Seedless black Vitis vinifera polyphenols suppress hepatocellular carcinoma in vitro and in vivo by targeting apoptosis, cancer stem cells, and proliferation.

Hepatocellular carcinoma (HCC) is an aggressive tumor and one of the most challenging cancers to treat. Here, we evaluated the in vitro and in vivo ameliorating impacts of seedless black Vitis vinifera (VV) polyphenols on HCC. Following the preparation of the VV crude extract (VVCE) from seedless VV (pulp and skin), three fractions (VVF1, VVF2, and VVF3) were prepared. The anticancer potencies of the prepared fractions, compared to 5-FU, were assessed against HepG2 and Huh7 cells. In addition, the effects of these fractions on p-dimethylaminoazobenzene-induced HCC in mice were evaluated. The predicted impacts of selected phenolic constituents of VV fractions on the activity of essential HCC-associated enzymes (NADPH oxidase "NADPH-NOX2", histone deacetylase 1 "HDAC1", and sepiapterin reductase "SepR") were analyzed using molecular docking. The results showed that VVCE and its fractions induced apoptosis and collapsed CD133+ stem cells in the studied cancer cell lines with an efficiency greater than 5-FU. VVF1 and VVF2 exhibited the most effective anticancer fractions in vitro; therefore, we evaluated their influences in mice. VVF1 and VVF2 improved liver morphology and function, induced apoptosis, and lowered the fold expression of various crucial genes that regulate cancer stem cells and other vital pathways for HCC progression. For most of the examined parameters, VVF1 and VVF2 had higher potency than 5-FU, and VVF1 showed more efficiency than VVF2. The selected phenolic compounds displayed competitive inhibitory action on NADPH-NOX2, HDAC1, and SepR. In conclusion, these findings declare that VV polyphenolic fractions, particularly VVF1, could be promising safe anti-HCC agents.

The black Vitis vinifera seed oil saponifiable fraction ameliorates hepatocellular carcinoma in vitro and in vivo via modulating apoptosis and ROS/NF-κB signaling.

To date, no total curative therapy for hepatocellular carcinoma (HCC) is available. This study aimed to evaluate the anticancer effect of black Vitis vinifera (VV) seed oil saponifiable (Sap) fraction (BSap) using five different cancer cell lines. The apoptotic and anti-inflammatory impacts of BSap on the cell line with the highest cytotoxic effect were studied. Furthermore, its therapeutic effect on p-dimethylaminoazobenzene (p-DAB)-induced HCC in mice was investigated. The phenolic and vitamin content, as well as the antiradical activities of BSap, were assessed. BSap demonstrated a greater cytotoxic effect on HepG-2 cells (lowest IC50 and highest SI values) than did the other tested cell lines. BSap showed superior anticancer efficacy to 5-FU on all examined cancer cells, particularly HepG-2 cells, by inducing apoptosis and downregulating NF-κB. In HCC-bearing mice, BSap reduced hepatic lipid peroxidation and boosted GSH levels due to its potent antiradical activities and high reducing power. In addition, it had an apoptotic effect by upregulating p53 and BAX and downregulating Bcl-2 fold expression. Moreover, BSap lowered the fold expression of various crucial HCC-related genes: CD133, ALAD1α1, COX-2, ABCG1, AKT1, Gli, Notch1, and HIF1α. Liver function markers and histopathology showed significant improvements in HCC-bearing mice after BSap administration compared to 5-FU. In silico analysis revealed that the most abundant phenolic and fatty acid ingredients of BSap exhibited competitive inhibitory effects on valuable HCC-associated enzymes (NADPH oxidase, histone deacetylase 1, and sepiapterin reductase). Thus, BSap fraction may be a promising treatment of HCC.

Major royal jelly proteins elicited suppression of SARS-CoV-2 entry and replication with halting lung injury.

