Juglone Research Articles

An integrated approach of homogenization-ultrasound-assisted extraction followed by high-speed countercurrent chromatography for rapidly preparative separation naphthoquinones and diarylheptanes from Juglands mandshurica Maxim. exocarp.

This research investigated the effectiveness of an integrated method for the extraction and separation of naphthoquinones and diarylheptanes from exocarp of Juglands mandshurica Maxim. (namely, green walnut husks). The target compounds were obtained by ultra-turrax homogenization (UTH) coupled with ultrasound-assisted extraction (UAE) technology followed by high-speed countercurrent chromatography (HSCCC). The UTH-UAE extraction method achieved higher efficiency with 2.49- and 2.36-fold to those by UAE, and 1.39- and 1.34-fold to those by UTH in a short time. HSCCC was adopted for further separation and purification; six target compounds, namely, regiolone (RE), juglone (JU), myricatomento-genin (MG), galleon (GA), 2-oxatrycyclo[13.2.2.13,7]eicosa-3,5,7(20),15,17,18-hexaen-10-16-diol (OE), and juglanin A (JA), were separated with more than 95.37% purities and more than 84.71% final recovery rates, respectively. In this study, the integrated strategy of extraction and separation could get high purity compounds quickly, which would provide time and solvent saved method for the natural products separation from plants.

The protective role of 5-hydroxy-1,4-naphthoquinone against the harmful effects of 50 Hz electric field in rat lung tissue

ABSTRACT There is strong scientific evidence that the electric field is harmful to life. Exposure to an electric field (EF) can cause lung toxicity and respiratory disorders. In addition, the electric field has been shown to cause tissue damage through inflammation and apoptosis. Juglone (JUG) is one of the powerful antioxidants with anti-apoptotic and anti-inflammatory, various pharmacological properties in the biological system. In this study, we evaluated the efficacy of JUG against the potential adverse effects of electric field on the lung. Twenty-four Wistar albino rats were randomly divided into three groups; control group (Cont), EF group, and EF exposure+JUG-treated group (EJUG). After routine histological procedures, sections stained with hematoxylin-eosin (H&E) showed significant changes in lung tissues in the EF group compared to the Cont group. Significant protective effects were observed in the building volumes and histopathology in the EJUG group. Our immunohistochemical and gene expression results increased the expression of caspase-3 and tumor necrosis factor alpha (TNF-α) in the EF group (p < 0.05). Juglon increased cytokine signal suppressor (SOCS) expression (p < 0.001). These findings were consistent with the antioxidant effect of JUG treatment. We reasoned that exposure to EF damaged rat lung tissues and administration of JUG alleviated the complications caused by 50 Hz EF.

Juglone and 1,4-Naphthoquinone-Promising Nematicides for Sustainable Control of the Root Knot Nematode Meloidogyne luci.

The scarce availability of efficient and eco-friendly nematicides to control root-knot nematodes (RKN), Meloidogyne spp., has encouraged research toward the development of bionematicides. Naphthoquinones, juglone (JUG) and 1,4-naphthoquinone (1,4-NTQ), are being explored as alternatives to synthetic nematicides to control RKN. This study expands the knowledge on the effects of these natural compounds toward M. luci life cycle (mortality, hatching, penetration, reproduction). M. luci second-stage juveniles (J2)/eggs were exposed to each compound (250, 150, 100, 50, and 20 ppm) to monitor nematode mortality and hatching during 72 h and 15 days, respectively. Tomato seedlings were then inoculated with 200 J2, which had been exposed to JUG/1,4-NTQ for 3 days. The number of nematodes inside the roots was determined at 3 days after inoculation, and the final population density was assessed at 45 days after inoculation. Moreover, the potential mode of action of JUG/1,4-NTQ was investigated for the first time on RKN, through the assessment of reactive oxygen species (ROS) generation, acetylcholinesterase (AChE) in vitro inhibitory activity and expression analysis of ache and glutathione-S-transferase (gst) genes. 1,4-NTQ was the most active compound, causing ≥50% J2 mortality at 250 ppm, within 24 h. At 20 and 50 ppm, hatching was reduced by ≈50% for both compounds. JUG showed a greater effect on M. luci penetration and reproduction, decreasing infection by ≈80% (50 ppm) on tomato plants. However, 1,4-NTQ-induced generation of ROS and nematode vacuolization was observed. Our study confirms that JUG/1,4-NTQ are promising nematicidal compounds, and new knowledge on their physiological impacts on Meloidogyne was provided to open new avenues for the development of innovative sustainable nematicides.

