Fractionation of polyphenolic extract from walnut male flowers (Juglans regia L.) and evaluation of biological activity towards cancer cells

BackgroundThe genus Artemisia of the Asteraceae family has different species that are used in the treatment of a wide range of diseases, including cancers due to the presence of valuable compounds and important medicinal properties. Various studies on the anti-tumor effect of different species of Artemisia have proven the cytotoxic properties of these plants in cancer treatment, and several anti-cancer compounds of this genus have been purified.ObjectiveThe objective of this study was to investigate the cytotoxicity and related mortality mechanisms of Artemisia marschalliana essential oil and extracts.MethodsThe essential oil and various extracts of Artemisia marschalliana were elicited using a Soxhlet extractor. Anti-cancer to anti-proliferative activity as MTT assay is measuring cancerous and non-cancerous cell viability. In the next step, the strongest extract fractions were obtained by using the vacuum liquid chromatography method. Flow cytometry was applied to identify the mechanism of cell death, and a Real-time polymerase chain reaction test of apoptosis genes, which encode apoptosis-regulating proteins, was measured to confirm the flow cytometry results.ResultsThe strongest extract belonged to dichloromethane extract 60% fraction of the extract on breast cancer cells and 80% fraction on liposarcoma cancer cells showed the most cytotoxicity within 48 h, while, the fractions did not notable cytotoxicity of non-cancerous cells cell. Flow cytometry analysis illustrated the mentioned extract and its fractions kill cancer cell lines through the apoptosis mechanism. Our findings confirmed the flow cytometry results. In addition, the essential oil of Artemisia marschalliana showed a considerable cytotoxic property.ConclusionDichloromethane extract of Artemisia marschalliana shoot and its 60 and 80% fraction selectively inhibited the growth of cancer cells by inducing the apoptosis mechanism. Regarding obtained results, 60 and 80% fractions of dichloromethane extract can be a good candidate for future studies in the field of identification and separation of pure cytotoxic compounds.

BackgroundJuglans regia has been found to exhibit significant anticancer activity against various human cancer cell lines. This study was undertaken to isolate the active chemical constituent (Juglone) and to investigate its cytotoxic activity along with its various analogs against different human cancer cell lines.MethodsIsolation of juglone, a napthoquinone, from the chloroform extract of the root part of Juglans regia was executed by flash chromatography using silica gel as stationary phase. The isolated Juglone was used as starting material for the further synthesis of a novel series of triazolyl analogs using click chemistry approach to investigate their cytotoxic potential against different human cancer cell lines using 3-(4,5-Dimethylthiazol-yl)-diphenyl tetrazoliumbromide (MTT) assay.ResultsThe different extracts of Juglans regia and the isolated compound (juglone) exhibited satisfactory cytotoxic activity against a panel of eight different human cancer cell lines namely, prostate colon (Colo-205 and HCT-116), breast (T47D), prostate (PC-3 and DU-145), skin (A-431) and lung (NCI-H322 and A549). Interestingly, all the synthesised analogs displayed enhanced and selective cytotoxic activity against lung cancer cell lines only. Of the synthesized derivatives, 15a and 16a displayed the best activity with IC50 of 4.72 and 4.67 μM against A549 cells. Both these derivatives exhibited superior potency to BEZ-235 against both the lung cancer cell lines. So far as the structural aspects are concerned, electron withdrawing substituents at the ortho position of R moiety of the triazolyl analogs seem to be essential for attaining better activity.ConclusionThe present study demonstrates the selective and enhanced cytotoxic activity of the triazolyl analogs of juglone against NCI-H322 and A549 human lung cancer cell lines. Some derivatives exhibited superior potency to BEZ-235, a commercially available anticancer agent.

