Synergistic effects of phosphorylation modification and protocatechuic acid copolymerization improve the physical and oxidation stability of high internal phase emulsion stabilized by perilla protein isolate.

A compact antioxidant interfacial layer was fabricated by combining phosphorylation treatment with protocatechuic acid (PA) copolymerization to enhance the physical and oxidative stability of high internal phase emulsions (HIPEs) prepared using perilla protein isolate (PPI). The covalent binding between PPI and phosphate groups induced conformational changes, facilitating the interaction between PPI and PA. The formed phosphorylated PPI-PA conjugates (LPPI-PA) exhibited a reduced particle size of 196.75 nm, promoting their adsorption at the interface. HIPEs prepared by LPPI-PA conjugates showed higher storage stability due to decreased droplet size, increased interfacial protein adsorption content (90.48%), and the formation of an interconnected network within the system. Additionally, the combination of LPPI and PA anchored PA to the interface, significantly inhibiting lipid oxidation in HIPEs as evidenced by low levels of lipid hydroperoxide (30.33 µmol/g oil) and malondialdehyde (379.34 nmol/g oil). This study holds significant implications for improving the stability of HIPEs.

Modulation the Synergistic Effect of Chitosan-Sodium Alginate Nanoparticles with Ca2+: Enhancing the Stability of Pickering Emulsion on D-Limonene.

Pickering emulsions (PEs) have been regarded as an effective approach to sustaining and preserving the bioactivities of essential oils. The aim of this research is to prepare a PE stabilized by chitosan/alginate nanoparticles (CS-SA NPs) for the encapsulation and stabilization of D-limonene. In this work, the influence of calcium ions (Ca2+) on the morphology and interaction of nanoparticles was studied, and then the preparation technology of CS-SA/Ca2+ NPs was optimized. The results showed that the presence of Ca2+ reduced the size of the nanoparticles and made them assume a spherical structure. In addition, under the conditions of 0.2 mg/mL CaCl2, 0.6 mg/mL SA, and 0.4 mg/mL CS, the CS-SA/Ca2+ NPs had the smallest size (274 ± 2.51 nm) and high stability (-49 ± 0.69 mV). Secondly, the PE was prepared by emulsifying D-limonene with CS-SA/Ca2+ NPs, and the NP concentrations and homogenization speeds were optimized. The results showed that the small droplet size PE could be prepared with 2 mg/mL NP and a homogenization speed of 20,000 r/min, and it had excellent antibacterial and antioxidant activities. Most importantly, the emulsion showed higher activity, higher resistance to ultraviolet (UV) and a higher temperature than free D-limonene. This research provides a feasible solution for the encapsulation, protection and delivery of essential oils.

Biopolymer-based pickering high internal phase emulsions: Intrinsic composition of matrix components, fundamental characteristics and perspective.

Pickering HIPEs have received tremendous attention in recent years due to their superior stability and unique solid-like and rheological properties. Biopolymer-based colloidal particles derived from proteins, polysaccharides and polyphenols have been demonstrated to be safety stabilizers for the construction of Pickering HIPEs, which can meet the demands of consumers for "all-natural" products and provide "clean-label" foods. Furthermore, the functionality of these biopolymers can be further extended by forming composite, conjugated and multi-component colloidal particles, which can be used to modulate the properties of the interfacial layer, thereby adjusting the performance and stability of Pickering HIPEs. In this review, the factors affecting the interfacial behavior and adsorption characteristics of colloidal particles are discussed. The intrinsic composition of matrix components and fundamental characteristics of Pickering HIPEs are emphatically summarized, and the emerging applications of Pickering HIPEs in the food industry are reviewed. Inspired by these findings, future perspectives concerning this field are also put forward, including (1) the exploration of the interactions between biopolymers used to produce Pickering HIPEs and target food ingredients, and the influence of the added biopolymers on the flavor and mouthfeel of the products, (2) the investigation of the digestion properties of Pickering HIPEs under oral administration, and (3) the fabrication of stimulus-responsive or transparent Pickering HIPEs. This review will give a reference for exploring more natural biopolymers for Pickering HIPEs application development.

