Computational Docking as a Tool in Guiding the Drug Design of Rutaecarpine Derivatives as Potential SARS-CoV-2 Inhibitors.

COVID-19 continues to spread around the world. This is mainly because new variants of the SARS-CoV-2 virus emerge due to genomic mutations, evade the immune system and result in the effectiveness of current therapeutics being reduced. We previously established a series of detection platforms, comprising computational docking analysis, S-protein-based ELISA, pseudovirus entry, and 3CL protease activity assays, which allow us to screen a large library of phytochemicals from natural products and to determine their potential in blocking the entry of SARS-CoV-2. In this new screen, rutaecarpine (an alkaloid from Evodia rutaecarpa) was identified as exhibiting anti-SARS-CoV-2 activity. Therefore, we conducted multiple rounds of structure-activity-relationship (SAR) studies around this phytochemical and generated several rutaecarpine analogs that were subjected to in vitro evaluations. Among these derivatives, RU-75 and RU-184 displayed remarkable inhibitory activity when tested in the 3CL protease assay, S-protein-based ELISA, and pseudovirus entry assay (for both wild-type and omicron variants), and they attenuated the inflammatory response induced by SARS-CoV-2. Interestingly, RU-75 and RU-184 both appeared to be more potent than rutaecarpine itself, and this suggests that they might be considered as lead candidates for future pharmacological elaboration.

The Ethanol Extract of Evodiae Fructus and Its Ingredient, Rutaecarpine, Inhibit Infection of SARS-CoV-2 and Inflammatory Responses.

COVID-19, derived from SARS-CoV-2, has resulted in millions of deaths and caused unprecedented socioeconomic damage since its outbreak in 2019. Although the vaccines developed against SARS-CoV-2 provide some protection, they have unexpected side effects in some people. Furthermore, new viral mutations reduce the effectiveness of the current vaccines. Thus, there is still an urgent need to develop potent non-vaccine therapeutics against this infectious disease. We recently established a series of detecting platforms to screen a large library of Chinese medicinal herbs and phytochemicals. Here, we reveal that the ethanolic extract of Evodiae Fructus and one of its components, rutaecarpine, showed promising potency in inhibiting the activity of 3C-like (3CL) protease, blocking the entry of the pseudo-typed SARS-CoV-2 (including wild-type and omicron) into cultured cells. In addition, inflammatory responses induced by pseudo-typed SARS-CoV-2 were markedly reduced by Evodiae Fructus extract and rutaecarpine. Together our data indicate that the herbal extract of Evodiae Fructus and rutaecarpine are potent anti-SARS-CoV-2 agents, which might be considered as a treatment against COVID-19 in clinical applications.

Tectoridin Stimulates the Activity of Human Dermal Papilla Cells and Promotes Hair Shaft Elongation in Mouse Vibrissae Hair Follicle Culture.

To search hair growth-promoting herbal extract, a screening platform of having HEK293T fibroblast being transfected with pTOPFLASH DNA construct was developed over a thousand of herbal extracts and phytochemicals were screened. One of the hits was ethanolic extract of Rhizoma Belamcandae, the rhizome of Belamcanda chinensis (L.) DC. Tectoridin, an isoflavone from Rhizoma Belamcandae, was shown to be responsible for this activation of promoter construct, inducing the transcription of pTOPFLASH in the transfected fibroblasts in a dose-dependent manner. The blockage by DKK-1 suggested the action of tectoridin could be mediated by the Wnt receptor. The hair growth-promoting effects of tectoridin were illustrated in human follicular dermal papilla cells and mouse vibrissae organ cultures. In tectoridin-treated dermal papilla cultures, an activation of Wnt signaling was demonstrated by various indicative markers, including TCF/LEF1 transcriptional activity, nuclear translocation of ß-catenin, expressions level of mRNAs encoding axin-related protein, (AXIN2), ß-catenin, lymphoid enhancer-binding factor-1 (LEF-1), insulin-like growth factor 1 (IGF-1) and alkaline phosphatase (ALP). In addition, an increase of hair shaft elongation was observed in cultured mouse vibrissae upon the treatment of tectoridin. Tectoridin, as well as the herbal extract of Rhizoma Belamcandae, possesses hair promoting activity, which deserves further development.