For reasons of high transmissibility and virulence, Alpha (UK, B.1.1.7) and Beta (South African, B.1.351) SARS-CoV-2 variants are classified with other types as variants of concern. Here we report on the influence of royal jelly (RJ) protein fraction (PF)50 (major RJ protein 2 and its isoform X1) on the entry of these variants into the ACE2-human embryonic kidney (HEK) 293 cells using the lentiviral system. The efficiency of PF50 on SARS-CoV-2 replication (RNA-dependent RNA polymerase "RdRp" activity), as well as its impact on bleomycin-induced lung injury in vitro, were also assessed. The PF50 efficiently inhibited infection of kidney cells with the UK and S. African variant spikes of pseudotyped lentivirus particles (IC50 = 7.25 µM and 16.92 µM, respectively) and suppressed the RdRp activity (IC50 = 29.93 µM). Moreover, PF50 displayed protective and therapeutic efficacy against lung injury due to its antioxidant, anti-inflammatory, and angiotensin II blocking activities. The current findings, taken together, offer a novel perspective on PF50 as a promising agent against COVID-19.

Targeting apoptosis in MCF-7 and Ehrlich ascites carcinoma cells by saponifiable fractions from green and black Vitis vinifera seed oil.

Grape seed (GS) oil is one of the potential functional foods. For the first time, we evaluated the therapeutic effects of GS oil saponifiable (Sap)-fraction from black (BSap) and green (GSap) grapes on MCF-7 cells and Ehrlich ascites carcinoma (EAC) in mice. The fatty acid composition of BSap and GSap was determined using gas chromatography-mass spectrometry analysis. Approximately twelve distinct fatty acids were detected in BSap and eleven in GSap. BSap showed a greater cytotoxic effect on MCF-7 cells than GSap did by inducing apoptosis and reducing inflammation, while both grape fractions had superior potency to 5-FU. Furthermore, BSap massively boosted apoptosis and lowered redox potential (Eh) and CD44+ cells in EAC cells of EAC-bearing mice more than GSap, and both fractions were more efficient than 5-FU. Blood tests and liver histopathology revealed significant improvement in EAC-induced pathological alterations with these fractions. The in silico analysis implied the competitive inhibitory impacts of the most abundant fatty acid composites in BSap and GSap on cancer-metastasis-associated proteases (cathepsin B and MMP9). Also, this analysis predicted that the apoptotic action of these Sap fractions is independent of the 5'AMP-activated protein kinase. Therefore, grape Sap-fraction, especially BSap, may be a useful agent for cancer prevention.

Regulation of the NF-κB signaling pathway and IL-13 in asthmatic rats by aerosol inhalation of the combined active constituents of Punica granatum juice and peel.

Bronchial asthma is a chronic inflammatory airway illness. For the first time, we evaluated the proposed anti-asthmatic protective and therapeutic potency of inhaling Punica granatum juice (PJE) and peel (PPE) extract mixture (PM). Rats were challenged with ovalbumin (OVA) for 23 days and aerosolized with PM before each OVA challenge (protected group) or following the final OVA challenge for 3 days (therapeutic group). Considerable concentrations of phenolics were detected in PJE and PPE. Therefore, PM demonstrated synergistic scavenging abilities of NO and DPPH radicals. It also showed synergistic anti-inflammatory activities against lipopolysaccharide (LPS)-induced inflammation in the white blood cells by lowering the gene expression of CXCR1, CXCR2, IL-6, and IL-8. In addition, PM increased IL-10 gene expression while decreasing NO and TNF-α levels in LPS-exposed cells. Regarding the rats that were protected with PM, they exerted pulmonary pro-oxidant effects but prevented the OVA-induced upregulation of NF-κB, IKK, TNF-α, COX-2, iNOS, IL-13, and COL1A1, as well as MUC5AC and mucin over-secretion. While PM in the therapeutic group improved reactive oxygen species levels and normalized most of the investigated inflammatory and fibrotic mediators and mucin formation, but slightly improved the antioxidant indices. In addition, OVA-induced morphological alterations were massively improved after PM inhalation for short or long periods. Thus, PM inhalation prevented and treated OVA-induced pulmonary inflammation and fibrosis, while the inhalation period between 3 and 23 days needs to be optimized to acquire a better impact on the antioxidant indices.