High internal phase emulsion gels stabilized by phosphorylated perilla protein isolate for protecting hydrophobic nutrients: Adjusting emulsion performance by incorporating chitosan-protocatechuic acid conjugate.

The delivery vehicles based on protein-polysaccharide-polyphenol are promising methods to encapsulate bioactive components with the aim of improving their solubility and bioavailability. In this study, chitosan-protocatechuic acid (CSPA) conjugate interacted with phosphorylated perilla protein isolate (LZPI) to engineer a composite antioxidant interfacial architecture to delay lipid oxidation and regulate the stability and digestion profiles of ß-carotene loaded in high internal phase emulsions (HIPEs). Compared to LZPI, the LZPI-CSPA complexes formed by hydrogen bond and electrostatic interaction showed improved wettability and reduced interfacial tension, which facilitated their adsorption at the interface. Furthermore, the addition of CSPA conjugate promoted the formation of interconnected network structure of LZPI-stabilized HIPEs, thereby endowing them with excellent viscoelasticity and storage stability. Moreover, the denser interfacial film based on LZPI-CSPA complexes effectively decreased the contents of lipid hydroperoxide and malondialdehyde in HIPEs, thus improving their oxidation stability. The encapsulation of ß-carotene by LZPI-CSPA complex-stabilized HIPEs could further enhance its retention rate against different environmental stresses. After in vitro simulated digestion, the bioaccessibility of ß-carotene also improved, reaching the highest value in HIPEs containing 1.5 % CSPA conjugate. These findings will give a reference for the fabrication of delivery vehicles to enhance the stability and bioaccessibility of bioactive components.

Effect of chitosan-protocatechuic acid conjugate on stability and encapsulation capacity of polysaccharide-based high internal phase emulsion.

The aim of this work was to fabricate chitosan-protocatechuic acid (CSPA) conjugates by free radical grafting method and use them as novel emulsifiers to inhibit lipid oxidation and delay the photodegradation rate of curcumin in polysaccharide-based high internal phase emulsions (HIPEs). Results of UV-vis, FT-IR and 1H NMR spectra demonstrated that PA had been successfully bonded to chitosan (CS) through ester and amino linkages. CSPA conjugates (especially those with the ratio of CS to PA of 1:0.75) showed significantly increased water solubility and antioxidant activity than CS monomer. Furthermore, compared with sole OSA starch (OSAS), the electrostatic combination of CS and CSPA conjugate with OSAS could further reduce the interfacial tension, which was conducive to their adsorption at the oil-water interface. The introduction of CS and CSPA conjugate also increased the viscosity of aqueous phase and promoted the formation of gel-like percolating network structure, thereby effectively preventing droplets coalescence and endowing HIPEs with ideal viscoelasticity. More importantly, the contents of lipid hydroperoxide (24.09 µmol/g oil) and malondialdehyde (166.71 nmol/g oil) in HIPEs prepared by OSAS-CS-CSPA complexes were lower than those stabilized by OSAS, OSAS-CS and OSAS-CSPA complexes during accelerated storage. In addition, HIPEs prepared by OSAS-CS-CSPA complexes showed stronger protection capacity on curcumin against ultraviolet irradiation and natural light degradation. This study will provide useful information and technical reference for the fabrication of antioxidant polysaccharide-based HIPEs delivery vehicles.

Recent advances on food-grade water-in-oil emulsions: Instability mechanism, fabrication, characterization, application, and research trends.