The Extracts of Polygonum cuspidatum Root and Rhizome Block the Entry of SARS-CoV-2 Wild-Type and Omicron Pseudotyped Viruses via Inhibition of the S-Protein and 3CL Protease.

COVID-19, resulting from infection by the SARS-CoV-2 virus, caused a contagious pandemic. Even with the current vaccines, there is still an urgent need to develop effective pharmacological treatments against this deadly disease. Here, we show that the water and ethanol extracts of the root and rhizome of Polygonum cuspidatum (Polygoni Cuspidati Rhizoma et Radix), a common Chinese herbal medicine, blocked the entry of wild-type and the omicron variant of the SARS-CoV-2 pseudotyped virus into fibroblasts or zebrafish larvae, with IC50 values ranging from 0.015 to 0.04 mg/mL. The extracts were shown to inhibit various aspects of the pseudovirus entry, including the interaction between the spike protein (S-protein) and the angiotensin-converting enzyme II (ACE2) receptor, and the 3CL protease activity. Out of the chemical compounds tested in this report, gallic acid, a phytochemical in P. cuspidatum, was shown to have a significant anti-viral effect. Therefore, this might be responsible, at least in part, for the anti-viral efficacy of the herbal extract. Together, our data suggest that the extracts of P. cuspidatum inhibit the entry of wild-type and the omicron variant of SARS-CoV-2, and so they could be considered as potent treatments against COVID-19.

Corylin, a flavonoid derived from Psoralea Fructus, induces osteoblastic differentiation via estrogen and Wnt/ß-catenin signaling pathways.

Corylin is a naturally occurring flavonoid isolated from the fruit of Psoralea corylifolia L. (Fabaceae), which is a Chinese medicinal herb in treating osteoporosis. Although a variety of pharmacological activities of corylin have been reported, its osteogenic action and the underlying mechanism in bone development remain unclear. In the present study, the involvement of bone-specific genes in corylininduced differentiated osteoblasts was analyzed by RT-PCR, promoter-reporter assay, and Western blotting. In cultured osteoblasts, corylin-induced cell differentiation and mineralization, as well as increased the expressions of vital biological markers for osteogenesis, such as Runx2, Osterix, Col1, and ALP. Corylin was proposed to have dual pathways in triggering the osteoblastic differentiation. First, the osteogenic function of corylin acted through the activation of Wnt/ß-catenin signaling. The nuclear translocation of ß-catenin of cultured osteoblasts, as determined by flow cytometry and confocal microscopy, was triggered by applied corylin, and which was blocked by DKK-1, an inhibitor of Wnt/ß-catenin signaling. Second, the application of corylin-induced estrogenic response in a dose-dependent manner, and which was blocked by ICI 182 780, an antagonist of estrogen receptor. Furthermore, the activation of Runx2 promoter by corylin was abolished by both DKK-1 and ICI 182,780, indicating that the corylin exhibited its osteogenic effect via estrogen and Wnt/ß-catenin signaling pathways. In addition, corylin regulated the metabolic profiles, as well as the membrane potential of mitochondria, in cultured osteoblasts. Corylin also stimulated the osteogenesis in bone micromass derived from mesenchymal progenitor cells. This study demonstrated the osteogenic activities of corylin in osteoblasts and micromass, suggesting that corylin has the potential to be developed as a novel pro-osteogenic agent in targeting for the treatment of osteoblast-mediated osteoporosis.

Biological Evaluation and Transcriptomic Analysis of Corylin as an Inhibitor of Osteoclast Differentiation.

