Integrated analysis and separation of 5-lipoxygenase inhibitors from triterpenes of Poria cocos using bioaffinity ultrafiltration UPLC-Q-Exactive, molecular docking and target-based multiple complex networks.

Poria cocos (Schw.) Wolf (P. cocos) has been widely used as medical plant in East Asia with remarkable anti-Alzheimer's disease (anti-AD) activity. However, the underlying mechanisms are still confused. In this study, based on the ß-Amyloid deposition hypothesis of AD, an integrated analysis was conducted to screen and separation 5-lipoxygenase (5-LOX) inhibitors from triterpenoids of P. cocos and investigate the anti-AD mechanisms, containing bioaffinity ultrafiltration UPLC-Q-Exactive, molecular docking, and multiple complex networks. Five triterpenoids were identified as potential 5-LOX inhibitors, including Tumulosic acid, Polyporenic acid C, 3-Epi-dehydrotumulosic acid, Pachymic acid and Dehydrotrametenolic acid. Five potential 5-LOX inhibitors were screened by ultrafiltration affinity assay in P. cocos. The molecular docking simulation results are consistent with the ultrafiltration experimental results, which further verifies the accuracy of the experiment. The commercial 5-LOX inhibitor that Zileuton was used as a positive control to evaluate the inhibitory effect of active ingredients on 5-LOX. Subsequently, the established separation method allowed the five active ingredients (Pachymic acid, 3-Epi-dehydrotumulosic acid, Dehydrotrametenolic acid, Tumulosic acid and Polyporenic acid C) with high purity to be isolated. Targeting network pharmacology analysis showed that five active ingredients correspond to a total of 286 targets. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis found that target cells were mainly enriched in Pathways in cancer, Lipid and atherosclerosis. Our results indicate that P. cocos extract has the potential to be used in the prevention and treatment of neurodegenerative diseases. This will help elucidate the mechanisms of action of various medicinal plants at the molecular level and provide more opportunities for the discovery and development of new potential treatments from health food resources.

Efficient and fast screening and separation based on computer-aided screening and complex chromatography methods for lipoxygenase inhibitors from Ganoderma lucidum.

INTRODUCTION: Accurate screening and targeted preparative isolation of active substances from natural medicines have long been technical challenges in natural medicine research. OBJECTIVES: This study outlines a new approach for improving the efficiency of natural product preparation, focusing on the rapid and accurate screening of potential active ingredients in Ganoderma lucidum and efficient preparation of lipoxidase inhibitors, with the aim of providing new ideas for the treatment of Alzheimer's disease with G. lucidum. METHODS: The medicinal plant G. lucidum was selected through ultrafiltration coupled with liquid chromatography and mass spectrometry (UF-LC-MS) and computer-assisted screening for lipoxygenase (LOX) inhibitors. In addition, the inhibitory effect of the active compounds on LOX was studied using enzymatic reaction kinetics, and the underlying mechanism is discussed. Finally, based on the earlier activity screening guidelines, the identified ligands were isolated and purified through complex chromatography (high-speed countercurrent chromatography and semi-preparative high-performance liquid chromatography). RESULTS: Five active ingredients, ganoderic acids A, B, C2, D2, and F, were identified and isolated from G. lucidum. We improved the efficiency and purity of active compound preparation using virtual computer screening and enzyme inhibition assays combined with complex chromatography. CONCLUSION: The innovative methods of UF-LC-MS, computer-aided screening, and complex chromatography provide powerful tools for screening and separating LOX inhibitors from complex matrices and provide a favourable platform for the large-scale production of bioactive substances and nutrients.

Screening 5-lipoxygenase inhibitors from selected traditional Chinese medicines and isolation of the active compounds from Polygoni Cuspidati Rhizoma by an on-line bioactivity evaluation system.