Preparation and Antiproliferative Activity Evaluation of Juglone-Loaded BSA Nanoparticles.

Purpose: Today, the discovery of novel and effective chemotherapeutic compounds is the main challenge in cancer therapy. In recent years, the anti-tumoral activity of natural naphthoquinone juglone (JUG), present in different parts of walnut trees, has received considerable interest. The purpose of the current study was to prepare and evaluate the in vitro antiproliferative activity of JUG-loaded bovine serum albumin nanoparticles (JUG-BSA NPs). Methods: BSA NPs and JUG-BSA NPs were prepared using the desolvation technique. The NPs were characterized for their particle size (PS), zeta potential (ZP), drug loading (DL) capacity and encapsulation efficiency (EE). The anti-proliferative activity of JUG-BSA NPs was evaluated on A431 and HT29 cancer cell lines using cellular uptake and MTT assays. Results: The PS and ZP values of JUG-BSA NPs were 85 ± 6.55 nm and -29.6 mV, respectively. The DL capacity and EE were 3.7% to 5% and 50.4% to 94.6%, respectively. The cytotoxicity of JUG-BSA NPs was significantly less on both cultured A431 and HT29 cells at the studied concentrations when compared to free JUG. However, the effect was not very substantial, particularly at high levels. Conclusion: In conclusion, BSA NPs can be used as a suitable and safe carrier for the delivery of JUG, a cytotoxic hydrophobic natural compound.

Juglone-loaded metal-organic frameworks for H2O2 self-modulating enhancing chemodynamic therapy against prostate cancer

As an innovative treatment strategy for cancer treatment, Fenton reaction-mediated Chemodynamic therapy (CDT) dependent on the conversion of endogenous hydrogen peroxide (H2O2) into cytotoxic hydroxyl radical (•OH) to destroy tumor cells has attracted increasing interest. However, the insufficient supply of H2O2 has greatly hindered the anticancer efficacy of CDT in the tumor microenvironment, and this inherent disadvantage has rarely attracted attention. In order to develop a strategy with high-efficiency H2O2 self-supply ability to enhance the CDT efficacy, we constructed a Fe-based metal–organic framework (MOF) as a carrier for the drug Juglone (JUG), which can not only increase the intracellular H2O2 concentration, but also serve as an inhibitor of the peptidyl-prolyl cis–trans isomerase NIMA-interacting 1 (Pin1), which is a key regulator for multiple physiological processes in cancer cells. Once internalized by tumor cells, the acidic environment of the tumor promotes the release of JUG for drug therapy, and the released Fe ions trigger the Fenton reaction to produce •OH from sufficient H2O2, thereby achieving significant antitumor effects. Furthermore, the coating of cancer cell membranes onto the JUG-loaded MOF could enable it with homologous tumor targeting ability. Both in vitro and in vivo results show that the use of drugs can activate the cascade to provide sufficient H2O2 with outstanding anti-tumor efficacy, which paves a new avenue for the realization of drug/CDT synergistic therapy.

Juglone Suppresses Inflammation and Oxidative Stress in Colitis Mice.