Corylin, a flavonoid isolated from the fruit of Psoralea corylifolia, has an osteogenic effect on osteoblasts in vitro and bone micromass ex vivo. However, the effect and mechanism of corylin in regulating osteoclastogenesis remain unknown. By using murine bone marrow macrophages as the osteoclast precursor, corylin was found to inhibit the receptor activator of nuclear factor (NF) κB ligand (RANKL)-induced osteoclast differentiation via down-regulating osteoclastic marker genes. In parallel, F-actin formation and osteoclast migration were diminished in corylin-treated cultured osteoclasts, and subsequently the expressions of osteoclastic proteins were suppressed: the suppression of protein expression was further illustrated by transcriptomic analysis. Furthermore, corylin inhibited the nuclear translocation of p65, giving rise to a restraint in osteoclastic differentiation through the attenuation of transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and nuclear factor of activated T cells c1 (NFATc1). There was no obvious change in apoptosis when the RANKL-induce osteoclasts were cultured in the presence of corylin. The finding supports the potential development of corylin as an osteoclast inhibitor against osteoporosis.

Polydatin suppresses VEGF-induced angiogenesis through binding with VEGF and inhibiting its receptor signaling.

Polydatin, also called piceid, is a stilbenoid glucoside of a resveratrol derivative. It derives mainly from the root and rhizome of Polygonum cuspidatum Sieb. et Zucc. Although the role of P. cuspidatum root in angiogenesis has been reported, the active chemical or chemicals responsible for such function is not known. Here, polydatin was proposed to bind VEGF, which therefore altered the functions of VEGF in angiogenesis. Several lines of evidence supported the pharmaceutical effects of polydatin in VEGF-induced angiogenesis. In human umbilical vein endothelial cells, polydatin inhibited VEGF-stimulated cell proliferation, cell migration, and tube formation. Moreover, polydatin showed suppressive effects on the subintestinal vessel formation in zebrafish embryos. In signaling cascades, polydatin application attenuated VEGF-induced phosphorylations of VEGF receptor 2 and JNK. Moreover, the VEGF-induced phosphorylations of Akt, eNOS, and Erk were significantly decreased in the presence of polydatin. In parallel, the formation of reactive oxygen species, triggered by VEGF, was markedly decreased under polydatin application. Thus, our results supported the angiogenic roles of polydatin, as well as its signaling mechanism in blocking VEGF-mediated responses. The current study provides support for the possible development of polydatin as a potential therapeutic agent for treatment and prevention of angiogenesis-related diseases.-Hu, W.-H., Wang, H.-Y., Kong, X.-P., Xiong, Q.-P., Poon, K. K.-M., Xu, L., Duan, R., Chan, G. K.-L., Dong, T. T.-X., Tsim, K. W.-K. Polydatin suppresses VEGF-induced angiogenesis through binding with VEGF and inhibiting its receptor signaling.

The herbal extract deriving from aerial parts of Scutellaria baicalensis shows anti-inflammation and anti-hypoxia responses in cultured fin cells from rabbit fish.

A new cell line derived from dorsal fin of rabbit fish Siganus fuscescens was developed and characterized. The cell line was isolated from the dorsal fin, named as rabbit fish fin (RFF) cell line, and which was sub-cultured for 50 cycles since the development. This cell line was tested for growth in different temperatures and serum concentrations, and the best growing condition was at 20% serum at 28 °C. In cultured RFF cells, amplification of 18S rRNA from genomic DNA and immunostaining of cellular cytokeratin confirmed the proper identity of S. fuscescens fish. After 30th passage of cultures, the cells were exposed to challenge of inflammation, triggered by LPS, and hypoxia, mimicked by CoCl2. Cultured RFF cells showed robust sensitive responses to inflammation and hypoxia in directing the expressions of cytokines and hypoxia inducible factor-1α (HIF-1α). The water extract of aerial part of Scutellaria baicalensis (SBA) has been shown in rabbit fish to prevent inflammation. Here, we extended this notion of testing the efficacy of SBA extract in the developed cultured RFF cells. Application of SBA extract inhibited the expression of LPS-induced inflammatory cytokines, i.e. IL-1ß, IL-6, as well as the signaling of NF-κB. The application of CoCl2 in cultured RFF cells triggered the hypoxia-induced cell death and up regulation of HIF-1α. As expected, applied SBA extract in the cultures prevented the hypoxia-induced signaling. Our results show the established RFF cell line may be served as an ideal in vitro model in drug screening relating to inflammation and hypoxia. Additionally, we are supporting the usage of SBA herbal extract in fish aquaculture, which possesses efficacy against inflammation and hypoxia.