To identify natural products as new prototypes for 5-lipoxygenase (5-LOX), 12 traditional Chinese medicines (TCMs) were selected for screening their 5-LOX inhibition activities. The results showed that the methanol extracts of all selected TCMs (n = 12) possessed inhibitory activities against 5-LOX at 200 µg/mL, of which six extracts of the TCMs showed significant inhibitory effects with IC50 values in the range from 33.2 ± 1.4 µg/mL to 153.5 ± 1.7 µg/mL, and the extract of Polygoni Cuspidati Rhizoma (RPC) was the most active sample. An on-line ultra-performance liquid chromatography-photodiode array-MSn -5-LOX-fluorescence detector (UPLC-PDA-MSn -5-LOX-FLD) method was applied to further identify the potential 5-LOX inhibitory constituents in RPC extracts, which resulted in the identification of seven components with 5-LOX-binding activities. Finally, four compounds (polydatin, resveratrol, emodin-8-O-glucoside, and emodin) were successfully purified from RPC extracts. The 5-LOX inhibition action was assayed in vitro, and the results showed that these compounds possessed potent inhibitory effects against 5-LOX with IC50 values of 15.3 ± 2.1, 4.5 ± 1.2, 23.8 ± 0.4, and 11.8 ± 1.5 µg/mL, respectively. This was the first study to reveal the 5-LOX inhibitory constituents of RPC, and the present investigation might provide a valuable approach for the rapid discovery of natural inhibitors from TCMs.

The bioactive alkaloids identified from Cortex Phellodendri ameliorate benign prostatic hyperplasia via LOX-5/COX-2 pathways.

BACKGROUND: The bioactive alkaloids identified from Cortex Phellodendri (CP) were highly effective in treating rats with benign prostatic hyperplasia (BPH). Specifically, lipoxygenase-5 (LOX-5) and cyclooxygenase-2 (COX-2) were identified as two primary targets for alleviating inflammation in BPH rats. However, it remains unknown whether the alkaloid components in CP can interact with the two target proteins. PURPOSE: To further identify bioactive alkaloids targeting LOX/COX pathways. METHODS: An affinity-ultrafiltration mass spectrometry approach was employed to screen dual-target LOX-5/COX-2 ligands from alkaloid extract. The structures of bioactive alkaloids were characterized by high-resolution Fourier transform ion cyclotron resonance mass spectrometry. To understand the molecular mechanisms underlying the effects of bioactive alkaloids, the expression levels of LOX-5 and COX-2 in BPH model rats were investigated at both protein and mRNA levels. The LOX-5/COX-2 enzymes activity experiments and molecular docking analysis were performed to fully evaluate the interactions between bioactive alkaloids and LOX-5/COX-2. RESULTS: After comprehensive analysis, the results showed that bioactive alkaloids could suppress the expression of LOX-5 and COX-2 simultaneously to exert an anti-inflammatory effect on the progression of BPH. In addition, the screened protoberberine, demethyleneberberine was found to exhibit prominent inhibitory activities against both LOX-5 and COX-2 enzymes, palmatine and berberine with moderate inhibitory activities. Molecular docking analysis confirmed that demethyleneberberine could interact well with LOX-5/COX-2. CONCLUSION: This study is the first to explore the inhibitory effects of bioactive alkaloids from CP on LOX-5 and COX-2 activities in BPH rats. Our findings demonstrate that the bioactive alkaloids from CP can ameliorate BPH via dual LOX-5/COX-2 pathways, which serves as an efficient approach for the discovery of novel drug leads from natural products with reduced side effects.

In vitro pharmacological attributes and metabolite's fingerprinting of Conocarpus lancifolius / Atributos farmacológicos in vitro y huellas dactilares de metabolitos de Conocarpus lancifolius

