Hemin competitively inhibits HSPA8 ATPase activity mitigating its foldase function.

Hemolysis in red blood cells followed by hemoglobin degradation results in high hemin levels in the systemic circulation. Such a level of hemin is disastrous for cells and tissues and is considerably responsible for the pathologies of diseases like severe malaria. Hemin's hydrophobic chemical nature and structure allow it to bind several proteins leading to their functional modification. Such modifications in physiologically relevant proteins can have a high impact on various cellular processes. HSPA8 is a chaperone that has a protective role in oxidative stress by aiding protein refolding. Through ATPase activity assays we found that hemin can competitively inhibit ATP hydrolysis by the chaperone HSPA8. Hemin as such does not affect the structural integrity of the protein which is inferred from CD spectroscopy and Gel filtration but it hinders the ATP-dependent foldase function of the chaperone. HSPA8 was not able to cause the refolding of the model protein lysozyme in the presence of hemin. The loss in HSPA8 function was due to competition between hemin and ATP as the chaperone was able to regain the foldase function when the concentration of ATP was gradually increased with hemin present at the inhibitory concentration. In-silico studies to establish the competition for the specific binding site revealed that ATP was unable to replace hemin from the ATP binding pocket of HSPA8 and was forced to form a non-specific and unstable complex. In-vitro isothermal calorimetry revealed that the affinity of ATP for binding to HSPA8 was reduced 22 folds in the presence of hemin. The prevention of HSPA8's cytoprotective function by hemin can be a major factor contributing to the overall cellular damage during hemin accumulation in the case of severe malaria and other hemolytic diseases.

Platelet matrix metalloprotease-1 mediates thrombogenesis by activating PAR1 at a cryptic ligand site.

Matrix metalloproteases (MMPs) play important roles in normal and pathological remodeling processes including atherothrombotic disease, inflammation, angiogenesis, and cancer. MMPs have been viewed as matrix-degrading enzymes, but recent studies have shown that they possess direct signaling capabilities. Platelets harbor several MMPs that modulate hemostatic function and platelet survival; however their mode of action remains unknown. We show that platelet MMP-1 activates protease-activated receptor-1 (PAR1) on the surface of platelets. Exposure of platelets to fibrillar collagen converts the surface-bound proMMP-1 zymogen to active MMP-1, which promotes aggregation through PAR1. Unexpectedly, MMP-1 cleaves PAR1 at a distinct site that strongly activates Rho-GTP pathways, cell shape change and motility, and MAPK signaling. Blockade of MMP1-PAR1 curtails thrombogenesis under arterial flow conditions and inhibits thrombosis in animals. These studies provide a link between matrix-dependent activation of metalloproteases and platelet-G protein signaling and identify MMP1-PAR1 as a potential target for the prevention of arterial thrombosis.

Isolation and characterization of Newcastle disease virus from biological fluids using column chromatography.

Newcastle disease virus (NDV), belonging to the species avian orthoavulavirus 1, genus Orthoavulavirus, and family Paramyxoviridae, is responsible for Newcastle disease in poultry and other avian species. It has shown significant potential as an oncolytic virus and as a vector for vaccine delivery. NDV from infected biological serum is usually isolated or purified using density gradient ultracentrifugation. However, it has many disadvantages, including the fact that it is time consuming and can process only a limited quantity of sample at one time. In our study, native agarose gel electrophoresis and dynamic light scattering (DLS) analysis showed that NDV carried a net negative surface charge. Thus, we purified the virus using a HiTrap Q Sepharose Fast Flow anion exchange column with salt elution. Hemagglutination assay and plaque assay showed that the procedure yielded high-purity NDV particles with a recovery of more than 80%, and the process was fast and simple. The purity of the virus was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The hydrodynamic volume and 'dry state' diameter of the purified NDV were analyzed using dynamic light scattering and transmission electron microscopy and were to be in the range of 200-300 nm. The viruses did not exhibit any deviation from their known physical properties. The genome of the virus was also detected by amplifying a 423-bp region using reverse transcription-polymerase chain reaction. Our study confirmed that NDV could be effectively purified using an anion exchange column. In addition, the procedure could be easily upscaled or downscaled based on the experimental requirements.

Delivery of Small Molecules by Syndiotactic Peptides for Breast Cancer Therapy.