Piceatannol, a Natural Analog of Resveratrol, Exerts Anti-angiogenic Efficiencies by Blockage of Vascular Endothelial Growth Factor Binding to Its Receptor.

Piceatannol is also named as trans-3,4,3',5'-tetrahydroxy-stilbene, which is a natural analog of resveratrol and a polyphenol existing in red wine, grape and sugar cane. Piceatannol has been proved to possess activities of immunomodulatory, anti-inflammatory, antiproliferative and anticancer. However, the effect of piceatannol on VEGF-mediated angiogenesis is not known. Here, the inhibitory effects of piceatannol on VEGF-induced angiogenesis were tested both in vitro and in vivo models of angiogenesis. In human umbilical vein endothelial cells (HUVECs), piceatannol markedly reduced the VEGF-induced cell proliferation, migration, invasion, as well as tube formation without affecting cell viability. Furthermore, piceatannol significantly inhibited the formation of subintestinal vessel in zebrafish embryos in vivo. In addition, we identified the underlying mechanism of piceatannol in triggering the anti-angiogenic functions. Piceatannol was proposed to bind with VEGF, thus attenuating VEGF in activating VEGF receptor and blocking VEGF-mediated downstream signaling, including expressions of phosphorylated eNOS, Erk and Akt. Furthermore, piceatannol visibly suppressed ROS formation, as triggered by VEGF. Moreover, we further determined the outcome of piceatannol binding to VEGF in cancer cells: piceatannol significantly suppressed VEGF-induced colon cancer proliferation and migration. Thus, these lines of evidence supported the conclusion that piceatannol could down regulate the VEGF-mediated angiogenic functions with no cytotoxicity via decreasing the amount of VEGF binding to its receptors, thus affecting the related downstream signaling. Piceatannol may be developed into therapeutic agents or health products to reduce the high incidence of angiogenesis-related diseases.

Prenylated Flavonoids from Roots of Glycyrrhiza uralensis Induce Differentiation of B16-F10 Melanoma Cells.

Roots of Glycyrrhiza uralensis have been used as herbal medicine and natural sweetener. By activity-guided phytochemical investigation of the extracts from G.uralensis root, ten flavonoids, namely GF-1⁻GF-10, of which five were prenylated flavonoids, were found to show antiproliferative effects in melanoma B16-F10 cells. Three of the prenylated flavonoids, namely GF-1, GF-4 and GF-9, significantly induced the differentiation of B16-F10 cells; the inductions included increase of tyrosinase activity, tyrosinase protein, and melanin content. In GF-1 and GF-9 induced melanoma differentiation, the phosphorylation of p38 MAPK (mitogen activated potein kinase) was identified; while GF-4 could trigger the phosphorylation of PI3K/AKT (phosphatidylinositol 3-kinase/Protein Kinase B) signaling. However, application of GF-6 to the melanoma cells did not induce differentiation; but which promoted cell apoptotic signaling, i.e., increase levels of cleaved-PRAP, cleaved-caspase 3, and cleaved-caspase 9. These results suggested that different types of prenylated flavonoids from G.uralensis might have potential anticancer effects against melanoma cells by acting through different signaling pathways.

The Extracts and Major Compounds Derived from Astragali Radix Alter Mitochondrial Bioenergetics in Cultured Cardiomyocytes: Comparison of Various Polar Solvents and Compounds.