Search for safe antioxidants and novel nutraceuticals urged to evaluate the antioxidant, anti-acetylcholine esterase and anti-lipoxygenase activity of various leaf extracts of Conocarpus lancifolius. Extraction was optimized from freeze dried plant extracts quenched with liquid nitrogen using water, ethanol, methanol, hexane, ethyl acetate and chloroform. Maximum extract yield, total phenolic contents and total flavonoid contents were obtained in case of ethanolic extraction. The highest 2,2-diphenyl-1-picrylhydrazylradical scavenging in terms of IC50 value of 55.26 µg/mL was observed for ethanolic leaf extract. The acetylcholine esterase and lipoxygenase inhibitory activities (IC50) were also observed for ethanolic extract. These findings for ethanolic extract were statistically significant when compared with other extracts (ρ<0.05). The haemolytic % values indicated that all extracts were associated with very low or negligible toxicity. The epicatechin, isorhamnetin, rutin, scopoleptin, skimmianine, quercetin-3-O-α-rhamnoside, quercetin-3-O-ß-glucoside, cornoside, creatinine, choline, pyruvic acid, α-hydroxybutyric acid, phyllanthin and hypophyllanthin were identified as major functional metabolites in ethanolic leaf extract of C. lancifoliusby 1H-NMR. The identified metabolites were probably responsible for the pharmacological properties of C.lancifolius. The findings may be utilized as pharmacological leads for drug development and food fortification.

Se insta a la búsqueda de antioxidantes seguros y nuevos nutracéuticos para evaluar la actividad antioxidante, anti-acetilcolina esterasa y anti-lipoxigenasa de varios extractos de hojas de Conocarpus lancifolius. La extracción se optimizó a partir de extractos de plantas liofilizados enfriados con nitrógeno líquido usando agua, etanol, metanol, hexano, acetato de etilo y cloroformo. En el caso de extracción etanólica se obtuvo el rendimiento máximo de extracto, el contenido de fenoles totales y el contenido de flavonoides totales. La mayor eliminación de radicales 2,2-difenil-1-picrilhidrazilo en términos de valor de CI50 de 55,26 µg/mL se observó para el extracto de hoja etanólico. También se observaron las actividades inhibidoras de la acetilcolina esterasa y lipoxigenasa (CI50) para el extracto etanólico. Estos hallazgos para el extracto etanólico fueron estadísticamente significativos en comparación con otros extractos (ρ<0.05). Los valores del % hemolítico indicaron que todos los extractos estaban asociados con una toxicidad muy baja o insignificante. Se identificaron la epicatequina, isorhamnetina, rutina, escopoleptina, skimmianina, quercetina-3-O-α-ramnosido, quercetina-3-O-ß-glucósido, cornosido, creatinina, colina, ácido pirúvico, ácido α-hidroxibutírico, filantrina e hipofillantina. como metabolitos funcionales principales en el extracto etanólico de hojas de C. lancifoliuspor 1H-NMR. Los metabolitos identificados probablemente fueron responsables de las propiedades farmacológicas de C. lancifolius. Los hallazgos pueden utilizarse como pistas farmacológicas para el desarrollo de fármacos y la fortificación de alimentos.

Chemical Variability and In Vitro Anti-Inflammatory Activity of Leaf Essential Oil from Ivorian Isolona dewevrei (De Wild. & T. Durand) Engl. & Diels.

The chemical variability and the in vitro anti-inflammatory activity of the leaf essential oil from Ivorian Isolona dewevrei were investigated for the first time. Forty-seven oil samples were analyzed using a combination of CC, GC(RI), GC-MS and 13C-NMR, thus leading to the identification of 113 constituents (90.8-98.9%). As the main components varied drastically from sample to sample, the 47 oil compositions were submitted to hierarchical cluster and principal components analyses. Three distinct groups, each divided into two subgroups, were evidenced. Subgroup I-A was dominated by (Z)-ß-ocimene, ß-eudesmol, germacrene D and (E)-ß-ocimene, while (10ßH)-1ß,8ß-oxido-cadina-4-ene, santalenone, trans-α-bergamotene and trans-ß-bergamotene were the main compounds of Subgroup I-B. The prevalent constituents of Subgroup II-A were germacrene B, (E)-ß-caryophyllene, (5αH,10ßMe)-6,12-oxido-elema-1,3,6,11(12)-tetraene and γ-elemene. Subgroup II-B displayed germacrene B, germacrene D and (Z)-ß-ocimene as the majority compounds. Germacrene D was the most abundant constituent of Group III, followed in Subgroup III-A by (E)-ß-caryophyllene, (10ßH)-1ß,8ß-oxido-cadina-4-ene, germacrene D-8-one, and then in Subgroup III-B by (Z)-ß-ocimene and (E)-ß-ocimene. The observed qualitative and quantitative chemical variability was probably due to combined factors, mostly phenology and season, then harvest site to a lesser extent. The lipoxygenase inhibition by a leaf oil sample was also evaluated. The oil IC50 (0.020 ± 0.005 mg/mL) was slightly higher than the non-competitive lipoxygenase inhibitor NDGA IC50 (0.013 ± 0.003 mg/mL), suggesting a significant in vitro anti-inflammatory potential.