The utilization of peptide-based drug delivery systems has been suboptimal due to their poor proteolytic susceptibility, poor cell permeability, and limited tumor homing capabilities. Earlier attempts in using d-enantiomers in peptide sequences increased proteolytic stability but have compromised the overall penetration capability. We designed a series of peptides (STRAPs) with a syndiotactic polypeptide backbone that can potentially form a spatial array of cationic groups, an important feature that facilitates cellular uptake. The peptides penetrate cell membranes through a combination of active and passive modes. Furthermore, the cellular uptake of the peptides was unaffected by the presence of or treatment with bovine serum and human plasma. The designed peptides successfully delivered methotrexate, an anticancer drug, to the in vitro and in vivo models of breast cancer, with the best performing peptide STRAP-4-MTX conjugate having an EC50 value of 1.34 µM. Peptide drug delivery in mouse xenograft models showed a greater reduction of primary tumor and metastasis of breast cancer, in comparison to methotrexate of the same dose. The in vivo biodistribution assay of the STRAP-4 peptide suggests that the peptide accumulates at the tumor site after 2 h of treatment, and in the absence of tumors, the peptide gets metabolized and excreted from the system.

Insulin signalling in RBC is responsible for growth stimulation of malaria parasite in diabetes patients.

A cross-talk between diabetes and malaria within-host is well established. Diabetes is associated with modulation of the immune system, impairment of the healing process and to disturb the host metabolism to contribute towards propagation of parasite infection. Glucose metabolism in host is maintained by insulin and RBC has 2000 insulin receptor present on plasma membrane. These receptors are robust to relay down-stream signaling in RBCs but role of intracellular signaling in parasite growth is not been explored. The malaria parasite treated with insulin (100 ng/ml) is giving stimulation in parasite growth. The effect is lasting for several generations resulting into high parasitemia. Insulin signaling is phosphorylating protein in infected RBCs and level is high in parasite RBCs compared to uninfected RBCs. It is phosphorylating Spectrin-(α/ß), Band-4.2, Ankyrin and the other proteins of RBC cytoskeleton. It in-turn induces enhanced glucose uptake inside infected RBCs. There is a high level of infection of normal RBCs by merozoites. In summary, insulin and glucose metabolism plays a crucial role in parasite propagation, disease severity and need consideration while treating patients.

Conformationally constrained peptides for drug delivery.

Peptides have shown great potential in acting as template for developing versatile carrier platforms in nanomedicine, aimed at selective delivery of drugs to only pathological tissues saving its normal neighbors. Cell-penetrating peptides (CPPs) are short oligomeric peptides capable of translocating across the cell membrane while simultaneously employing multiple mechanisms of entry. Most CPPs exist as disordered structures in solution and may adopt a helical conformation on interaction with cell membrane, vital to their penetrative capability. Herein, we report a series of cationic helical amphipathic peptides (CHAPs), which are topologically constrained to be helical. The peptides were tested against cervical and breast cancer cells for their cell penetration and drug delivery potential. The cellular uptake of CHAP peptides is independent of temperature and energy availability. The activity of the peptides is biocompatible in bovine serum. CHAPs delivered functional methotrexate (MTX) inside the cell as CHAP-MTX conjugates. CHAP-MTX conjugates were more toxic to cancer cells than MTX alone. However, the CHAP-MTX conjugates were less toxic to HEK-293 cells compared with the cancer cells suggesting higher affinity towards cancer cells.

Severe malaria: Biology, clinical manifestation, pathogenesis and consequences.

Every year, millions of people are infected with malaria, resulting in significant economic losses to the developing and developed nations. The malaria parasite pursues a complicated life cycle in an invertebrate, mosquito and vertebrate host with several distinct stages. In the human host, it invades the liver and red blood cells to complete its life cycle. It is surprising that not only these two organs are under pressure and exhibit functional abnormalities; a large number of clinical studies also support the notion that malaria parasite propagation in the host affects several other organs and modulates functional outcomes of individual cells. Moreover, patients recovered from severe malaria may suffer throughout their life from impairments in organ function such as loss of eyesight, kidney failure, and much more. Thus, malaria infection leads to several pathological outcomes involving different organs and individual cells in the host. The sole purpose of the present article was to give an overview of pathological outcomes during severe malaria along with their molecular mechanisms. A large proportion of deaths associated with disease is contributed by the pathological effect in host due to parasite propagation and toxicity of antimalarials or combination of both. Hence, there is a need, not only to develop antiparasitic agents but also to discover lead molecules to take care of pathophysiological effects in the host. This may help a beginner to get involved with the topic and initiate research work towards improving adjuvant therapy or avoiding serious complications.