Juglans regia L. is an economically important crop cultivated worldwide for its high quality and quantity of wood and nuts. Phenylalanine ammonia-lyase (PAL) is the first enzyme in the phenylpropanoid pathway that plays a critical role in plant growth, development, and adaptation, but there have been few reports of the PAL gene family in common walnut. Here, we report a genome-wide study of J. regia PAL genes and analyze their phylogeny, duplication, microRNA, and transcriptional expression. A total of 12 PAL genes were identified in the common walnut and clustered into two subfamilies based on phylogenetic analysis. These common walnut PALs are distributed on eight different pseudo-chromosomes. Seven of the 12 PALs (JrPAL2-3, JrPAL4-2, JrPAL2-1, JrPAL4-1, JrPAL8, JrPAL9, and JrPAL6) were specific found in J. regia, and JrPAL3, JrPAL5, JrPAL1-2, JrPAL7, and JrPAL2-2 were found to be closely associated with the woody plant Populus trichocarpa. Additionally, the expression patterns of JrPAL3, JrPAL7, JrPAL9, and JrPAL2-1 showed that they had high expression in female and male flowers. The miRNA ath-miR830-5p regulates two genes, JrPAL5 and JrPAL1, such that they have low expression in the male and female flowers of the common walnut. Our research provides useful information for further research into the function of PAL genes in common walnut and Juglans.

The common walnut (or Persian walnut), Juglans regia L., is an economically important temperate tree species valued for both its edible nut and high-quality wood. F-box gene family members are involved in plant development, which includes regulating plant development, reproduction, cellular protein degradation, response to biotic and abiotic stresses, and flowering. However, in common walnut (J. regia), there are no reports about the F-box gene family. Here, we report a genome-wide identification of J. regia F-box genes and analyze their phylogeny, duplication, microRNA, pathway, and transcriptional expression profile. In this study, 74 F-box genes were identified and clustered into three groups based on phylogenetic analysis and eight subfamilies based on special domains in common walnut. These common walnut F-box genes are distributed on 31 different pseudo-chromosomes. The gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and microRNA profiles showed that the F-box gene family might play a critical role in the flowering of common walnut. The expressions were significantly higher in female flowers and male flowers compared with leaf and hull tissues at a transcriptome level. The results revealed that the expressions of the F-box gene in female flowers were positively correlated with male flowers, but there was no correlation between any other tissue combinations in common walnut. Our results provided insight into the general characteristics of the F-box genes in common walnut.

Context: Cancer chemopreventive action of walnut [Juglans regia L. (Juglandaceae)] has been explored.Objective: This study evaluated antiproliferative and antioxidant activities of walnut.Materials and methods: Various fractions of walnut extract have been screened for antiproliferative activity against human cancer cell lines using the MTT assay. All these fractions have also been evaluated for total phenolic content, antioxidant activity, and reducing power capacity.Results and discussion: Chloroform and ethyl acetate fractions exhibited a high level of antiproliferation against HepG-2, liver cancer cell line (IC50 = 9 and 15 µg/mL, respectively).Conclusion: Exhibiting high phenolic content, antioxidant activity, and potent antiproliferative activity, walnut may act as a cancer chemopreventive agent.

Purpose: To elucidate the anticancer activity of Juglans regia leaf extract and its effect on cell cycle analysis, apoptosis and cancer cell morphology.Methods: Hexane extract of the leaves of Juglans regia was prepared by hot extraction. The anticancer activity of Juglans regia extract against human prostate cancer (PC3) cells was evaluated by MTT assay. Flow cytometry, using propidium iodide as a staining agent, was used to study the effect of the extract on cell cycle phase distribution. Apoptosis induced by the extract was evaluated by Annexin V binding assay using flow cytometer. Alterations in cell morphology following apoptosis were studied by inverted phase contrast microscope.Results: The extract of Juglans regia exhibited a potent and dose-dependent anti-proliferative activity against human prostate cancer cells in vitro. The extract also induced significant apoptosis in these PC3 cancer cells as revealed by annexin V binding assay as well as inverted phase contrast microscopy. It triggered a significant formation of apoptotic bodies after treatment with varying concentrations of the extract. Within 48 h of incubation, approximately 9.5, 15.5 and 26.3 % of the cells underwent early apoptosis after treatment with 5, 50 and 100 μg/mL of the extract, respectively. Similarly, 5.2, 11.2 and 18.9 % of the cells underwent late apoptosis after treatment with 5, 50 and 100 μg/mL of the extract, respectively. Treatment with different concentrations of the extract for 48 h induced an increase in the population of cells in the sub-G1 phase and a slight decrease in the G2/M phase.Conclusion: The hexane extract of Juglans regia inhibits growth of human prostate cancer cells by inducing apoptosis with concomitant alterations in cell cycle phase distribution.Keywords: Prostate cancer, Juglans regia, Apoptosis, Flow cytometry, Cell cycle phase, Sub-G1 phase