A Cornflower Extract Containing N-Feruloylserotonin Reduces Inflammation in Human Skin by Neutralizing CCL17 and CCL22 and Inhibiting COX-2 and 5-LOX.

Thymus and Activation-Regulated Chemokine (TARC/CCL17) and Macrophage-Derived Chemokine (MDC/CCL22) are two key chemokines exerting their biological effect via binding and activating a common receptor CCR4, expressed at the surface of type 2 helper T (Th2) cells. By recruiting Th2 cells in the dermis, CCL17 and CCL22 promote the development of inflammation in atopic skin. The aim of this research was to develop a plant extract whose biological properties, when applied topically, could be beneficial for people with atopic-prone skin. The strategy which was followed consisted in identifying ligands able to neutralize the biological activity of CCL17 and CCL22. Thus, an in silico molecular modeling and a generic screening assay were developed to screen natural molecules binding and blocking these two chemokines. N-Feruloylserotonin was identified as a neutraligand of CCL22 in these experiments. A cornflower extract containing N-feruloylserotonin was selected for further in vitro tests: the gene expression modulation of inflammation biomarkers induced by CCL17 or CCL22 in the presence or absence of this extract was assessed in the HaCaT keratinocyte cell line. Additionally, the same cornflower extract in another vehicle was evaluated in parallel with N-feruloylserotonin for cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzymatic cellular inhibition. The cornflower extract was shown to neutralize the two chemokines in vitro, inhibited COX-2 and 5-LOX, and demonstrated anti-inflammatory activities due mainly to the presence of N-feruloylserotonin. Although these findings would need to be confirmed in an in vivo study, the in vitro studies lay the foundation to explain the benefits of the cornflower extract when applied topically to individuals with atopic-prone skin.

Discovery of natural 15-LOX small molecule inhibitors from Chinese herbal medicine using virtual Screening, biological evaluation and molecular dynamics studies.

Chinese herbal medicines (CHM) are frequently used to treat different types of inflammatory diseases and 15-Lipoxygenase (15-LOX) is a critical target enzyme for treating various inflammatory diseases. In this study, natural 15-LOX inhibitors were identified in CHM using an approach of virtual screening combined with the biological assays. First, an in-house Chinese medicine database containing 360 compounds was screened using a virtual screening approach based on pharmacophore and molecular docking to uncover several novel potential 15-LOX inhibitors. Secondly, the inhibitory effect of virtual screening hits against the 15-LOX enzyme was validated in an in vitro enzyme inhibition assay. Then, a tumor necrosis factor-α (TNF-α) release assay was carried out to explore the anti-inflammatory response of the active compounds. Furthermore, molecular dynamics (MD) simulation and binding free energy calculation were applied to analyze the process of inhibitors binding and also compared the mode of binding of the inhibitors by using the Molecular Mechanics-Generalized Born Surface Area (MM/GBSA) method. Finally, licochalcone B and eriodictyol were confirmed as inhibitors of the 15-LOX enzyme with IC50 values of 9.67 and 18.99 µM, respectively. In vitro cell-based assay showed that licochalcone B and eriodictyol inhibited the release of TNF-α factor in RAW264.7 cells stimulated by lipopolysaccharides (LPS) in a dose-dependent manner. Molecular dynamics and binding free energy analysis showed that the two 15-LOX-ligand systems immediately attained equilibrium with almost 1 Å fluctuation, the calculated binding free energies were found around -18.89 and -12.96 kcal/mol for licochalcone B and eriodictyol, respectively. Thr412, Arg415, Val420, Thr429, Ile602 and Trp606 were the main amino acid residues for the inhibition of 15-LOX enzyme activity. The current study confirms that licochalcone B and eriodictyol are 15-LOX inhibitors and can suppress the release of the TNF-α factor in RAW264.7 cells stimulated by LPS, thus providing a basis for the follow-up research and development for 15-LOX inhibitors.