Suppressing crucial oncogenes of leukemia initiator cells by major royal jelly protein 2 for mediating apoptosis in myeloid and lymphoid leukemia cells.

Relapse of leukemia and drug resistance are still the major obstacles to therapy due to leukemia-initiating stem/progenitor cells (LICs); thus, targeting them using safe compounds is crucial. Here, we evaluated the anti-leukemic effect of royal jelly (RJ) components, which had a higher safe concentration (EC100 values) than the chemotherapeutic drug doxorubicin (DOX). The RJ-protein fraction 50 (PF50, precipitated at 40-50% ammonium sulfate saturation) and its constituents, major RJ protein (MRJP) 2 and its isoform X1, exhibited the highest growth inhibitory effect against myeloid NFS-60 and lymphoid Jurkat cell lines. MRJP2 has a nanosize, which may be the reason for its higher anti-leukemic activity than its isoform. These RJ proteins, particularly MRJP2, suppressed LIC-associated oncogenes (GATA2 and Evi-1) and eliminated CD34+ LICs, in contrast to the low anti-LIC efficacy of DOX. MRJP2 demonstrated higher apoptotic activity than its isoform by upregulating p53 and p21-mediated cell cycle arrest. This study also reported the potent inhibitory effect of RJ-proteins on matrix metallopeptidase 10 (metastatic marker) and histone deacetylase 8 (mediates LIC survival) activities. Thus, MRJP2 can be considered a promising novel therapeutic agent for both myeloid and lymphoid leukemia.

Augmenting apoptosis-mediated anticancer activity of lactoperoxidase and lactoferrin by nanocombination with copper and iron hybrid nanometals.

There is an urgent need in the medicinal fields to discover biocompatible nanoformulations with low cytotoxicity, which provide new strategies for promising therapies for several types of tumors. Bovine lactoperoxidase (LP) and lactoferrin (LF) have recently attracted attention in medicine for their antitumor activities with recognized safety pattern. Both LP and LF are suitable proteins to be coated or adsorbed to Cu and Fe nanometals for developing stable nanoformulations that boost immunity and strong anticancer effects. New nanometals of Cu and Fe NPs embedded in LP and LF forming novel nanocombinations of LP-CNPs and LF-FNPs had a spherical shape with an average nanosize of about 21 nm. The combination of LP-CNPs and LF-FNPs significantly exhibited the highest growth inhibitory efficacy, in terms of effectively lowering the half-maximal inhibitory concentration (IC50) values, against Caco-2, HepG2 and MCF7 cells comparing to nanometals, LP, LF and individual nanoproteins (LP-CNPs or LF-FNPs). The highest apoptotic effect of this nanocombination (LP-CNPs and LF-FNPs) was confirmed by the highest percentages of annexin-stained apoptotic cells and G0 population with the strongest alteration in the expression of two well-characterized apoptosis guards (p53 and Bcl-2) and the maximum suppression in the proliferation marker (Ki-67). Also, the in silico analysis predicted that LP-CNPs and LF-FNPs enhanced AMP-activated protein kinase (AMPK, p53 activator) activity and inhibited cancer migration-related proteases (cathepsin B and matrix metalloproteinase (MMP)-9). Our results offer for the first time that these novel nanocombinations of LP and LF were superior in their selectivity and apoptosis-mediating anticancer activity to Cu and Fe nanometals as well as the free form of these proteins or their individual nanoforms.

Inhibitory Effects of Bacterial Silk-like Biopolymer on Herpes Simplex Virus Type 1, Adenovirus Type 7 and Hepatitis C Virus Infection.