Astragali Radix (AR) is a widely used "Qi-invigorating" herb in China for its tonic effects in strengthening biological tissues. The extract of AR contains abundant antioxidants, including astragalosides and isoflavonoids. However, very few reports have systematically measured the effects of the major components of AR on cell mitochondrial bioenergetics. Here, a systemic approach employing an extracellular flux analyzer was developed to evaluate mitochondrial respiration in cultured cardiomyocyte cells H9C2. The effects of different polar extractives, as well as of the major compounds of AR, were compared. The contents of astragaloside IV, calycosin, formononetin, and genistein in the AR extracts obtained by using water, 50% ethanol, and 90% ethanol were measured by liquid chromatograph-mass spectrometer (LC⁻MS). The antioxidant activities of the AR extracts, as well as of their major compounds, were determined by measuring the free radical scavenging activity and protective effects in tert-butyl hydroperoxide (tBHP)-treated H9C2 cells. By monitoring the real-time oxygen consumption rate (OCR) in tBHP-treated cardiomyocytes with a Seahorse extracellular flux analyzer, the tonic effects of the AR extracts and of their main compounds on mitochondrial bioenergetics were evaluated. AR water extracts possessed the strongest antioxidant activity and protective effects in cardiomyocytes exposed to oxidative stress. The protection was proposed to be mediated via increasing the spare respiratory capacity and mitochondrial ATP production in the stressed cells. The major compounds of AR, astragaloside IV and genistein, showed opposite effects in regulating mitochondrial bioenergetics. These results demonstrate that highly polar extracts of AR, especially astragaloside-enriched extracts, possess better tonic effects on mitochondrial bioenergetics of cultured cardiomyocytes than extracts with a lower polarity.

20(S)-Protopanaxadiol Phospholipid Complex: Process Optimization, Characterization, In Vitro Dissolution and Molecular Docking Studies.

20(S)-Protopanaxadiol (PPD), a bioactive compound extracted from ginseng, possesses cardioprotective, neuroprotective, anti-inflammatory, antiestrogenic, anticancer and anxiolytic effects. However, the clinical application of PPD is limited by its weak aqueous solubility. In this study, we optimized an efficient method of preparing its phospholipid complex (PPD-PLC) using a central composite design and response surface analysis. The prepared PPD-PLC was characterized by differential scanning calorimetric, powder X-ray diffraction, Fourier-transformed infrared spectroscopy and nuclear magnetic resonance analyses associated with molecular docking calculation. The equilibrium solubility of PPD-PLC in water and n-octanol increased 6.53- and 1.53-times, respectively. Afterwards, using PPD-PLC as the intermediate, the PPD-PLC-loaded dry suspension (PPD-PLC-SU) was prepared with our previous method. In vitro evaluations were conducted on PPD-PLC and PPD-PLC-SU, including dissolution behaviors and stability properties under different conditions. Results of in vitro dissolution behavior revealed the improved dissolution extents and rates of PPD-PLC and PPD-PLC-SU (p < 0.05). Results of the formulation stability investigation also exposed the better stability of PPD-PLC-SU compared with free PPD. Therefore, phospholipid complex technology is a useful formulation strategy for BCS II drugs, as it could effectively improve their hydrophilicity and lipophilicity.

Fast Simultaneous Determination of 13 Nucleosides and Nucleobases in Cordyceps sinensis by UHPLC-ESI-MS/MS.