Assessment of the in-vitro toxicity and in-vivo therapeutic capabilities of Juglans regia on human prostate cancer and prostatic hyperplasia in rats

Juglans regia is a medicinal plant widely used in traditional medicine and it. It is applied for healing a numerous ailment such us diabetes, digestive problems, hypertension, and gout diseases. This review aims to highlight the importance of Juglans regia as source of nutritional and bioactive compounds. Mechanisms of antimicrobial, antioxidant, anti-inflammatory, antidiabetic, and anticancer activities of Juglans regia leaves, fruit, septum kernels, flowers, bark, and husk have been discussed. Juglans regia fruits have an important nutritive value allowed by their richness in lipids and proteins. Medicinal properties of Juglans regia are certainly due to its bioactive compounds. Indeed, Juglans regia leaves, husks, flowers, bark, and roots contain several secondary metabolites including polyphenols, flavonoids, and glycosides. Pharmacological investigations reported by the literature demonstrated that Juglans regia exhibit different biological activities such as antioxidant, anti-inflammatory, anti-diabetic, antitumoral, antifungal, and antibacterial effects, in vitro, in vivo and in clinical trials. Mechanisms of bioactive compound action include ROS neutralization, proinflammatory mediators and enzyme inhibition, insulin levels increase, regulation of apoptosis, and cell cycle disruption. Biological properties were remarkably correlated with bioactive compounds containing in Juglans regia. Therefore, with its important nutritive values and its bioactive compounds, Juglans regia could be considered as a relevant functional food.

Context: Currently, natural products have been shown to present interesting biological and pharmacological activities and are used as chemotherapeutic agents. Plants have historically been used in treating cancer and are recognized for their ability to produce secondary metabolites. Juglans regia L. (Juglandaceae) has medicinal applications to treat a wide range of diseases such as cancer.Objective: The current study was designed to evaluate the antiproliferative activity of total extract as well as several fractions from the leaves of J. regia.The total phenolics, flavonoids, and condensed tannins content of these extracts were also determined to obtain further information on the correlation between the contents of phenolic compounds and antiproliferative effects as well as the leaf developmental stages.Materials and methods: Antiproliferative activity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometry methods against human oral cancer, breast adenocarcinoma and colon adenocarcinoma cell lines. The total phenolics, flavonoids, and condensed tannins were determined by Folin-Ciocalteu, aluminum chloride and butanol-HCl colorimetric methods.Results: Our present study has shown that chloroform fraction has the lowest IC50 values (0.36–0.81 mg/mL) and also induces cell cycle arrest (G0\\G1 phase) after a 24 h treatment. The colorimetric methods showed the highest amount of total phenolics, flavonoids, and condensed tannins in the methanol fraction (120.28 ± 2.32, 59.44 ± 0.87, 227.00 ± 4.91 mg/g of dry weight of extract).Discussion and conclusion: The results obtained herein indicate that walnut chloroform fraction may contain effective compounds which can be used as a chemotherapeutic agent.