Owing to their promising application prospects, water-in-oil (W/O) emulsions have aroused continuous attention in recent years. However, long-term stability of W/O emulsions remains a particularly challenging problem in colloid science. With the increasing demand of consumers for natural, green, and healthy foods, the heavy reliance on chemically synthesized surfactants to achieve long-term stability has become the key technical defect restricting the application of W/O emulsions in food. To design and manufacture W/O emulsions with long-term stability and clean label, a comprehensive understanding of the fundamentals of the W/O emulsion system is required. This review aims to demystify the field of W/O emulsions and update its current research progress. We first provide a summary on the essential basic knowledge regarding the instability mechanisms, including physical and chemical instability in W/O emulsions. Then, the formulation of the W/O emulsion system is introduced, particularly focusing on the use of natural stabilizers. Besides, the characterization and application of W/O emulsions are also discussed. Finally, we propose promising research trends, including (1) developing W/O high internal phase emulsions (HIPEs) as fat mimetic and substitute, (2) promising formulation routine for long-term stable double emulsions, and (3) searching for novel plant-derived stabilizers of W/O emulsions.

Fabrication of chitosan-protocatechuic acid conjugates to inhibit lipid oxidation and improve the stability of ß-carotene in Pickering emulsions: Effect of molecular weight of chitosan.

In this study, chitosan (CS) with different molecular weights was functionalized with protocatechuic acid (PA) by free-radical grafting reaction, and used for the inhibition of lipid oxidation and the enhancement of stability of ß-carotene in Pickering emulsions. The order of grafting ratio of PA in CS-PA conjugates was CS400 (400 kDa CS) > CS200 (200 kDa CS) > CS100 (100 kDa CS). UV-vis, FT-IR and 1H NMR spectra proved that PA was covalently bonded to CS through amino and ester linkages. Compared with native CS, three CS-PA conjugates exhibited reduced crystallinity and thermal stability and improved antioxidant activity, with a molecular weight-dependent relationship. Besides, CS-PA-conjugate particles formed by ionic gelling procedure were spherically shaped and homogeneously dispersed, which substantially improved the stability of ß-carotene in Pickering emulsions than CS particles under ultraviolet irradiation, natural light exposure and heat treatment, and the retention rates of ß-carotene were in the following order: CS200-PA- > CS400-PA- > CS100-PA-conjugate particles. Furthermore, the oxidation stability of Pickering emulsions fabricated by CS-PA-conjugate particles was also higher than that of CS particles. These results will provide valuable information for the application of CS-PA conjugates to protect bioactive components and inhibit lipid oxidation in emulsion systems.

Fabrication and characterization of oleogels and temperature-responsive water-in-oil emulsions based on candelilla (Euphorbia cerifera) wax.

Developing novel fats with zero trans and low saturated fatty acids represents a research hotspot in the colloid field today. Herein, natural candelilla (Euphorbia cerifera) wax was used as an oleogelator to construct oleogel systems, and can make strong oleogels at low concentrations (3 wt%). These oleogels were further employed as continuous phases to fabricate surfactant-free W/O emulsions with excellent stability (at least 30 days). Microstructural observation confirmed that the stability of emulsions was attributed to the interface and bulk phase crystallization of wax. All oleogels and emulsions were pseudoplastic fluids whose gel properties could be tuned via regulating oleogelator concentration. Water content also influenced the emulsion rigidity, denoting the droplets acted as "active fillers". Additionally, the emulsions displayed a temperature-responsive property, beneficial in mimicking the "fat-like" melt-in-the-mouth effect. These findings greatly enrich the formulation of surfactant-free W/O emulsions, providing technical support for the development of novel fats.

Recent advances on formation mechanism and functionality of chitosan-based conjugates and their application in o/w emulsion systems: A review.

Chitosan is very attractive in the food industry due to its good biocompatibility and high biodegradability. In particular, it can be used as a preferred material for the fabrication of stabilizers in emulsion-based foods. However, poor solubility and antioxidant activity limit its wide application. The functionality of chitosan can be extended by forming chitosan-based conjugates, which can be used to modulate the characteristics of the oil-water interface, thereby improving the stability and performance of the o/w emulsions. This review highlights the recent progress of chitosan-based conjugates, focusing on the classification, formation mechanism and functional properties, and the applications of these conjugates in o/w emulsions are summarized. Lastly, the promising research trends and challenges of chitosan-based conjugates and their emulsion systems in this field are also discussed. This review will provide a theoretical basis for the wide application of chitosan-based conjugates in emulsion systems.