Juglone (JUG), a natural product found in walnut trees and other plants, shows potent antioxidant, antimicrobial, and immunoregulatory activities. However, it remains unknown whether JUG can alleviate ulcerative colitis. This study aims to explore the effect of JUG on dextran sulfate sodium (DSS)-induced colitis in mice. The mice were randomly assigned into three groups: the vehicle group, the DSS group, and the JUG group. The experiments lasted for 17 days; during the experiment, all mice received dimethyl sulfoxide (DMSO, 0.03% v/v)-containing water, while the mice in the JUG group received DMSO-containing water supplemented with JUG (0.04 w/v). Colitis was induced by administering DSS (3% w/v) orally for 10 consecutive days. The results showed that the JUG treatment significantly ameliorated body weight loss and disease activity index and improved the survival probability, colon length, and tissue damage. JUG reversed the DSS-induced up-regulation of proinflammatory cytokines, including interleukin (IL)-6, 12, 21, and 23, and tumor necrosis factor-alpha, and anti-inflammatory cytokines, such as IL-10 and transforming growth factor-beta, in the serum of the colitis mice. Additionally, the activation of mitochondrial uncoupling protein 2 and phospho-Nuclear Factor-kappa B p65 and the inhibition of the kelch-like ECH-associated protein 1 and NF-E2-related factor 2 induced by DSS were also reversed under JUG administration. Although the JUG group possessed a similar microbial community structure as the DSS group, JUG enriched potential beneficial microbes such as Lachnospiraceae_NK4A136_group but not pathogens such as Escherichia Shigella, which was dominative in DSS group, at the genus level. In conclusion, our results demonstrated that JUG could be a promising agent for UC prevention to regulate inflammatory cytokines and oxidative stress.

Crossover-free hydroxy-substituted quinone anolyte and potassium ferrocyanide catholyte for aqueous alkaline organic redox flow battery

Among the aqueous organic redox flow batteries, quinone derivatives based redox molecules are commonly explored, due to their facile electron transfer ability. In this work, we have screened three redox active quinones (anthrarufin (AN), naphthazarin (NN), and juglone (JN)) for anolyte and cells were fabricated against K4Fe(CN)6 catholyte. The anolyte with more number of aromatic rings, i.e., AN is capable of better delocalizing the electrons and show more negative redox potential, thereby enhancing the operating voltage of the battery. AN exhibited a single step two-electron reduction process and delivered a capacity of 1.32 Ah L−1 at 20 mA cm-2 current density.

ROS -mediated p53 activation by juglone enhances apoptosis and autophagy in vivo and in vitro

Juglone (JG) exhibits a broad-spectrum of cytotoxicity against some cancer cells. However, its molecular mechanisms have not been investigated well. Here, the present results showed that JG significantly inhibited tumor growth in vivo. CCK-8 assays, flow cytometric analysis, western blotting and immunohistochemistry revealed that JG effectively inhibited cell proliferation and induced apoptosis through extrinsic pathways. We also observed that JG treatment induced autophagy flux via activiting the AMPK-mTOR signaling pathway. In addition, we found that JG enhanced p53 activation by increasing down-regulation of ubiquitin-mediated degradation. Inhibition of p53 by siRNA attenuated JG-induced cell death and autophagy. Moreover, JG enhanced the generation of hydrogen peroxide (H2O2) and superoxide anion radical (O2• −). Further experiments proved that H2O2 was a major factor since the H2O2 scavenger catalase (CAT) reduced both autophagy and cell death to a greater extent than the O2• − scavenger SOD. Overall, our results illustrated that JG caused apoptosis and autophagy via activating the ROS-mediated p53 pathway in human liver cancer cells in vitro and in vivo, which provided basic scientific evidence that JG serves as a potential anti-cancer agent.

Binding Interaction of Juglone with Lysozyme: Spectroscopic Studies Aided by In Silico Calculations

Binding interactions between the drug Juglone (JUG) and Lysozyme (LYZ) have been explored in details from spectroscopic studies aided by in silico calculations. UV–Vis, steady state and time resolved fluorescence spectroscopic studies indicate the formation of LYZ–JUG complex in the ground state. Quenching of corrected fluorescence spectra of LYZ in presence of JUG at varied concentrations in different temperature range have been estimated from Stern–Volmer (SV) plots. Time resolved fluorescence spectroscopic studies confirm the mechanism of quenching to be of static type. Binding constant associated with the LYZ–JUG complex has been estimated from Scatchard plot. The number of binding sites, thermodynamic parameters and the modes of interaction are also estimated. Synchronous fluorescence spectra monitored at two discrete wavelength windows confirm the prominent role of Tryptophan residues towards quenching of fluorescence in LYZ. The circular dichroism (CD) spectra signify alterations in the population of α–helical content within the secondary structure of LYZ in presence of JUG molecules. REES of LYZ in the presence of JUG further signify definite impact of the drug JUG molecule on the Trp residues of the protein. The experimental observations are supported by in silico molecular docking and molecular dynamics simulations.