Key message NAC transcription factors may play important roles in the biological processes in Persian walnut. A total of 102 JrNAC s were identified in Persian walnut. The conserved domains, transcriptome profile, expression analysis, and interaction network suggest that JrNAC1-4 plays potential roles in Persian walnut flowering development. Context NACs are plant-specific transcription factors that participate in various plant developmental processes such as flowering, plant growth regulation, and development. We identified and analyzed the evolution and expression profiles of NAC genes in Juglans regia. Aims The main objectives were to identify the NAC transcription factors and verify the expression levels in different tissues and female flowers in developmental stages in Persian walnut. Methods We identified NAC transcription factors in J. regia based on the genome-wide analysis. We analyzed the phylogenetic relationships, conserved domain, chromosome location, gene structure, and gene collinearity of JrNACs. We also verified the JrNAC expression levels based on transcriptome analysis and qRT-PCR. Results We identified 102 NAC genes in J. regia and divided them into ten subfamilies. A total of 30 pairs of JrNAC genes were expanded by whole-genome duplications (WGDs) and one pair of genes (JrNAC2-10 and JrNAC9-8) as a tandem duplication in Persian walnut. Collinearity analysis results indicate that a large number of syntenic relationship events existed between J. regia and Populus trichocarpa. We found that JrNAC1-4 and JrNAC2-6 were expressed significantly higher in female flowers based on both transcriptome and qPCR analysis. We further identified that JrNAC2-9 and JrNAC9-6 were highly expressed at the end period of flowering stages. Conclusion A total of 102 JrNACs were identified in the Persian walnut genome. These genes were conserved in plants for collinearity analysis, which was performed within the genome and other genomes (P. trichocarpa, Olea europaea, and Quercus robur). A total of 24 NAC transcription factors were highly expressed in female and male flowers, and these transcription factors play a role in J. regia flowering.

Cytotoxicity assay of biosynthesis gold nanoparticles mediated by walnut (Juglans regia) green husk extract

Walnut (Juglans regia L.) is considered to be a 'superfood' for its multiple protective actions on human health. Walnut extracts have proven antitumor activity in different cancer cell lines. However, the efficacy of septum extract against glioblastoma has still not been investigated. Glioblastoma is the most difficult type of brain cancer to treat. The standard therapy, based on temozolomide, causes several side effects, including neutropenia and lymphocytopenia, which often favor the onset of opportunistic infections. In the present study, the chemical profile of the Sicilian walnut septum ethanolic extract was analyzed using high-performance liquid chromatography (HPLC)-diode array detection and HPLC-electrospray ionization tandem mass spectrometry. The potential cytostatic activity of the extract against the human A172 glioblastoma cell line was investigated and the results showed that the extract could decrease cancer cell proliferation and migration. Using cytofluorimetric analyses and caspase-3 assays, the pro-apoptotic action of walnut extract was demonstrated. Furthermore, the evaluation of the antibacterial activity high-lighted the efficacy of the extract in reducing Gram-positive and Gram-negative bacterial growth, most of which were resistant to the antibiotic, ciprofloxacin. Finally, Prediction of Activity Spectra for Substances analysis showed the predicted antitumor and antibacterial activity of HPLC detected compounds. The promising results could provide novel perspective in the field of chemotherapeutic co-adjuvants.

This study evaluated the extraction methods for optimizing lipid and phenolic yields from Juglans regia L. (walnut) green husk and assessed the extracts’ cytotoxic effects on cancer cells (liver cancer cell line, HepG2) and normal human cells (HUVEC). Among the various lipid extraction techniques, the Bligh and Dyer method was most efficient (3.4% dry weight), followed by Soxhlet extraction with n-hexane (2.4%), Folch (2.3%), and sonication (1.7%). For phenolic extraction, sonication with 70% ethanol achieved the highest yield (18.8%), with Soxhlet extraction close behind (17.7%). Total phenolic content varied, with reflux extraction yielding 0.07 ± 0.05 mg GAE/g and hexane Soxhlet extraction at 0.023 ± 0.03 mg GAE/g. Cytotoxicity assays showed dose-dependent inhibitory effects on HepG2 cells for both 70% ethanol maceration and Bligh and Dyer extracts, with IC50 values of 277.3 and 237.5 μg/mL, respectively. The Bligh and Dyer extract had an IC50 of 185.3 μg/mL on HUVEC cells. Microscopy and DAPI staining indicated apoptotic changes in HepG2 cells, while DCFH-DA staining revealed elevated ROS levels, suggesting oxidative stress. Gas Chromatography-Mass Spectrometry identified unique compounds with potential bioactivity, and molecular docking highlighted 5-hydroxy-1-tetralone as a strong binder with protein 3TZM (−8.0 kcal/mol). This study underscores the influence of extraction methods on yield and bioactivity.