Solubility and emulsifying properties of perilla protein isolate: Improvement by phosphorylation in the presence of sodium tripolyphosphate and sodium trimetaphosphate.

To investigate the mechanisms of phosphates affecting the solubility and emulsifying properties of perilla protein isolate (PPI), PPI was treated with sodium trimetaphosphate (STMP) and sodium tripolyphosphate (STPP) at different concentrations. FTIR and XPS showed that phosphate groups were bonded to PPI through COP bonds. The introduction of STPP increased the particle size and electronegativity of PPI, resulting in the exposure of hydrophobic groups and free -SH groups, as well as the changes of secondary structure and tertiary conformation. All these variations induced increased oil holding capacity, thermal stability, solubility, emulsifying and foaming properties of PPI, especially at STPP concentration of 8%. Furthermore, o/w emulsions stabilized by STPP-modified PPI exhibited higher stability, which could be attributed to the higher interfacial adsorption protein amount and smaller droplet size. These results indicated that PPI modified by phosphate (especially STPP) can be used in the construction of food-grade emulsion systems.

Modification of functional properties of perilla protein isolate by high-intensity ultrasonic treatment and the stability of o/w emulsion.

This study investigated the effects of ultrasonic treatment on the structural characteristics and functional properties of perilla protein isolate (PPI). Besides, the performance of the emulsions stabilized by ultrasonic-treated PPI was analyzed, aiming at exploring the potential mechanism of ultrasonic technology to improve emulsion stability. Results showed that ultrasonic treatment reduced the particle size, induced the exposure of hydrophobic groups and changes in the secondary structure and tertiary conformation of PPI. However, the molecular weight and the crystalline regions were remained unchanged. Apart from this, ultrasonic treatment improved the solubility, water/oil holding capacity, foaming and emulsifying capacity of PPI. Furthermore, the emulsions prepared by ultrasonic-treated PPI possessed the highest stability, which might be due to the smaller droplets size and reduced droplets attraction by higher proportion of interfacial adsorbed protein. This findings will provide a new insight into the application of ultrasonic to improve the stability of PPI-stabilized emulsions.

Modulation of the structural and functional properties of perilla protein isolate from oilseed residues by dynamic high-pressure microfluidization.

Dynamic high-pressure microfluidization (DHPM) is an alternative method to physically modify proteins to improve their functional properties. In this study, perilla protein isolate (PPI) was treated by DHPM at different pressures. Results showed that DHPM treatment reduced the particle size and absolute potential of PPI by 75.90% and 22.28%. The increased surface hydrophobicity and free sulfhydryl content were observed in DHPM-treated PPI, which may be caused by the comformation changes of PPI. Furthermore, DHPM treatment would not cause the degradation of the main subunits and the variation of crystalline regions in PPI, but enhancing the thermal stability of PPI at 90 MPa and 120 MPa. Functional properties analysis indicated that DHPM treatment at 120 MPa was more effective in improving the solubility, foaming and emulsifying capacities of PPI. The results suggested that DHPM can be used to enhance the functional properties of PPI and expand its application in food systems.

Recent advances on protein-based Pickering high internal phase emulsions (Pickering HIPEs): Fabrication, characterization, and applications.