Extracellular methemoglobin promotes cyto-adherence of uninfected RBC to endothelial cells: Insight into cerebral malaria pathology.

The endothelial cell barrier is tightly regulated, and disruption or the leaky behavior of the barrier leads to pathology. Disturbance of blood-brain barrier is observed during viral infection, cerebral malaria, and acute hemorrhagic encephalitis. Red blood cells (RBCs) bind to the endothelial cells (ECs) and their affinity towards ECs enhances in the presence of Plasmodium falciparum infection. ECs stimulated with methemoglobin (MetHb; 20 µM) for 1 hour exhibit high levels of cyto-adherence receptors CD36 and ICAM-1 on their cell surface compared with unstimulated cells. These ECs have acquired affinity towards uninfected RBCs in flow at arterial shear stress. SEM analysis indicates that EC-RBC cyto-adherence involved multiple attachment points. Initially, ECs bind single layer of RBCs and the number of RBCs increases over time to give high-order cyto-adherence with more than 30 RBCs adhered to each endothelial cell. The cyto-adherence complexes are stable to high shear stress and can withstand shear stress up to 450 dyne/cm 2 . MetHb-treated ECs exhibited high reactive oxygen species level, and preincubation of ECs with antioxidant (NAC or mannitol) abolished the formation of EC-RBC cyto-adherence complexes. In addition, gallic acid (present in red wine) and green tea extract has inhibited the formation of EC-RBC cyto-adherence complex. A better understanding of gallic acid and tea polyphenol targeting pathological cyto-adherence may allow us to develop a better adjuvant therapy for cerebral malaria and other noninfectious diseases.

Azidophosphonate Chemistry as a Route for a Novel Class of Vesicle-Forming Phosphonolipids.

Membrane forming synthetic lipids constitutes a new class of biomaterials with impressive applications in the field of biological and pharmaceutical sciences. Interestingly, alteration(s) in the headgroup region of the lipids offer a wide chemical space to investigate their specific properties. In this regard, we have utilized ß-azidophosphonate chemistry to gain access to a novel class of triazole-phosphonate (TP) amphiphiles with fascinating physicochemical properties of lipids. TP lipids form stable vesicles that exhibit negative surface potential across a broad pH range. These anionic lipids have high phase-transition temperatures, phospholipase resistance, slow vesicle leakage profiles, and doxorubicin delivery efficacy. We hypothesize that these readily synthesizable phosphonolipids could find several applications as phospholipid substituents.

Mapping of phosphatidylserine recognition region on CD36 ectodomain.

CD36-PS interaction is an important affair to identify and remove dead/aged cells to control inflammation. CD36 ectodomain was cloned, over-expressed in bacterial expression system and purified to homogeneity. The dot-blot analysis shows that the CD36_ecto selectively binds PS vesicles blotted on the nitrocellulose membrane. PS binds strongly to CD36_ecto with a dissociation constant KD of 53.7 ±â€¯0.48 µM. The stoichiometry of interaction between CD36 and PS is 1:2. The hCD36_ecto-PS thermogram revealed that the hydrophobic and salt bridge interactions play crucial role in their interactions. PS docked nicely into the predicted pharmacophoric site with a binding energy of 5.1 kcal/mol. Analysis of CD36-PS molecular model showed that the residues R63, R96, N118, D270 and E418 were forming hydrogen bonds with PS. Molecular dynamics simulations indicate that R63 mutation has disrupted the integrity of biophoric constituents, directly affecting the hydrogen bonding from R96, N118 and D270. ITC thermogram analysis of mutant protein with PS vesicles indicate complete loss of binding with R63A and very low affinity of PS vesicles with D270A. Dot blot analysis further confirmed the ITC results. These finding may help to design suitable agents mimicking PS biophore with potentials in diagnostics of apoptotic cells and cardiovascular intervention.

A dinitro-functionalized metal-organic framework featuring visual and fluorogenic sensing of H2S in living cells, human blood plasma and environmental samples.