Bacterial polymeric silk is produced by Bacillus sp. strain NE and is composed of two proteins, called fibroin and sericin, with several biomedical and biotechnological applications. In the current study and for the first time, the whole bacterial silk proteins were found capable of exerting antiviral effects against herpes simplex virus type-1 (HSV-1), adenovirus type 7 (AD7), and hepatitis C virus (HCV). The direct interaction between bacterial silk-like proteins and both HSV-1 and AD7 showed potent inhibitory activity against viral entry with IC50 values determined to be 4.1 and 46.4 µg/mL of protein, respectively. The adsorption inhibitory activity of the bacterial silk proteins showed a blocking activity against HSV-1 and AD7 with IC50 values determined to be 12.5 and 222.4 ± 1.0 µg/mL, respectively. However, the bacterial silk proteins exhibited an inhibitory effect on HSV-1 and AD7 replication inside infected cells with IC50 values of 9.8 and 109.3 µg/mL, respectively. All these results were confirmed by the ability of the bacterial silk proteins to inhibit viral polymerases of HSV-1 and AD7 with IC50 values of 164.1 and 11.8 µg/mL, respectively. Similarly, the inhibitory effect on HCV replication in peripheral blood monocytes (PBMCs) was determined to be 66.2% at concentrations of 100 µg/mL of the bacterial silk proteins. This antiviral activity against HCV was confirmed by the ability of the bacterial silk proteins to reduce the ROS generation inside the infected cells to be 50.6% instead of 87.9% inside untreated cells. The unique characteristics of the bacterial silk proteins such as production in large quantities via large-scale biofermenters, low costs of production, and sustainability of bacterial source offer insight into its use as a promising agent in fighting viral infection and combating viral outbreaks.

Inhibition of oxidative stress, IL-13, and WNT/ß-catenin in ovalbumin-sensitized rats by a novel organogel of Punica granatum seed oil saponifiable fraction.

Bronchial asthma is a chronic inflammatory disease marked by inflammation, oxidative stress, and structural remodeling. Here, we prepared two pomegranate fractions from the seed oil, saponifiable (Sap) and unsaponifiable (UnSap). Two organogels (Orgs) were also formulated with the Sap (Org1) or the UnSap (Org2) fraction and beeswax (BW). All preparations were evaluated in vitro for their antioxidant and anti-inflammatory impacts. The transdermal delivery of the most efficient one was evaluated against ovalbumin (OV)-induced bronchial asthma in rats compared to dexamethasone (DEX). The results showed that the prepared pomegranate fractions and BW had considerable amounts of phenolics (flavonoids and tannins) and triterpenoids. Org1 was shown to be the most effective antioxidant and anti-inflammatory fraction with synergistic activities (combination index, 1), as well as having protective and therapeutic influences on OV-sensitized rats. Org1 inhibited the multiple OV-induced signaling pathways, comprising ROS, WNT/ß-catenin, and AKT, with an efficiency superior to DEX. Subsequently, the pro-inflammatory (COX-2, NO, and IL-13), and pro-fibrotic (COL1A1) mediators, oxidative stress, and mucin secretion, were all down-regulated. These outcomes were verified by the histopathological results of lung tissue. Collectively, these outcomes suggest that the transdermal delivery of Org1 to OV-sensitized rats shows promise in the protection and treatment of the pathological hallmarks of asthma.

Targeting ROS/NF-κB sigaling pathway by the seedless black Vitis vinifera polyphenols in CCl4-intoxicated kidney, lung, brain, and spleen in rats.

