Human Hepatocellular Liver Carcinoma Research Articles

Lactobionic acid-functionalized hollow mesoporous silica nanoparticles for cancer chemotherapy and phototherapy

The combination of chemotherapy and phototherapy has become a promising cancer treatment strategy. In this study, a pH/NIR dual-responsive hollow mesoporous silica nanoparticles (HMSNs) based nanocarrier for co-delivering doxorubicin hydrochloride (DOX) and indocyanine green (ICG) was developed. DOX was loaded for chemotherapy and ICG for photothermal therapy (PTT) and photodynamic therapy (PDT). This system targets the asialoglycoprotein receptor (ASGPR) in over-expressing human hepatocellular liver carcinoma cells (HepG2) by linking HMSNs and lactobionic acid (LA)-modified dialdehyde chitosan (DACS) via Schiff base bonds. It is demonstrated that HMSNs-DACS-LA have been successfully prepared through various physical characterizations. Drug release tests revealed that it had the characteristics of properties of pH/NIR dual-responsive. In vitro cellular uptake and cytotoxicity assay showed that ID@HMSNs-DACS-LA could target HepG2 cells and reduced the activity of tumor cells. Therefore, co-delivery of ICG and DOX via HMSNs-DACS-LA nanocarriers may provide a feasible strategy for tumor-targeted combined chemotherapy and phototherapy.

Two La(III) complexes containing pyridine-2,6-dicarboxylate as in vitro potent cytotoxic agents toward human lymphocyte cells

Two La(III) complexes (C1) and (C2) were designed througha one-pot reaction between pyridine-2,6-dicarboxylic acid, 2-aminobenzimidazole and/or 2-aminopyridine with lanthanum(III) nitrate hexahydrate. The structure of compounds was confirmed by single-crystal X-ray diffraction and distorted tricapped triangular prism geometry was suggested for them. The inhibitory potent of the compounds was tested in vitro against four cell lines including HL60 (a human lymphocyte), HepG2 (a human liver hepatocellular carcinoma), HT29 (a human colon adenocarcinoma), and HFF (a human foreskin fibroblast). The most prominent antiproliferative effect was indicated for both C1 (IC50 = 0.69 μM, MAE = 85.34 %) and C2 (IC50 = 62.50 μM, MAE = 61.71 %) against the HL60 cell line. The assay of the reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) confirmed that apoptosis through a mitochondria pathway may be the main mechanism for the death of HL60 cells treated with the complexes.

New Benzil and Isoflavone Derivatives with Cytotoxic and NO Production Inhibitory Activities from Placolobium vietnamense.

The phytochemical investigation of Placolobium vietnamense stems led to the isolation of a new isoflavone derivative (1) and three new benzil derivatives (2–4), together with four known pyranoisoflavones (5–8). The structures of all isolated compounds were determined on the basis of extensive spectroscopic analyses, including NMR and HRMS spectral data, as well as comparison of their spectroscopic data with those reported in the literature. The cytotoxicity of all isolated compounds was assessed against the human liver hepatocellular carcinoma (Hep G2) cell line, and compound 1 displayed the most significant cytotoxicity with an IC50 value of 8.0 μM. Furthermore, all isolated compounds were also tested for their inhibitory activity against NO production in RAW 264.7 macrophages. Of these, compound 1 exhibited the strongest inhibitory efficacy against the LPS-induced NO production with the IC50 value of 13.7 μM.

Toxicological effects of polystyrene nanoplastics and perfluorooctanoic acid to Gambusia affinis