Here, we describe a new dinitro-functionalized Zr(iv) MOF (MOF = metal-organic framework) having a UiO-66 (UiO = University of Oslo) framework topology called UiO-66-(NO2)2 (1). It shows fluorescence turn-on behavior towards H2S in simulated biological medium (HEPES buffer, pH = 7.4). By employing solvothermal conditions, 1 was successfully synthesized by reacting ZrCl4, H2BDC-(NO2)2 [H2BDC-(NO2)2 = 2,5-dinitro-1,4-benzenedicarboxylic acid] ligand and benzoic acid with a molar ratio of 1 : 1 : 10 in DMF (DMF = N,N-dimethylformamide) at 130 °C for 24 h. The material was characterized by infrared spectroscopy, X-ray powder diffraction (XRPD) and thermogravimetric (TG) analyses. The compound not only displays highly sensitive fluorometric sensing of H2S but also exhibits a visually detectable colorimetric change towards H2S in daylight. Moreover, the high selectivity of 1' towards H2S is retained even when several other biologically intrusive species co-exist in the sensing medium. The limit of detection (LOD) of the compound is 14.14 µM which lies in the range of the H2S concentration found in biological systems. Fluorescence microscopy studies on J774A.1 cells revealed the efficacy of the probe for imaging H2S in living cells. Moreover, this material can detect H2S in human blood plasma (HBP) and monitor the sulfide concentration in real water samples. All these features clearly demonstrate that the material has huge potential for highly selective sensing of both extracellular and intracellular H2S.

Metal-Organic Framework Showing Selective and Sensitive Detection of Exogenous and Endogenous Formaldehyde.

In this work, we report a new hydrazine-functionalized Al(III)- based metal-organic framework having MIL-53 (MIL = Material of Institute Lavoisier) framework topology for the sensitive and selective detection of formaldehyde (FA). The phase purity of the thermally activated and as-synthesized forms of the material was examined by X-ray powder diffraction experiments, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The desolvated material (1') showed great potential for the selective sensing of FA in the existence of other potentially competitive aldehydes in both aqueous and 10 mM HEPES buffer (pH = 7.4) media. The fluorescence "turn-on" behavior of the reaction-based probe can be ascribed to the inhibition of the photoinduced electron transfer process (from the hydrazine group to the phenyl ring) because of the formation of the hydrazone moiety. The detection limit of the probe toward FA in HEPES buffer is 8.37 µM (0.25 ppm), which lies below the intracellular concentration of FA (100-400 µM). A very short response time (1 min) has been displayed by 1' for FA sensing. Moreover, a remarkable enhancement in the emission intensity (sevenfold and fourfold in aqueous and HEPES buffer media, respectively) of 1' was observed after 1 min of FA addition. Furthermore, the ability of the probe to detect FA in the vapor phase was demonstrated. Interestingly, the material is also capable to detect endogenous FA in cancer cells. All the above discussed features clearly reveal that the present material has a huge potential for selective recognition of FA in both real water and biological samples.

Selective Sensing of Peroxynitrite by Hf-Based UiO-66-B(OH)2 Metal-Organic Framework: Applicability to Cell Imaging.

The first boronic acid functionalized Hf-based UiO-66 (UiO = University of Oslo) metal-organic framework (MOF) having the ability to detect both extracellular and intracellular peroxynitrite is presented. The Hf-UiO-66-B(OH)2 material (1) was synthesized under solvothermal conditions from a mixture of HfCl4 and 2-borono-1,4-benzenedicarboxylic acid [H2BDC-B(OH)2] ligand in DMF in the presence of formic acid (modulator) at 130 °C for 48 h. The desolvated material (1') was utilized as a fluorescent turn-on probe for the rapid sensing of extracellular peroxynitrite (ONOO-) under conditions mimicking those of biological medium (10 mM HEPES buffer, pH 7.4). Selective sensing of ONOO- over other ROS/RNS was also achieved by 1'. The oxidative cleavage of attached boronic acid groups forming corresponding hydroxy-functionalized ligands can be accounted for the fluorescent increment phenomenon in the presence of ONOO-. The probe showed extraordinary sensitivity (detection limit = 9.0 nM) toward ONOO- in 10 mM HEPES buffer at pH 7.4. Probe-loaded cells did not exhibit cytotoxicity and morphological deformities. It is remarkable that the probe inside the cells responded toward the peroxynitrite solution to give an intense blue fluorescent signal. The fluorescence microscopy study with J774A.1 macrophage cells unambiguously demonstrated that probe 1' is suitable to image peroxynitrite in living cells.

Selective and Sensitive Sensing of Hydrogen Peroxide by a Boronic Acid Functionalized Metal-Organic Framework and Its Application in Live-Cell Imaging.