A reliable ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) method for the fast simultaneous determination of 13 nucleosides and nucleobases in Cordyceps sinensis (C. sinensis) with 2-chloroadenosine as internal standard was developed and validated. Samples were ultrasonically extracted in an ice bath thrice, and the optimum analyte separation was performed on an ACQUITY UPLC(TM) HSS C18 column (100 mm × 2.1 mm, 1.8 µm) with gradient elution. All targeted analytes were separated in 5.5 min. Furthermore, all calibration curves showed good linear regression (r > 0.9970) within the test ranges, and the limits of quantitation and detection of the 13 analytes were less than 150 and 75 ng/mL, respectively. The relative standard deviations (RSDs) of intra- and inter-day precisions were <6.23%. Recoveries of the quantified analytes ranged within 85.3%-117.3%, with RSD < 6.18%. The developed UHPLC-ESI-MS/MS method was successfully applied to determine nucleosides and nucleobases in 11 batches of C. sinensis samples from different regions in China. The range for the total content in the analyzed samples was 1329-2057 µg/g.

Authentication of Bulbus Fritillariae Cirrhosae by RAPD-derived DNA markers.

Bulbus Fritillariae is the most commonly used antitussive herb in China. Eleven species of Fritillaria are recorded as Bulbus Fritillariae in the Chinese Pharmacopoeia. Bulbus Fritillariae Cirrhosae is a group of six Fritillaria species with higher efficiency and lower toxicity derived mainly from wild sources. Because of their higher market price, five other Fritillaria species are often sold deceptively as Bulbus Fritillariae Cirrhosae in the herbal market. To ensure the efficacy and safety of medicinal herbs, the authentication of botanical resources is the first step in quality control. Here, a DNA based identification method was developed to authenticate the commercial sources of Bulbus Fritillariae Cirrhosae. A putative DNA marker (0.65 kb) specific for Bulbus Fritillariae Cirrhosae was identified using the Random Amplified Polymorphic DNA (RAPD) technique. A DNA marker representing a Sequence Characterized Amplified Region (SCAR) was developed from a RAPD amplicon. The SCAR marker was successfully applied to differentiate Bulbus Fritillariae Cirrhosae from different species of Fritillaria. Additionally, the SCAR marker was also useful in identifying the commercial samples of Bulbus Fritillariae Cirrhosae. Our results indicated that the RAPD-SCAR method was rapid, accurate and applicable in identifying Bulbus Fritillariae Cirrhosae at the DNA level.

Sophoricoside, a genistein glycoside from Fructus Sophorae, promotes hair growth via activation of M4 muscarinic AChR in dermal papilla cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Alopecia, or hair loss, refers to ongoing decline of mature hair on the scalp or any other region of the body. Fructus Sophorae, a fruit from Sophora japonica L., contains various phytochemicals, e.g., sophoricoside, that exhibit a broad range of pharmacological effects. The potential functions of herbal extracts deriving from Fructus Sophorae and/or its major phytochemical, sophoricoside, in treating alopecia are probed here. AIM OF STUDY: The objective was to determine the ability of Fructus Sophorae extract and sophoricoside in promoting hair growth and it signalling mechanism. METHODS: Molecular docking studies were conducted to measure the binding affinities between sophoricoside and M4 mAChR in the allosteric binding site. The mechanism of Fructus Sophorae and sophoricoside in activating the signalling involving Wnt/ß-catenin and muscarinic AChR was evaluated by using immortalized human dermal papilla cell line (DPC), as well as their roles in promoting hair growth. The activity of pTOPflash-luciferase in transfected DPCs was used to examine the transcriptional regulation of Wnt/ß-catenin-mediated genes. RT-PCR was applied to quantify mRNA expressions of the biomarkers in DPCs responsible for hair growth. The phosphorylated protein levels of Wnt/ß-catenin and PI3K/AKT in DPC were revealed by using Western blot analysis. The culture of ex vivo mouse vibrissae hair follicle was used to evaluate the hair growth after the treatments. RESULTS: The ethanol extract of Fructus Sophorae and sophoricoside activated Wnt/ß-catenin signalling. The result of molecular docking showed a high binding affinity between sophoricoside and M4 mAChR. The effect of sophoricoside was blocked by specific inhibitor of M4 mAChR, but not by other inhibitors of mAChRs. Sophoricoside promoted hair growth in cultured ex vivo mouse vibrissae hair follicle by acting through M4 mAChR. CONCLUSION: The ethanol extract of Fructus Sophorae and sophoricoside activated Wnt/ß-catenin signalling via activation of M4 mAChR. The results suggested beneficial functions of Fructus Sophorae and sophoricoside as a potential candidate in treating alopecia.

