extract and Its Isolated Compounds.

Angelica shikokiana is a Japanese medicinal plant that is used traditionally in several ailments of cardiovascular diseases. However, there is no report regarding its anticoagulant or antiplatelet activities. So this study was designed to screen for such activities (anticoagulant by prothrombin time [PT], activated partial thromboplastin time, and thrombin time assays and antiplatelet activities against adenosine 5'-diphosphate [ADP] and arachidonic acid-induced platelet aggregations) for the methanol extract of the aerial part (Angelica methanol extract [AME]), its isolated coumarins, flavonoids, and flavonoid metabolites. The AME had potent anticoagulant and antiplatelet activities, and the flavonoid compounds were evidenced to be responsible for such activities. Among coumarins compounds, hyuganin C showed significant prolongation of only PT, while other coumarins were inactive. Similarly, hyuganin C and bergapten were the only active coumarins against ADP-induced platelet aggregation. Compared to the parent compounds, colonic metabolites of the flavonoids had similar anticoagulant and antiplatelet activities, while glucuronides showed sharp decreases in all studied activities. This is the first report showing that the medicinal plant A shikokiana has potent antiplatelet and anticoagulant activities.

Acetylcholine esterase inhibitors and melanin synthesis inhibitors from Salvia officinalis.

BACKGROUND: Salvia officinalis is a traditionally used herb with a wide range of medicinal applications. Many phytoconstituents have been isolated from S. officinalis, mainly phenolic diterpenes, which possess many biological activities. PURPOSE: This study aimed to evaluate the ability of the phenolic diterpenes of S. officinalis to inhibit acetylcholine esterase (AChE) as well as their ability to inhibit melanin biosynthesis in B16 melanoma cells. METHODS: The phenolic diterpenes isolated from the aerial parts of S. officinalis were tested for their effect on melanin biosynthesis in B16 melanoma cell lines. They were also tested for their ability to inhibit AChE using Ellman's method. Moreover, a molecular docking experiment was used to investigate the binding affinity of the isolated phenolic diterpenes to the amino acid residues at the active sites of AChE. RESULTS: Seven phenolic diterpenes-sageone, 12-methylcarnosol, carnosol, 7b-methoxyrosmanol, 7a-methoxyrosmanol, isorosmanol and epirosmanol-were isolated from the methanolic extract of the aerial parts of S. officinalis. Isorosmanol showed a melanin-inhibiting activity as potent as that of arbutin. Compounds 7a-methoxyrosmanol and isorosmanol inhibited AChE activity by 50% and 65%, respectively, at a concentration of 500 µM. CONCLUSIONS: The results suggest that isorosmanol is a promising natural compound for further studies on development of new medications which might be useful in ageing disorders such as the declining of cognitive functions and hyperpigmentation.

Improved Biological Activities of Isoepoxypteryxin by Biotransformation.

Isoepoxypteryxin is the major coumarin of a Japanese medicinal plant Angelica shikokiana. This research was designed to study the effect of structural changes through fungal biotransformation on the reported biological activities of isoepoxypteryxin. Among the tested microorganisms, only Cordyceps sinensis had enzymes that could catalyze the ester hydrolysis and the reductive cleavage of the epoxide ring of isoepoxypteryxin, separately, to give two more polar metabolites (+)-cis-khellactone (P1) and a new coumarin derivative (+)-cis-3'-[(2-methyl-3-hydroxybutanoyl)oxy]-4'-acetoxy-3',4'-dihydroseselin (P2), respectively. The polar metabolite P2 showed stronger cytotoxicity and higher selectivity than isoepoxypteryxin. On the molecular level, P2 showed more in vitro inhibition of both tubulin polymerization and histone deacetylase 8 (HDAC8). Similarly, P2 showed more neuroprotection against amyloid beta fragment 1 - 42 (Aß1 - 42 )-induced neurotoxicity in human neuroblastoma cells (SH-SY5Y) and exhibited more inhibition of the in vitro aggregation of Aß1 - 42 . Both metabolites showed stronger antiplatelet aggregation by increased inhibition of thromboxane-A2 synthase (TXS) activity and thromboxane-A2 (TXA2) production. This study is the first to describe the improved cytotoxic, neuroprotective, and antiplatelet aggregation activities of isoepoxypteryxin through its biotransformation by C. sinensis.

Biological Activities of Oleanolic Acid Derivatives from Calendula officinalis Seeds.