A new boronic acid functionalized Zr(IV) metal-organic framework having the capability of sensing H2O2 in live cells is reported. The Zr-MOF bears a UiO-66 structure and contains 2-boronobenzene-1,4-dicarboxylic acid (BDC-B(OH)2) as a framework linker. The activated Zr-UiO-66-B(OH)2 compound (called 1') is highly selective for the fluorogenic detection of H2O2 in HEPES buffer at pH 7.4, even in the presence of interfering ROS (ROS = reactive oxygen species) and other biologically relevant analytes. The fluorescent probe was found to display extraordinary sensitivity for H2O2 (detection limit 0.015 µM) in HEPES buffer, which represents a lower value in comparison to those of the MOF probes documented so far for sensing H2O2 using other analytical methods. Taking advantage of its high selectivity and sensitivity for H2O2 in HEPES buffer, the probe was successfully employed for the imaging of intracellular H2O2. Imaging studies with MDAMB-231 cells revealed the emergence of bright blue fluorescence after loading with probe 1' and subsequent treatment with H2O2 solution.

Improving the frying performance of RBD palm olein oil using NaturFORT™ TRLG 101 liquid as on-top of TBHQ in deep-fat frying of potato chips.

Improvement in the frying performance of palmolein oil with NaturFORT™ TRLG 101 (TRLG 101) liquid in addition to tert-butyl hydroquinone (TBHQ) has been evaluated. Four treatment groups (Negative control, 200 ppm TBHQ, 200 ppm TBHQ + 400 ppm TRLG 101 liquid and 200 ppm TBHQ + 750 ppm TRLG 101 liquid) were added to RBD (Refined, Bleached and Deodorized) palm olein oil which was used to produce potato chips. Frying trials were conducted for 120th frying cycles. The oil samples were analyzed for peroxide value, free fatty acid, p-anisidine value, oxidative stability index, %total polar compounds and Chroma (C*) values after every 20 frying cycles. Potato chips were analyzed for Chroma (C*) values after every 20 frying cycles. The results of oxidative stability index, total polar compounds and p-anisidine value showed that the addition of NaturFORT™ TRLG 101 liquid at 750 ppm had showed significantly better performance followed by the addition of NaturFORT™ TRLG 101 liquid at 400 ppm. Thus, the addition of NaturFORT™ TRLG 101 liquid as on top of TBHQ contributes to the improvement in the frying performance of palm olein oil with subject to their dosage and this could be a better solution for frying industries which would like to get extra frying cycles for their products without encountering the regulatory hurdles posed by the limitations of using such additives.

An actin-like protein is involved in regulation of mitochondrial and flagellar functions as well as in intramacrophage survival of Leishmania donovani.

Actin-related proteins are ubiquitous actin-like proteins that show high similarity with actin in terms of their amino acid sequence and three-dimensional structure. However, in lower eukaryotes, such as trypanosomatids, their functions have not yet been explored. Here, we show that a novel actin-related protein (ORF LmjF.13.0950) is localized mainly in the Leishmania mitochondrion. We further reveal that depletion of the intracellular levels of this protein leads to an appreciable decrease in the mitochondrial membrane potential as well as in the ATP production, which appears to be accompanied with impairment in the flagellum assembly and motility. Additionally, we report that the mutants so generated fail to survive inside the mouse peritoneal macrophages. These abnormalities are, however, reversed by the episomal gene complementation. Our results, for the first time indicate that apart from their classical roles in the cytoplasm and nucleus, actin-related proteins may also regulate the mitochondrial function, and in case of Leishmania donovani they may also serve as the essential factor for their survival in the host cells.

An anionic conjugated polymer as a multi-action sensor for the sensitive detection of Cu(2+) and PPi, real-time ALP assaying and cell imaging.

A Cu(2+) ensemble polyfluorene derivative, poly[5,5'-(((9H-fluorene-9,9-diyl)bis(hexane-6,1-diyl))bis(oxy))diisophthalate] sodium salt (PFT), displays unprecedented selectivity for PPi (LOD = 2.26 ppb) in aqueous solution as well as in random urine samples at physiological pH vis-a-vis monitoring ALP activity. Furthermore, intracellular imaging of Cu(2+) and PPi in mouse macrophage (J774A.1) and human breast cancer cells (MDA-MB231) was achieved to confirm the viability of PFT in biological systems.

MUC1 oncoprotein activates the IkappaB kinase beta complex and constitutive NF-kappaB signalling.