Flavonoids from Seabuckthorn (Hippophae rhamnoides L.) restore CUMS-induced depressive disorder and regulate the gut microbiota in mice.

Seabuckthorn (Hippophae rhamnoides L.), which is enriched with flavonoids, including isorhamnetin, quercetin and kaempferol, is a representative example of "medicine food homology" targeting several diseases. Major depressive disorders seriously threaten mental health worldwide and may even lead to death. Chronic unpredictable mild stress (CUMS)-induced depressive-like symptoms in mice are usually considered as the highest similarity to the situation in humans. Herein, we determined the potential functions of the flavonoid-enriched fraction from Seabuckthorn, which was named SBF, in treating major depressive disorder in mice. In the CUMS-induced mouse model, the intake of SBF reversed their depressive behaviors and relieved the CUMS-disturbed levels of neurotrophins, neurotransmitters, stress-related hormones, and inflammation-related cytokines. Additionally, the treatment of depressive mice with SBF showed ability to regulate the gut microbiota, especially in decreasing the abundance of Lactobacillaceae, while increasing the abundance of Lachnospiraceae at the family level. The results suggest the beneficial effects of Seabuckthorn flavonoids in functioning as a health food supplement to treat major depressive disorders.

Flavonoids from Seabuckthorn (Hippophae rhamnoides L.) mimic neurotrophic functions in inducing neurite outgrowth in cultured neurons: Signaling via PI3K/Akt and ERK pathways.

BACKGROUND: Various brain disorders, including neurodegenerative diseases and major depressive disorders, threaten an increasing number of patients. Seabuckthorn, a fruit from Hippophae rhamnoides L., is an example of "medicine food homology". The fruit has enriched flavonoids that reported to have benefits in treating cognitive disorders. However, the studies on potential functions of Seabuckthorn and/or its flavonoid-enriched fraction in treating neurodegenerative disorders are limited. PURPOSE: This study aimed to determine the ability and mechanism of the flavonoid-enriched fraction of Seabuckthorn (named as SBF) in mimicking the neurotrophic functions in inducing neurite outgrowth of cultured neurons. METHODS: Cultured PC12 cell line, SH-SY5Y cell line and primary neurons (cortical and hippocampal neurons isolated from E17-19 SD rat embryos) were the employed models to evaluate SBF in inducing neurite outgrowth by comparing to the effects of NGF and BDNF. Immuno-fluorescence staining was applied to identify the morphological change during the neuronal differentiation. Luciferase assay was utilized for analyzing the transcriptional regulation of neurofilaments and cAMP/CREB-mediated gene. Western blot assay was conducted to demonstrate the expressions of neurofilaments and phosphorylated proteins. RESULTS: The application of SBF induced neuronal cell differentiation, and this differentiating activation was blocked by the inhibitors of PI3K/Akt and ERK pathways. Additionally, SBF showed synergy with neurotrophic factors in stimulating the neurite outgrowth of cultured neurons. Moreover, the major flavonoids within SBF, i.e., isorhamnetin, quercetin and kaempferol, could account for the neurotrophic activities of SBF. CONCLUSION: Seabuckthorn flavonoids mimicked neurotrophic functions in inducing neuronal cell differentiation via activating PI3K/Akt and ERK pathways. The results suggest the beneficial functions of Seabuckthorn as a potential health food supplement in treating various brain disorders, e.g., neurodegenerative diseases.

Novel phenylpropanoids and isoflavone glycoside are isolated and identified from the carob pods (Ceratonia siliqua L.).