Most trans fats in processed foods come from partially hydrogenated oils (PHOs). Numerous studies have shown that the excessive intake of trans fats may cause some adverse effects on human health. An effective alternative to PHOs is to construct Pickering high internal phase emulsions (HIPEs). In recent years, considerable attention has been paid to Pickering HIPEs stabilized by protein-based particles because of their high stability and promising applications in the fields of food, cosmetics, and pharmaceutical industries. This review summarizes the recent progress of Pickering HIPEs stabilized by protein-based particles focusing on the methods for their preparation and characterization, and discusses the applications of Pickering HIPEs in the food industry. Promising research trends in this field are also proposed, including (a) developing protein-based antioxidant Pickering HIPEs and Pickering nanoemulsions, and (b) expanding the potential applications of protein-based Pickering HIPEs in the fields of delivery vehicles, the template for porous materials, and biodegradable packaging films. This review will provide a theoretical basis for future technological innovation and application development of protein-based Pickering HIPEs.

Polysaccharides from pineapple pomace: new insight into ultrasonic-cellulase synergistic extraction and hypoglycemic activities.

This study determined the optimal extraction conditions for ultrasonic-cellulase synergistic extraction of polysaccharides from pineapple pomace (PPP) using Plackett-Burman design and response surface methodology. The optimal hydrolysis temperature, ratio of material to water, pH value, hydrolysis time, ultrasonic power and the additive quantity of cellulase were 50 °C, 1:45 g/mL, 6.0, 100 min, 160 W and 2.0%, respectively, giving a extraction yield of 1.10 ±â€¯0.03%. PPP was further isolated and purified by DEAE-52 cellulose and Sephadex G-100 chromatography columns, revealing four main elution peaks, named PPF0, PPF1, PPP2 and PPF3, were obtained. The molecular weight, monosaccharide compositions, structural features and appearance morphology of polysaccharide fractions (PPFs) were analyzed by high-performance liquid chromatography (HPLC), gel permeation chromatography (GPC), UV spectroscopy, fourier transform infrared spectroscopy (FT-IR), and scanning electron microscopy (SEM). Furthermore, the hypoglycemic activities of PPFs with different concentrations were also investigated by insulin resistance HepG2 cells model in vitro. Results showed that PPF0, PPF1, PPF2 and PPF3 were composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose and fucose with molecular weight of 6.71 × 104, 1.11 × 104, 2.22 × 104 and 5.1 × 103 Da, respectively. All of them could alleviate the development of insulin resistance HepG2 cells with a dose-dependent relationship. The glucose consumption increased 46.4%, 50.5%, 82.1% and 53.6%; 86.8%, 81.6%, 86.8% and 84.2% at the concentration of 20 µg/mL, respectively, without or with insulin. These results suggested that PPFs can be explored as a potential hypoglycemic agent in biomedical and functional food.

Optimization extraction, characterization and anticancer activities of polysaccharides from mango pomace.

Response surface methodology was used to optimize the extraction conditions for ultrasonic-assisted extraction of polysaccharides from mango pomace. The Optimum extraction conditions consisted of extraction temperature of 74 °C, ultrasonic power of 170 W, extraction time of 100 min, and raw material-to-water ratio of 1:40 g/mL. Under these conditions, the extraction yield was 3.71 ±â€¯0.07%. Three novel polysaccharide fractions, MG-1, MG-2 and MG-3 were purified from the crude polysaccharides by using DEAE-52 cellulose and Sephadex G-100 column chromatography. The molecular weight and monosaccharide composition of polysaccharide fractions (MPFs) were analyzed by high performance liquid gel permeation chromatography (HPGPC) and HPLC analysis, respectively. The characterizations of MPFs were conducted with FT-IR, 1H NMR and SEM. Furthermore, the anticancer activities of the polysaccharide fractions were also investigated in vitro. Results showed that MG-1, MG-2 and MG-3 exhibited significant anticancer activities against HepG2, MCF-7, A549, HeLa, A2780, HCT-116 and BGC-823 cells in a dose-dependent manner. MPFs were also showed to promote apoptosis as seen in the nuclear morphological examination study using calcein acetyl methoxy methyl easter (calcein-AM) and propidium iodide (PI) staining. This research could serve as a theoretical reference for the efficient utilization of MPFs in biomedical and functional food.