Nuclear factor-kappaB (NF-kappaB) is constitutively activated in diverse human malignancies by mechanisms that are not understood. The MUC1 oncoprotein is aberrantly overexpressed by most human carcinomas and, similarly to NF-kappaB, blocks apoptosis and induces transformation. This study demonstrates that overexpression of MUC1 in human carcinoma cells is associated with constitutive activation of NF-kappaB p65. We show that MUC1 interacts with the high-molecular-weight IkappaB kinase (IKK) complex in vivo and that the MUC1 cytoplasmic domain binds directly to IKKbeta and IKKgamma. Interaction of MUC1 with both IKKbeta and IKKgamma is necessary for IKKbeta activation, resulting in phosphorylation and degradation of IkappaBalpha. Studies in non-malignant epithelial cells show that MUC1 is recruited to the TNF-R1 complex and interacts with IKKbeta-IKKgamma in response to TNFalpha stimulation. TNFalpha-induced recruitment of MUC1 is dependent on TRADD and TRAF2, but not the death-domain kinase RIP1. In addition, MUC1-mediated activation of IKKbeta is dependent on TAK1 and TAB2. These findings indicate that MUC1 is important for physiological activation of IKKbeta and that overexpression of MUC1, as found in human cancers, confers sustained induction of the IKKbeta-NF-kappaB p65 pathway.

Role Transformation of HSPA8 to Heme-peroxidase After Binding Hemin to Catalyze Heme Polymerization.

Hemin, a byproduct of hemoglobin degradation, inflicts oxidative insult to cells. Following its accumulation, several proteins are recruited for heme detoxification with heme oxygenase playing the key role. Chaperones play a protective role primarily by preventing protein degradation and unfolding. They also are known to have miscellaneous secondary roles during similar situations. To discover a secondary role of chaperones during heme stress we studied the role of the chaperone HSPA8 in the detoxification of hemin. In-silico studies indicated that HSPA8 has a well-defined biophoric environment to bind hemin. Through optical difference spectroscopy, we found that HSPA8 binds hemin through its N-terminal domain with a Kd value of 5.9 ± 0.04 µM and transforms into a hemoprotein. The hemoprotein was tested for exhibiting peroxidase activity using guaiacol as substrate. The complex formed reacts with H2O2 and exhibits classical peroxidase activity with an ability to oxidize aromatic and halide substrates. HSPA8 is dose-dependently catalyzing heme polymerization through its N-terminal domain. The IR results reveal that the polymer formed exhibits structural similarities to ß-hematin suggesting its covalent nature. The polymerization mechanism was tested through optical spectroscopy, spin-trap, and activity inhibition experiments. The results suggest that the polymerization occurs through a peroxidase-H2O2 system involving a one-electron transfer mechanism, and the formation of free radical and radical-radical interaction. It highlights a possible role of the HSPA8-hemin complex in exhibiting cytoprotective function during pathological conditions like malaria, sickle cell disease, etc.

Molecular modelling, docking and network analysis of phytochemicals from Haritaki churna: role of protein cross-talks for their action.

Phytochemicals are bioactive agents present in medicinal plants with therapeutic values. Phytochemicals isolated from plants target multiple cellular processes. In the current work, we have used fractionation techniques to identify 13 bioactive polyphenols in ayurvedic medicine Haritaki Churna. Employing the advanced spectroscopic and fractionation, structure of bioactive polyphenols was determined. Blasting the phytochemical structure allow us to identify a total of 469 protein targets from Drug bank and Binding DB. Phytochemicals with their protein targets from Drug bank was used to create a phytochemical-protein network comprising of 394 nodes and 1023 edges. It highlights the extensive cross-talk between protein target corresponding to different phytochemicals. Analysis of protein targets from Binding data bank gives a network comprised of 143 nodes and 275 edges. Taking the data together from Drug bank and binding data, seven most prominent drug targets (HSP90AA1, c-Src kinase, EGFR, Akt1, EGFR, AR, and ESR-α) were found to be target of the phytochemicals. Molecular modelling and docking experiment indicate that phytochemicals are fitting nicely into active site of the target proteins. The binding energy of the phytochemicals were better than the inhibitors of these protein targets. The strength and stability of the protein ligand complexes were further confirmed using molecular dynamic simulation studies. Further, the ADMET profiles of phytochemicals extracted from HCAE suggests that they can be potential drug targets. The phytochemical cross-talk was further proven by choosing c-Src as a model. HCAE down regulated c-Src and its downstream protein targets such as Akt1, cyclin D1 and vimentin. Hence, network analysis followed by molecular docking, molecular dynamics simulation and in-vitro studies clearly highlight the role of protein network and subsequent selection of drug candidate based on network pharmacology.Communicated by Ramaswamy H. Sarma.