Two new phenylpropanoids (1 and 2) and one new isoflavone glycoside (3), along with nine known compounds (4 - 12), were isolated from the pod of Ceratonia siliqua L. Their chemical structures were elucidated based on extensive spectroscopic analyses (1 D and 2 D NMR, UV, IR, and HRESIMS) and compared with the literature data. In addition, all isolated compounds were evaluated in vitro for inhibitory activity against acetylcholinesterase (AChE). Compounds 4, 5, and 12 showed inhibitory activity against acetylcholinesterase (AChE) with IC50 values ranging from 15.0 to 50.2 µM.

The neurotrophic activities of brain-derived neurotrophic factor are potentiated by binding with apigenin, a common flavone in vegetables, in stimulating the receptor signaling.

AIMS: We aimed to identify the neurotrophic activities of apigenin (4',5,7-trihydroxyflavone) via its coordination with brain-derived neurotrophic factor (BNDF) and an elevated signaling of tyrosine kinase receptor B (Trk B receptor). METHODS: The direct binding of apigenin to BDNF was validated by ultrafiltration and biacore assay. Neurogenesis, triggered by apigenin and/or BDNF, was determined in cultured SH-SY5Y cells and rat cortical neurons. The amyloid-beta (Aß)25-35 -induced cellular stress was revealed by propidium iodide staining, mitochondrial membrane potential, bioenergetic analysis, and formation of reactive oxygen species levels. Activation of Trk B signaling was tested by western blotting. RESULTS: Apigenin and BDNF synergistically maintained the cell viability and promoted neurite outgrowth of cultured neurons. In addition, the BDNF-induced neurogenesis of cultured neurons was markedly potentiated by applied apigenin, including the induced expressions of neurofilaments, PSD-95 and synaptotagmin. Moreover, the synergy of apigenin and BDNF alleviated the (Aß)25-35 -induced cytotoxicity and mitochondrial dysfunction. The synergy could be accounted by phosphorylation of Trk B receptor, and which was fully blocked by a Trk inhibitor K252a. CONCLUSION: Apigenin potentiates the neurotrophic activities of BDNF through direct binding, which may serve as a possible treatment for its curative efficiency in neurodegenerative diseases and depression.

The ethanolic extract of peanut shell attenuates the depressive-like behaviors of mice through modulation of inflammation and gut microbiota.

Peanut shell is an agricultural byproduct being wasted on a large scale, which is in urgent need to be recycled. To fully utilize its pharmacological ingredients, e.g. luteolin, eriodyctiol, and 5,7-dihydroxychromone, we evaluated the curative effect of ethanol extract deriving from peanut shell (PSE) in treating chronic unpredictable mild stress (CUMS)-induced depressive mice. The chronic stress lasted for 10 weeks, and PSE at 100-900 mg/kg/day was gavaged to mice in the last 2 weeks of modeling. The depressive behaviors were assessed by analyses of sucrose preference, tail suspension, and forced swimming. The brain injury was demonstrated by Hematoxylin and Eosin (H&E), Nissl body, and TdT-mediated dUTP nick end labeling (TUNEL) stainings in the mouse hippocampus. Biochemical indicators were analyzed, including levels of neurotrophic factors, neurotransmitters, stress hormones, and inflammatory mediators. The feces were collected for the 16S rDNA sequencing of gut microbiome. Administration of PSE improved the sucrose water consumption of depressive mice, while it decreased the immobile time in tail suspension and forced swimming tests. Meanwhile, the anti-depressive effect of PSE was supported by ameliorated histochemical staining, increased levels of neurotrophic factors and neurotransmitters, as well as down-regulated stress hormones. Furthermore, the treatment of PSE was able to mitigate the levels of inflammatory cytokines in brain, serum, and small intestine. Besides, the tight junction proteins, e.g., occludin and ZO-1, of gut showed elevated expressions, which coincided with the elevated abundance and diversity of gut microbiota upon PSE treatment. This study validated the therapeutic efficacy of PSE in fighting against depression, as well as its modulatory action on inflammation and gut microbiota, which promoted the recycling of this agricultural waste to be health supplements of added value.