Current research in drug discovery from medicinal plants involves the identification of safe and inexpensive lead molecule towards disease tagets. Cancer is a second major national burden leading to cause death. Reducing the nation's cancer burden is a great challenge to win the “War of cancer”. During the last decades, numerous novel compounds have been found from marine organisms with interesting pharmaceutical activities. Hence, a novel brown algae, Sargassum ilicifolium (Turner) C.Aardh belonging to Sargassaceae family has selected and the study was aimed to investigate the in vitro anti-proliferative activity of Ethanolic extract and Chloroform fraction of Sargassum ilicifolium (Turner) C.Agardh. against Colon Cancer (HT-29) and Lung Cancer (A549) cell lines. Extraction of seaweed using 70% ethanol and fractionated with various solvents. Based on phytochemical screening, chloroform fraction was selected, then it was subjected to GC-MS analysis. The 70% ethanolic exract and chloroform fraction were selected to in vitro MTT cell line assay using Colon cancer cell line (HT-29) and lung Cancer cell line (A549). The GC-MS analysis report confirmed the presence of 21 compounds and in the the invitro assay, IC 50 value showed the effective anti-proliferative activity role against colon and lung cancer cell lines. The results of the present study proved the anti proliferative effect of Sargassum ilicifolium in the area of cancer. Further, the active biomolecule in the fraction has to be isolated , charecterized the formulated product subjected to in vivo study to strengthen the anticancer activity. Keywords: Antiproliferative effect, Colon cancer, lung cancer, Sargassum ilicifolium , GC-MS analysis.

The extraction of bioactive compounds present in walnut (Juglans regia L.) male flowers (WMFs) was performed based on an experimental design using ultrasonic-assisted extraction. Solvent nature, extraction time, and water content were selected as experimental variables, and phenolic, flavonoidic, and condensed tannins contents and antioxidant properties were evaluated. Acetone was the solvent with the highest extraction performance, with the extracts obtained using this solvent displaying an increased concentration of bioactive compounds and increased antioxidant activities. For several extracts with high bioactive content, individual polyphenolic and tocopherolic compounds were evaluated by means of LC-MS and LC-MS/MS. The best extraction conditions for polyphenolic (2.86 mg gallic acid equivalents/g WMF) and tocopherolic compounds (29.4 µg/g WMF) were acetone with 40% water content (N20) and acetone with 20% water content (N15), respectively. Although the total tocopherol concentrations were lower than in other Juglans regia parts, most of the total tocopherol quantity was provided by the highly biologically active δ-tocopherol (84%). Significant quantities of quercetin (101.9 µg/g), hyperoside (2662.9 µg/g), quercitrin (405.7 µg/g), and isoquercitrin (1293.7 µg/g) were determined in WMF (N20). Both extracts inhibited the enzymatic activity of α-glucosidase and tyrosinase; however, an increased inhibition was observed for N20, the extract with the higher polyphenolic content. Conversely, N15 had higher anticancerous activity on the cell lines used, with a moderate selectivity towards the cancerous phenotype being observed for both extracts. At non-cytotoxic concentrations, both extracts displayed good antioxidant activities in cellular cultures, decreasing basal and H2O2-induced oxidative stress. This is the first characterization of both hydrophilic and lipophilic phytochemicals in WMF extracts. The outcomes of our study reveal that walnut male flowers have strong biological activities, thus justifying further research to demonstrate their usefulness in the food, pharmaceutical, and/or cosmetic industries.