Phytochemical examination of butanol fraction of Calendula officinalis seeds led to the isolation of two compounds identified as 28-O-ß-D-glucopyranosyl-oleanolic acid 3-O-ß-D-glucopyranosyl (1→3)-ß-D-glucopyranosiduronic acid (CS1) and oleanolic acid 3-O-ß-D-glucopyranosyl (1→3)-ß-D-glucopyranosiduronic acid (CS2). Biological evaluation was carried out for these two compounds such as melanin biosynthesis inhibitory, hyaluronic acid production activities, anti obesity using lipase inhibition and adipocyte differentiation as well as evaluation of the protective effect against hydrogen peroxide induced neurotoxicity in neuro-2A cells. The results showed that, compound CS2 has a melanin biosynthesis stimulatory activity; however, compound CS1 has a potent stimulatory effect for the production of hyaluronic acid on normal human dermal fibroblast from adult (NHDF-Ad). Both compounds did not show any inhibitory effect on both lipase and adipocyte differentiation. Compound CS2 could protect neuro-2A cells and increased cell viability against H2 O2 . These activities (melanin biosynthesis stimulatory and protective effect against H2 O2 of CS2 and hyaluronic acid productive activities of these triterpene derivatives) have been reported for the first time. Copyright © 2016 John Wiley & Sons, Ltd.

Lucidumol C, a new cytotoxic lanostanoid triterpene from Ganoderma lingzhi against human cancer cells.

A new oxygenated lanostane-type triterpene, named lucidumol C, together with six known compounds, was isolated from the chloroform extract of the fruiting bodies of Ganoderma lingzhi. Structures were established based on extensive spectroscopic and chemical studies. Potential cytotoxic activities of the isolated compounds were evaluated against human colorectal carcinoma (HCT-116, Caco-2), human liver carcinoma (HepG2), and human cervical carcinoma (HeLa) cell lines using WST-1 reagent. Selectivity was evaluated using normal human fibroblast cells (TIG-1 and HF19). Among the compounds, lucidumol C showed potent selective cytotoxicity against HCT-116 cells with an IC50 value of 7.86 ± 4.56 µM and selectivity index (SI) >10 with remarkable cytotoxic activities against Caco-2, HepG2 and HeLa cell lines.

In vitro neuroprotective activities of compounds from Angelica shikokiana Makino.

Angelica shikokiana is widely marketed in Japan as a dietary food supplement. With a focus on neurodegenerative conditions such as Alzheimer's disease, the aerial part was extracted and through bio-guided fractionation, fifteen compounds [α-glutinol, ß-amyrin, kaempferol, luteolin, quercetin, kaempferol-3-O-glucoside, kaempferol-3-O-rutinoside, methyl chlorogenate, chlorogenic acid, hyuganin E, 5-(hydroxymethyl)-2-furaldehyde, ß-sitosterol-3-O-glucoside, adenosine (isolated for the first time from A. shikokiana), isoepoxypteryxin and isopteryxin] were isolated. Isolated compounds were evaluated for in vitro neuroprotection using acetylcholine esterase inhibitory, protection against hydrogen peroxide and amyloid ß peptide (Aß25-35)-induced neurotoxicity in neuro-2A cells, scavenging of hydroxyl radicals and intracellular reactive oxygen species and thioflavin T assays. Quercetin showed the strongest AChE inhibition (IC50 value = 35.5 µM) through binding to His-440 and Tyr-70 residues at the catalytic and anionic sites of acetylcholine esterase, respectively. Chlorogenic acid, its methyl ester, quercetin and luteolin could significantly protect neuro-2A cells against H2O2-induced neurotoxicity and scavenge hydroxyl radical and intracellular reactive oxygen species. Kaempferol-3-O-rutinoiside, hyuganin E and isoepoxypteryxin significantly decreased Aß25-35-induced neurotoxicity and Th-T fluorescence. To the best of our knowledge, this is the first report about neuroprotection of hyuganin E and isoepoxypteryxin against Aß25-35-induced neurotoxicity.

In vitro Cytotoxic Activities and Molecular Mechanisms of Angelica shikokiana Extract and its Isolated Compounds.