Optimization extraction and functional properties of soluble dietary fiber from pineapple pomace obtained by shear homogenization-assisted extraction.

Response surface methodology (RSM) was used to optimize the extraction conditions for shear homogenization-assisted extraction of soluble dietary fiber from pineapple pomace (s-SDF), and the absorption capacities and antioxidant activities of the obtained s-SDF were also investigated. The optimum extraction conditions consisted of a cutting speed of 9000 rpm, a cutting time of 20 min, a cellulase content of 5.0%, a hydrolysis time of 120 min, a pH value of 4.5, a hydrolysis temperature of 50 °C, and a raw material to water ratio of 1 : 45 g mL-1. Under these conditions, the theoretical and actual extraction yields of s-SDF were 8.80% and 8.76%, respectively. An absorption capacity analysis indicated that s-SDF exhibited higher absorption abilities to sodium cholate, cholesterol and fat. In addition, s-SDF possessed higher antioxidant activities, showing a positive concentration effect relationship for DPPH˙, ABTS+, ·OH and O2 -˙. The concentration of 1.0 mg mL-1 scavenged 76.72% DPPH˙, 58.40% ABTS+, 23.47% ·OH and 48.47% O2 -˙, respectively, and the reduction power was 0.70. These results indicated that pineapple pomace is a potential source of natural dietary fiber and a potential functional food ingredient.

Network pharmacology of cancer: From understanding of complex interactomes to the design of multi-target specific therapeutics from nature.

Despite massive investments in drug research and development, the significant decline in the number of new drugs approved or translated to clinical use raises the question, whether single targeted drug discovery is the right approach. To combat complex systemic diseases that harbour robust biological networks such as cancer, single target intervention is proved to be ineffective. In such cases, network pharmacology approaches are highly useful, because they differ from conventional drug discovery by addressing the ability of drugs to target numerous proteins or networks involved in a disease. Pleiotropic natural products are one of the promising strategies due to their multi-targeting and due to lower side effects. In this review, we discuss the application of network pharmacology for cancer drug discovery. We provide an overview of the current state of knowledge on network pharmacology, focus on different technical approaches and implications for cancer therapy (e.g. polypharmacology and synthetic lethality), and illustrate the therapeutic potential with selected examples green tea polyphenolics, Eleutherococcus senticosus, Rhodiola rosea, and Schisandra chinensis). Finally, we present future perspectives on their plausible applications for diagnosis and therapy of cancer.

Biopiracy of natural products and good bioprospecting practice.

BACKGROUND: Biopiracy mainly focuses on the use of biological resources and/or knowledge of indigenous tribes or communities without allowing them to share the revenues generated out of economic exploitation or other non-monetary incentives associated with the resource/knowledge. METHODS: Based on collaborations of scientists from five continents, we have created a communication platform to discuss not only scientific topics, but also more general issues with social relevance. This platform was termed 'PhytCancer -Phytotherapy to Fight Cancer' (www.phyt-cancer.uni-mainz.de). As a starting point, we have chosen the topic "biopiracy", since we feel this is of pragmatic significance for scientists working with medicinal plants. RESULTS: It was argued that the patenting of herbs or natural products by pharmaceutical corporations disregarded the ownership of the knowledge possessed by the indigenous communities on how these substances worked. Despite numerous court decisions in U.S.A. and Europe, several international treaties, (e.g. from United Nations, World Health Organization, World Trade Organization, the African Unity and others), sharing of a rational set of benefits amongst producers (mainly pharmaceutical companies) and indigenous communities is yet a distant reality. In this paper, we present an overview of the legal frameworks, discuss some exemplary cases of biopiracy and bioprospecting as excellent forms of utilization of natural resources. CONCLUSIONS: We suggest certain perspectives, by which we as scientists, may contribute towards prevention of biopiracy and also to foster the fair utilization of natural resources. We discuss ways, in which the interests of indigenous people especially from developing countries can be secured.