Juglone induces apoptosis and autophagy via modulation of mitogen-activated protein kinase pathways in human hepatocellular carcinoma cells

Juglone (JG), a naturally-occurring naphthoquinone of Manchurian walnut (Juglans mandshurica) was shown to inhibit proliferation in various tumor types. However, the molecular mechanisms of JG on the induction of apoptosis and autophagy in HepG2 cells have not been examined. Herein, we investigated that JG could inhibit cell proliferation by induction of G2/M phase arrest. Also, occurrence of apoptosis was closely related with loss of mitochondrial membrane potential, the changes of apoptosis-related proteins after treatment with JG. In addition, we found that JG caused autophagy, as evidenced by increased expressions of LC3-II and Beclin-1. Interestingly, inhibition of JG-induced autophagy by 3-methyladenine (3-MA) and wortmannin (WT) significantly decreased apoptosis, whereas the apoptosis inhibitor z-VAD-fmk slightly enhanced autophagy. Furthermore, the induction of autophagy and apoptosis was associated with activation of MAPK family members (p38 and JNK) and production of reactive oxygen species (ROS). Both JNK inhibitor (SP600125) and ROS scavenger (N-acetylcysteine, NAC) could attenuate JG-induced autophagy and apoptosis. However, the p38-specific inhibitor SB203580 enhanced autophagic and apoptotic death. Moreover, the ROS scavenger NAC prevented phosphorylation of both p38 and JNK. Collectively, our data revealed that JG induced G2/M phase arrest, apoptosis, and autophagy through the ROS-dependent signaling pathway.

In vitro nematicidal activity of naphthoquinones against the root-lesion nematode Pratylenchus thornei

The root-lesion nematode Pratylenchus thornei is a widely distributed and important parasite of cereals and legumes. As migratory endoparasites, P. thornei are difficult to manage because they enter and leave host roots and may remain quiescent inside roots or in soil when conditions are unfavourable for plant growth. The number of available nematicides to manage these nematodes is restricted, so new, effective and eco-friendly sustainable management strategies are needed. The effects of naphthoquinones (juglone, 1,4-naphtoquinone and plumbagin) produced by some plants species, including walnut (Juglandaceae), were assessed against P. thornei . An additional treatment of a mixture of juglone and 1,4-naphtoquinone (2:1, w/w), was included because these compounds are frequently found at these propotion in walnut extracts. Juveniles and adult nematodes were exposed to different concentrations of each naphthoquinone and nematode mortality was assessed. Juglone and 1,4-naphthoquinone (at 500 ppm) were more effective than plumbagin, and gave 100% mortality after 24 h of exposure. A synergistic effect was not detected when juglone and 1,4-naphthoquinone (2:1, w/w) were combined. Estimated lethal concentrations causing 50% P. thornei mortality (LC 50 s) (72 h exposure) were: 134.7 ppm for juglone, 161.2 ppm for 1,4-naphthoquinone, 207.6 ppm for juglone + 1,4-naphthoquinone (2:1, w/w), and 178.8 ppm for plumbagin. This study has demonstrated the nematicidal potential of these naphthoquinones against P. thornei , and has shown that walnut residues may be valuable sources for extraction of these compounds.

Juglone alleviates pneumolysin-induced human alveolar epithelial cell injury via inhibiting the hemolytic activity of pneumolysin.

Streptococcus pneumoniae (the pneumococcus) is an opportunistic pathogen responsible for several human diseases, including acute otitis media, pneumonia, sepsis and bacterial meningitis, and possesses numerous virulence factors associated with pneumococcal infection and pathogenesis. With the capacity to form pores in cholesterol-rich membranes, pneumolysin (PLY) is a key virulence factor of S. pneumoniae and causes severe tissue damage during pneumococcal infection. Juglone (JG), a natural 1,4-naphthoquinone widely found in the roots, leaves, woods and fruits of Juglandaceae walnut trees, inhibits PLY-induced hemolysis via inhibition of the oligomerization of PLY and exhibits minimal anti-S. pneumoniae activity. In addition, when human alveolar epithelial (A549) cells were co-cultured with PLY and JG, PLY-mediated cell injury was significantly alleviated. These results indicate that JG directly interacts with PLY to reduce the cytotoxicity of the toxin in human alveolar epithelial cells. Hence, JG is an effective inhibitor of PLY and protects lung cells from PLY-mediated cell injury. This study also provides the basis for the development of anti-virulence drugs for the treatment of S. pneumoniae infections.