BACKGROUND: Angelica shikokiana is a Japanese medicinal herb that is included among food and drug preparations protecting against cancer; however, there is no previous report about the cytotoxicity of A. shikokiana or its bioactive compounds. OBJECTIVE: This study was designed to investigate the cytotoxic activities of A. shikokiana methanol extract (AME) and its isolated compounds and to identify the molecular mechanisms of the cytotoxicity. MATERIALS AND METHODS: Cytotoxicity and selectivity was investigated by measuring the IC50 values on five cancer cell lines; human hepatocellular carcinoma, rhabdomyosarcoma (RD), colorectal carcinoma, human epithelioma and human breast adenocarcinoma and one normal cell line; human lung fibroblasts. The effects on tubulin polymerization and histone deacetylase 8 (HDAC8), were examined to determine the mechanism of cytotoxicity. Docking study was designed to examine the binding affinity to the target molecules. RESULTS: Methanol extract and some of its isolated coumarins and flavonoids showed potent, selective cytotoxicity against cancer cell lines. AME and all isolated compounds inhibited tubulin polymerization. Angelicin and kaempferol-3-O-rutinoside were the most active compounds. Phenolic compounds and furanocoumarins showed binding affinity to colchicine binding site rather than the vinblastine binding site of tubulin microtubules. On the other side, quercetin, kaempferol, luteolin, chlorogenic acid, and methyl chlorogenate exhibited the strongest activity against HDAC8 and the highest affinity to trichostatin A binding site. CONCLUSION: These findings provide the first scientific evidence of the cytotoxicity of AME through inhibition of tubulin polymerization and HDAC8 activity through its coumarin and flavonoid content. SUMMARY: The present study provides for the first time a clue for the cytotoxic activities of the AME. Our results indicate that the cytotoxic activities are partially related to the ability of AME to inhibit tubulin polymerization and HDAC8 activity. Isolated compounds; Angelicin and kaempferol-3-O-rutinoside showed the strongest inhibition of tubulin polymerization through binding to colchicine binding domain of tubulin microtubules. Phenolic compounds; quercetin, luteolin, kaempferol, chlorogenic acid and methyl chlorogenate exhibited a strong inhibition of HDAC8 through binding to TSA binding site. This, however, further detailed pharmacological and in vivo studies should be the next step in evaluating the cytotoxic activities of AME and its active compounds that are currently ongoing. Abbreviations used: AME: Methanol extract of the aerial part of A. shikokiana, HDACs: Histone deacetylases,HDAC8: Histone deacetylase 8.

Comparative biological study of roots, stems, leaves, and seeds of Angelica shikokiana Makino.

ETHNOPHARMACOLOGICAL RELEVANCE: Angelica shikokiana has been used as a health food for its anticancer, anti-inflammatory, antibacterial, antiallergic, and blood vessel dilating effects in Japan. It can also be used to prevent and treat hepatitis, diabetes, hyperlipidemia, and arteriosclerosis. AIM OF THE STUDY: The present study was designed to compare the biological activities such as melanin synthesis inhibitory, anti-allergy, anti-lipase, anti-bacterial, anti-oxidant, and neuroprotective activities of different parts of the plant that may justify the use of this plant in folk medicine. MATERIAL AND METHODS: The roots, stems, leaves and, seeds of Angelica shikokiana were separately extracted with water and ethanol. Each extract was examined for melanin synthesis inhibitory and anti-allergy activity on B16-melanoma and RBL-2H3 cells using IgE and A23187 as a stimulant for ß-hexosaminidase release, respectively. We also evaluated the inhibition of two enzymes, lipase and acetylcholine esterase, and of the bacterial growth of two species, Escherichia coli and Staphylococcus aureaus. The anti-oxidant activity was determined using oxygen radical anti-oxidant capacity, ORAC assay and its relation to the phenolic content was estimated using the Folin-Ciocalteu method. Besides, the protective effect of the extracts against H2O2-induced oxidative stress in mouse neuroblastoma, Neuro-2A cells was investigated. RESULTS: The most active extract exhibiting melanin synthesis inhibition (63%) and at the same time with low cytotoxicity (15%) was the ethanol extract of roots at 20 µg/ml, followed by the ethanol extract of stems (57% inhibition, 5% cytotoxicity). On the other hand, the highest inhibitions of ß-hexosaminidase release were recorded for the ethanol extract of leaves with IC50 value of 6.89 µg/ml followed by the water extract of the seeds and leaves with IC50 value of 78.32 and 88.44 µg/ml, respectively. For anti-lipase assay, ethanol extracts of the stems and roots showed the strongest inhibition with IC50 values of 204.06 and 216.24 µg/ml, respectively. None of the examined extracts showed any activity against Escherichia coli. while the ethanol extract of the roots and stems showed moderate inhibition for Staphylococcus aureus with minimum inhibitory concentration of 400 µg/ml. Ethanol extract of the roots showed only 30% inhibition of acetylcholine esterase enzyme. The results of anti-oxidant, phenolic content and protective effect against H2O2-induced cytotoxicity assays showed highly correlated data. Ethanol extract of the stems (ORAC value of 1.08 µmol Trolox/mg and phenolic content 44.25 µg GAE/mg) increased the cell viability of H2O2-treated Neuro-2A cells by 28%.