Inhibition of c-MYC with involvement of ERK/JNK/MAPK and AKT pathways as a novel mechanism for shikonin and its derivatives in killing leukemia cells.

Leukemia remains life-threatening despite remarkable advances in chemotherapy. The poor prognosis and drug resistance are challenging treatment. Novel drugs are urgently needed. Shikonin, a natural naphthoquinone, has been previously shown by us to be particularly effective towards various leukemia cell lines compared to solid tumors. However, the underlying mechanisms are still poorly understood. Here, we investigated shikonin and 14 derivatives on U937 leukemia cells. Four derivatives (isobutyrylshikonin, 2-methylbutyrylshikonin, isovalerylshikonin and ß,ß-dimethylacrylshikonin) were more active than shikonin. AnnexinV-PI analysis revealed that shikonins induced apoptosis. Cell cycle G1/S check point regulation and the transcription factor c-MYC, which plays a vital role in cell cycle regulation and proliferation, were identified as the most commonly down-regulated mechanisms upon treatment with shikonins in mRNA microarray hybridizations. Western blotting and DNA-binding assays confirmed the inhibition of c-MYC expression and transcriptional activity by shikonins. Reduction of c-MYC expression was closely associated with deregulated ERK, JNK MAPK and AKT activity, indicating their involvement in shikonin-triggered c-MYC inactivation. Molecular docking studies revealed that shikonin and its derivatives bind to the same DNA-binding domain of c-MYC as the known c-MYC inhibitors 10058-F4 and 10074-G5. This finding indicates that shikonins bind to c-MYC. The effect of shikonin on U937 cells was confirmed in other leukemia cell lines (Jurkat, Molt4, CCRF-CEM, and multidrug-resistant CEM/ADR5000), where shikonin also inhibited c-MYC expression and influenced phosphorylation of AKT, ERK1/2, and SAPK/JNK. In summary, inhibition of c-MYC and related pathways represents a novel mechanism of shikonin and its derivatives to explain their anti-leukemic activity.

Shikonin and its derivatives inhibit the epidermal growth factor receptor signaling and synergistically kill glioblastoma cells in combination with erlotinib.

Overexpression and mutation of the epidermal growth factor receptor (EGFR) gene play a causal role in tumorigenesis and resistance to treatment of glioblastoma (GBM). EGFR inhibitors such as erlotinib are currently used for the treatment of GBM; however, their efficacy has been limited due to drug resistance. New treatment strategies are therefore urgently needed. Shikonin, a natural naphthoquinone, induces both apoptosis and necroptosis in human glioma cells, but the effectiveness of erlotinib-shikonin combination treatment as well as the underlying molecular mechanisms is unknown yet. In this study, we investigated erlotinib in combination with shikonin and 14 shikonin derivatives in parental U87MG and transfected U87MG.ΔEGFR GBM cells. Most of the shikonin derivatives revealed strong cytotoxicity. Shikonin together with five other derivatives, namely deoxyshikonin, isobutyrylshikonin, acetylshikonin, ß,ß-dimethylacrylshikonin and acetylalkannin showed synergistic cytotoxicity toward U87MG.ΔEGFR in combination with erlotinib. Moreover, the combined cytotoxic effect of shikonin and erlotinib was further confirmed with another three EGFR-expressing cell lines, BS153, A431 and DK-MG. Shikonin not only dose-dependently inhibited EGFR phosphorylation and decreased phosphorylation of EGFR downstream molecules, including AKT, P44/42MAPK and PLCγ1, but also together with erlotinib synergistically inhibited ΔEGFR phosphorylation in U87MG.ΔEGFR cells as determined by Loewe additivity and Bliss independence drug interaction models. These results suggest that the combination of erlotinib with shikonin or its derivatives might be a potential strategy to overcome drug resistance to erlotinib.