Drug-protein binding mechanism of juglone for early pharmacokinetic profiling: Insights from ultrafiltration, multi-spectroscopic and molecular docking methods

Juglone (JL), as one of the major bioactive components present in the bark of Juglans mandshruica Maxim, exhibits versatile bioactivities, especially anti-cancer activity. To better understand the pharmacokinetic properties of juglone, the protein binding rate of juglone was determined by ultrafiltration method, and the binding affinity and mechanism between JL and human serum albumin (HSA) was investigated in vitro through multi-spectroscopic, thermodynamic, and molecular modeling methods. The binding degree of JL was measured more than 99.0% which suggested that JL had high binding ability to serum albumin. Fluorescence data showed that juglone quench the intrinsic fluorescence of HSA upon forming the JL–HSA nonfluorescent complex at 1:1 stoichiometric proportion, and the complex formation had a high affinity of 104 L·mol−1. Meanwhile, the site marker competitive experiments and the thermodynamic parameters (ΔG=-26.08 kJ·mol−1, ΔH=–16.34 kJ·mol−1, ΔS=32.69 J·mol−1·K−1) indicated that juglone could spontaneously bound to the site I (subdomain IIA) of HAS through hydrophobic and hydrogen bonding interactions. As further revealed by the synchronous fluorescence, three-dimensional fluorescence, Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy, JL could cause conformational and structural alterations of HSA. Additionally, molecular docking was employed to further define the specific binding site and the result was in accordance with the conclusion of experimental analysis. The present work provided reasonable models helping us further understand the pharmacokinetics, pharmacological and toxic effects of JL in vivo and supplied an important insight for the applications of JL in the clinical research.

Cellular responses to naphthoquinones: juglone as a case study

The effects of juglone (JG) on the endogenous growth, growth in the presence of either indoleacetic acid (IAA) or fusicoccin (FC) and on proton extrusion were studied in maize coleoptile segments. In addition, membrane potential changes were also determined at chosen JG concentrations. It was found that JG, when added to the incubation medium, inhibited endogenous growth as well as growth in the presence of either IAA or FC. Simultaneous measurements of growth and external pH indicated that inhibition of either IAA-induced growth or proton extrusion by JG was a linear function of JG concentration. Addition of JG to the control medium caused depolarization of the membrane potential (Em), value of which was dependent on JG concentration and time after its administration. Hyperpolarization of Em induced by IAA was suppressed in the presence of JG. It was also found that for coleoptile segments initially preincubated with JG, although subsequently removed, addition of IAA was not effective in the stimulation of growth and medium acidification. Taken together, these results suggest that the mechanism by which JG inhibits the IAA-induced growth of maize coleoptile segments involves inhibition of PM H+-ATPase activity.

Plant extract anti-cancer research progress

Plant extracts are kinds of important anticancer drugs.Anticancer drugs research has focused on several important plant extracts such as resveratrol,ricin,epigallocatechin-3-gallate,oridonin,juglone,quercetin,matrine,nordihydroguaiaretic acid.In vitro and in vivo studies have shown that their antitumor mechanisms including induction of apoptosis,cytotoxicity,induced redifferentiation,antiangiogesis,antiinvasion and anti-metastasis.Plant extracts have multi-link and multi-target.Part of them have been used in preclinical studies.In-depth study of the anti-tumor mechanisms of plant extracts on cancer treatment is important,and the full mechanism needs further study. Key words: Plant extracts; Antineoplastic agents, phytogenic; Apoptosis

Correlations between Xanthomonas arboricola pv. juglandis severity and endogenous juglone and phenolic acids in walnut.