Carbon tetrachloride (CCl4) is an abundant environmental pollutant that can generate free radicals and induce oxidative stress in different human and animal organs like the kidney, lung, brain, and spleen, causing toxicity. The present study evaluated the alleviative mechanism of the isolated polyphenolic fraction from seedless (pulp and skin) black Vitis vinifera (VVPF) on systemic oxidative and necroinflammatory stress in CCl4-intoxicated rats. Here, we found that the administration of VVPF to CCl4-intoxicated rats for ten days was obviously ameliorated the CCl4-induced systemic elevation in ROS, NO and TBARS levels, as well as MPO activity. Also, it upregulated the cellular activities of the enzymatic (SOD, and GPx) and non-enzymatic (TAC and GSH) antioxidants. Furthermore, the gene expression of the ROS-related necroinflammatory mediators (NF-κB, iNOS, COX-2, and TNF-α) in the kidney, brain, and spleen, as well as IL-1ß, and IL-8 in the lung were greatly restored. The histopathological studies confirmed these biochemical results and showed a noticeable enhancing effect in the architecture of the studied organs after VVPF intake. Thus, this study indicated that VVPF had an alleviative effect on CCl4-induced necroinflammation and oxidative stress in rat kidney, lung, brain, and spleen via controlling the ROS/NF-κB pathway.

Synergistic protective effect of Beta vulgaris with meso-2,3-dimercaptosuccinic acid against lead-induced neurotoxicity in male rats.

Lead (Pb) toxicity is one of the most prevalent causes of human neurotoxicity. The available chelator drugs used now have many adverse effects. So, in this study, the protective role of Beta vulgaris juice (BVJ) on rat neurotoxicity induced by Pb was evaluated and the results were compared with the results of dimercaptosuccinic acid (DMSA, as used drug). Additionally, the synergistic effect of BVJ and DMSA against Pb-induced neurotoxicity was assessed. The study focused on the determination of the antioxidant, anti-inflammatory, and neurological potential of BVJ (alone, and with DMSA) towards lead-induced neurotoxicity. Also, the characterization of BVJ was studied. The results showed that BVJ contains considerable quantities of polyphenols, triterpenoids, and betalains which play an important role as antioxidants and anti-inflammatory. BVJ exhibited a protective effect against neurotoxicity via the reduction of Pb levels in blood and brain. Moreover, BVJ decreased the oxidative stress, inflammation, and cell death induced by Pb. Also, BVJ regulated the activities of acetylcholine esterase and monoamine oxidase-A which changed by Pb toxicity. BVJ and DMSA combination displayed a synergistic antineurotoxic effect (combination index ˂ 1). These results were in harmony with brain histopathology. Conclusion: BVJ has a powerful efficacy in the protection from brain toxicity via diminishing Pb in the brain and blood circulation, resulting in the prevention of the oxidative and inflammatory stress. Treatment with BVJ in combination with DMSA revealed a synergistic effect in the reduction of neurotoxicity induced by Pb. Also, the antioxidant and anti-inflammatory effects of the BVJ lead to the improvement of DMSA therapy.

Vitis vinifera polyphenols from seedless black fruit act synergistically to suppress hepatotoxicity by targeting necroptosis and pro-fibrotic mediators.

Human is subjected from his surrounding to various hepatotoxins, which aggravates his liver. Nowadays, natural polyphenols have attracted great interest in health improvement, especially liver health. The present research, therefore, assessed the hepatotherapeutic potency of the isolated polyphenols (VVF1) from seedless (pulp and skin) black Vitis vinifera (VV) against CCl4-induced hepatotoxicity in vitro and in vivo. Further, VVF1 was fractionated into resveratrol-enriched (VVF2) and phenolics-enriched (VVF3) fractions to study (in vitro) the possible synergism of their coexistence. The highest content of phenolics in VVF1 displayed in vitro synergistic antioxidant and anti-hepatotoxic activities comparing to VVF2, VVF3, and silymarin (SM, reference drug). More importantly, it exhibited multiple in vivo regulatory functions via diminishing oxidative stress and inflammation, which in turn decreased necroptosis and pro-fibrotic mediators (mixed lineage kinase domain-like protein (MLKL), collagen type I alpha 1 chain (COL1A1), and transforming growth factor (TGF)-ß1). In addition to these novel findings, VVF1 had higher anti-hepatotoxic potency than that of SM in most of the studied parameters. The histopathological analysis confirmed the improving role of VVF1 in the serious hepatic damage induced by CCl4. Thus, the synergistic functions of VVF1 polyphenols could be a promising new anti-hepatotoxic agent for targeting both necroptotic and profibrotic mediators.