Dihydroisocoumarins, Naphthalenes, and Further Polyketides from Aloe vera and A. plicatilis: Isolation, Identification and Their 5-LOX/COX-1 Inhibiting Potency.

The present study aims at the isolation and identification of diverse phenolic polyketides from Aloe vera (L.) Burm.f. and Aloe plicatilis (L.) Miller and includes their 5-LOX/COX-1 inhibiting potency. After initial Sephadex-LH20 gel filtration and combined silica gel 60- and RP18-CC, three dihydroisocoumarins (nonaketides), four 5-methyl-8-C-glucosylchromones (heptaketides) from A. vera, and two hexaketide-naphthalenes from A. plicatilis have been isolated by means of HSCCC. The structures of all polyketides were elucidated by ESI-MS and 2D 1H/13C-NMR (HMQC, HMBC) techniques. The analytical/preparative separation of 3R-feralolide, 3'-O-ß-d-glucopyranosyl- and the new 6-O-ß-d-glucopyranosyl-3R-feralolide into their respective positional isomers are described here for the first time, including the assignment of the 3R-configuration in all feralolides by comparative CD spectroscopy. The chromones 7-O-methyl-aloesin and 7-O-methyl-aloeresin A were isolated for the first time from A. vera, together with the previously described aloesin (syn. aloeresin B) and aloeresin D. Furthermore, the new 5,6,7,8-tetrahydro-1-O-ß-d-glucopyranosyl- 3,6R-dihydroxy-8R-methylnaphtalene was isolated from A. plicatilis, together with the known plicataloside. Subsequently, biological-pharmacological screening was performed to identify Aloe polyketides with anti-inflammatory potential in vitro. In addition to the above constituents, the anthranoids (octaketides) aloe emodin, aloin, 6'-(E)-p-coumaroyl-aloin A and B, and 6'-(E)-p-coumaroyl-7-hydroxy-8-O-methyl-aloin A and B were tested. In the COX-1 examination, only feralolide (10 µM) inhibited the formation of MDA by 24%, whereas the other polyketides did not display any inhibition at all. In the 5-LOX-test, all aloin-type anthranoids (10 µM) inhibited the formation of LTB4 by about 25-41%. Aloesin also displayed 10% inhibition at 10 µM in this in vitro setup, while the other chromones and naphthalenes did not display any activity. The present study, therefore, demonstrates the importance of low molecular phenolic polyketides for the known overall anti-inflammatory activity of Aloe vera preparations.

Selective Extraction of Piceatannol from Passiflora edulis by-Products: Application of HSPs Strategy and Inhibition of Neurodegenerative Enzymes.

Passiflora edulis by-products (PFBP) are a rich source of polyphenols, of which piceatannol has gained special attention recently. However, there are few studies involving environmentally safe methods for obtaining extracts rich in piceatannol. This work aimed to concentrate piceatannol from defatted PFBP (d-PFBP) by means of pressurized liquid extraction (PLE) and conventional extraction, using the bio-based solvents selected with the Hansen solubility parameters approach. The relative energy distance (Ra) between solvent and solute was: Benzyl Alcohol (BnOH) < Ethyl Acetate (EtOAc) < Ethanol (EtOH) < EtOH:H2O. Nonetheless, EtOH presented the best selectivity for piceatannol. Multi-cycle PLE at 110 °C was able to concentrate piceatannol 2.4 times more than conventional extraction. PLE exhibited a dependence on kinetic parameters and temperature, which could be associated with hydrogen bonding forces and the dielectric constant of the solvents. The acetylcholinesterase (AChE) and lipoxygenase (LOX) IC50 were 29.420 µg/mL and 27.682 µg/mL, respectively. The results reinforce the demand for processes to concentrate natural extracts from food by-products.