Endogenous phenolic compounds in walnut fruits were correlated with the severity of walnut blight caused by Xanthomonas arboricola pv. juglandis (Xaj) assessed in the field, to determine the possible role of phenolics in resistance to the disease. Healthy fruits of the cvs Franquette, Cisco, and Sampion with different susceptibilities to infection by Xaj were sampled from diseased trees three times during growth and analysed using HPLC with a PDA detector. An identical phenolic profile, consisting of juglone and six phenolic acids (ellagic, gallic, syringic, p-coumaric, caffeic, and chlorogenic), was detected in the studied cultivars. Juglone was the most abundant, ranging between 373 mg 100 g-1 and 5,074 mg 100 g-1 DW, compared to the least abundant caffeic and p-coumaric acids, which did not exceed 10 mg 100 g-1 DW. A negative correlation between the total amount of phenolics present in the fruit tissues and blight severity was found in all cultivars, indicating the role of these compounds in the fruit-bacteria interactions. As the major phenolic characterized by the strongest seasonal fluctuations, juglone seemed to have the main and negative relation with disease development during the year. Thus, its involvement into the defence mechanism of walnut against bacterial blight is strongly suspected. The same may apply to gallic acid, considering its seasonal variations with respect to disease incidence. Additional studies including in vitro determination of the anti-bacterial activity of some phenolics, and their response to artificial inoculation with Xaj seem desirable to clarify the role of phenolic compounds in walnut resistance against this bacterium.

Juglone: A therapeutic phytochemical from Juglans regia L.

Juglans regia L. is a monoecious, heterodichogamous, deciduous tree species valued for its high-quality timber as well as its nuts. It belongs to the family Juglandaceae. Though, it is native to Central Asia, India, Nepal China and some part of Europe, it is being grown commercially in the USA, France, Italy and Spain for nut production. The nuts and plant parts of J. regia contain the aromatic secondary metabolite juglone (C10H6O3) (1, 4 – naptha quinone, 5 hydroxy – 8CI). Juglone is a well-known allelopathic agent and being used in commercial hair dye and also has therapeutic properties. Juglone is commercially extracted from the husks of walnuts, but fresh leaves of J. regia contain significant amounts of juglone (up to 0.5 / 100 g dry weight) to be a good source of the chemical. Juglone is understudy for anti-carcinogenic effects. In human intestinal neoplasia juglone checked multiplicity of tumour cells and is also a strong inhibitor of peptidyl-prolyl isomerase Pin1 which is overexpressed in several types of cancer affected cells. Juglone increases efficacy of anti cancer drugs as well. Key words: Phytochemical, medicinal, anticarcinogenic, antitumor, allelopathic.

Phenolics of green husk in mature walnut fruits.

Qualitative and quantitative determinations of phenolic compounds were carried out on green husk (mesocarp) of mature walnut fruits collected in September of five walnut (Juglans regia L.) cultivars ('Germisara', 'Jupanesti', 'Franquette', 'Vina', 'Valcor') grown in an uniform agroclimatic condition. Six compounds (ferulic acid, vanillic acid, coumaric acid, syringic acid, myricetin and juglone) were identified in all the cultivars by using reverse phase-high performance liquid chromatography (HPLC-RP).

Effects of juglone on seedling growth in intact and coatless seeds of cucumber (Cucumis sativus cv. Beith Alpha)

The main aim of this study was to test if the seed coat is a barrier to restrict juglone’s entrance into the seed using intact and coatless seeds of cucumber (Cucumis sativus cv. Beith Alpha). Elongation, fresh and dry weights of cucumber seedlings were decreased significantly by juglone in both intact and coatless seeds. Protein content in cotyledons of cucumber seedling was also decreased by juglone. The result do not prove the seed coat barrier hypothesis Because juglone’s inhibitory effect on seedling growth and protein content was not so much different in coatless seeds than was in intact seeds. However, in the present study, catechol and dopa oxidizing activities of polyphenol oxidase enzyme were increased by juglone in both seedlings. The increase in the activities of this enzyme was attributed to a reaction against to allelochemical stress produced by juglone. Key words: Cucumber, juglone, polyphenol oxidase activity, protein content, seedling growth.

Antitumor and Immunostimulating Activity of 5-Hydroxy-1,4-naphthoquinone (Juglone) O- and S-Acetylglycosides

Antitumor and Immunostimulating Activity of 5-Hydroxy-1,4-naphthoquinone (Juglone) O- and S-Acetylglycosides