Two purified proteins from royal jelly with in vitro dual anti-hepatic damage potency: Major royal jelly protein 2 and its novel isoform X1.

Liver diseases are serious life-threating conditions that should be controlled. Here, we identify a protein fraction from royal-jelly (RJ) that represents the most effective composite against CCl4-induced hepatotoxicity and HepG2 cell growth. Two closely related proteins were purified from this fraction by a new simple method and identified by MALDI-TOF MS as major RJ protein 2 (MRJP2) and its predicted isoform X1. The in silico assessment (3D structures and functions) of these proteins were performed using Iterative Threading ASSEmbly Refinement (I-TASSER) analysis and RAMPAGE program. These two purified proteins were able to relieve the necrotic hepatocytes (by 60.4%) via reducing tumor necrosis factor (TNF)-α, mixed lineage kinase domain-like protein (MLKL) and intracellular reactive species. The latter effects associated with improving hepatocyte functions. Furthermore, they revealed the potent anticancer effect via induction of caspase-dependent apoptosis and controlling the expression of both Bcl-2 and p53 in HepG2 cells. Thus, MRJP2 and its isoform X1 can be a promising dual strategy for fighting hepatic injury and cancer in future animal and human studies.

In vitro evaluation of the synergistic antioxidant and anti-inflammatory activities of the combined extracts from Malaysian Ganoderma lucidum and Egyptian Chlorella vulgaris.

BACKGROUND: Since oxidative stress and inflammation are two linked factors in the pathogenesis of several human diseases. Thus identification of effective treatment is of great importance. Edible mushroom and microalgae are rich in the effective antioxidant phytochemicals. Hence, their beneficial effects on oxidative stress-associated inflammation are extremely required to be investigated. METHODS: This study evaluated the functional constituents, antioxidant and anti-inflammatory activities of Malaysian Ganoderma lucidum aqueous extract (GLE) and Egyptian Chlorella vulgaris ethanolic extract (CVE). Also, the synergistic, addictive or antagonistic activities of the combination between the two extracts (GLE-CVE) were studied. Expression of inducible nitric oxide synthase, cyclooxygenase-2, and nuclear factor-kappa B, as well as levels of nitric oxide, tumor necrosis factor (TNF)-α, lipid peroxidation, reduced glutathione and antioxidant enzymes were determined using in vitro model of lipopolysaccharide-stimulated white blood cells.

Protective role of Punica granatum (pomegranate) peel and seed oil extracts on diethylnitrosamine and phenobarbital-induced hepatic injury in male rats.

The present study is an attempt to reveal the protective role of Punica granatum peel and seed oil extracts against diethylnitrosamine (DEN) and phenobarbital (PB) induced hepatic injury in rats. DEN administration increased the levels of malondialdehyde (MDA), DNA fragmentation, caspase-3 and glutathione reductase (GSR) activities, while the level of reduced glutathione (GSH) and the activities of superoxide dismutase (SOD), glutathione S-transferase (GST) and total glutathione peroxidase (t-GPx) were decreased compared with the control. Treatment with peel and seed oil extracts pre, during and post DEN administration improved liver functions, decreased the levels of MDA, DNA fragmentation, caspase-3 and GSR activities with an elevation in levels of GSH, SOD, GST and t-GPx activities. This indicates that these extracts reduced the oxidative stress and apoptosis induced by DEN. Also the effect of administration of PE and SOE separately for a long time (23 weeks) on healthy rats was studied.