Plastic pollution has attracted huge attention from public and scientific community in recent years. In the environment, nanoplastics (NPs, <100 nm) can interact with persistent organic pollutants (POPs) such as perfluorooctanoic acid (PFOA) and may exacerbate associated toxic impacts. The present study aims to explore the single and combined ecotoxicological effects of PFOA and polystyrene nanoplastics (PS-NPs, 80 nm) on the PI3K/AKT3 signaling pathway using a freshwater fish model Gambusia affinis. Fish were exposed individually to PS-NPs (200 μg/L) and PFOA (50, 500, 5000 μg/L) and their chemical mixtures for 96 h. Our results showed that the co-exposure significantly altered the mRNA relative expression of PI3K, AKT3, IKKβ and IL-1β, compared to corresponding single exposure and control groups, indicating that the PFOA-NP co-exposure can activate the PI3K/AKT3 signaling pathway. The bioinformatic analyses showed that AKT3 had more probes and exhibited a significantly sensitive correlation with DNA methylation, compared to other genes (PIK3CA, IKBKB, and IL1B). Further, the mRNA expressions of PIK3CA, AKT3, and IKBKB had a significant correlation with copy number variation (CNV) in human liver hepatocellular carcinoma (LIHC). And PIK3CA had the highest mutation rate among other genes of interest for LIHC. Moreover, AKT3 showed a relatively lower expression in TAM and CAF cells, compared to PIK3CA, IKBKB, and IL1B. Besides, hsa-mir-155-5p was closely correlated with AKT3, PIK3CA, IKBKB, and IL1B. In summary, these results provide evidence that NPs could enhance the carcinogenic effects of POPs on aquatic organisms and highlight possible targets of LIHC induced by PFOA-NP co-exposure.

Sinensetin suppresses angiogenesis in liver cancer by targeting the VEGF/VEGFR2/AKT signaling pathway.

Sinensetin (SIN) is a polymethoxy flavone primarily present in citrus fruits. This compound has demonstrated anticancer activity. However, the underlying mechanism of its action has not been fully understood. The present study investigated the impact of SIN on angiogenesis in a liver cancer model. In a murine xenograft tumor model, SIN inhibited the growth of HepG2/C3A human liver hepatoma cell-derived tumors and reduced the expression levels of platelet/endothelial cell adhesion molecule-1 and VEGF. In HepG2/C3A cells, SIN repressed VEGF expression by downregulating hypoxia-inducible factor expression. In cultured human umbilical vein endothelial cells, SIN increased apoptosis and repressed migration and tube formation. In addition, SIN decreased the phosphorylation of VEGFR2 and inhibited the AKT signaling pathway. Molecular docking demonstrated that the VEGFR2 core domain effectively combined with SIN at various important residues. Collectively, these data suggested that SIN inhibited liver cancer angiogenesis by regulating VEGF/VEGFR2/AKT signaling.

In-Vitro Evaluation of Anticancer and Antibacterial Properties of Pseudocerastes Persicus Snake Venom Fractions

Background and Objectives In recent years the isolation of effective molecules from snake venom as drug to treatment of some incurable diseases is considered by many biological research centers. The aim of this investigation was to determine electrophorotic and chromatographic patterns of Pseudocerastes persicus venom and in-vitro study of bactericidal and anti-cancer properties of its fractions in human hepatocellular liver carcinoma cell line (HepG2). Subjects and Methods The fractions of venom were isolated and collected using preparative RP-HPLC. The Bactericidal activity of fractions in 20μg/ml of protein concentration was investigated toward Gramnegative and Gram-positive bacteria using MTT and MIC assays. Then, the cytotoxic effects of fractions with higher antibacterial properties were measured by MTT reduction, neutral uptake and comet assays following exposure of HepG2 cancer cells to 20 and 40 μg/ml of protein concentration. Results SDS-PAGE pattern of the crude venom revealed 10 major bands with molecular weight ranging from 13.2 to 99.25 kDa and the separation of fractions from the venom by HPLC resulted in collection of 11 different fractions. The results of this study showed that two fractions (4 &amp; 8) have a significant antibacterial activity against Gram-positive as compared to Gram- negative bacteria. The study of the anti-cancer effect of these two fractions also showed that fraction 8 was more toxic in HepG2 cells. Conclusion We for the first time reported bactericidal and anti cancer effects of isolated fractions of Persian horned viper venom. These properties make components of these fractions a favorable source for isolation of molecule (s) with antibacterial and anti tumor activities.

Metabolomic Analysis Reveals the Mechanisms of Hepatotoxicity Induced by Aflatoxin M1 and Ochratoxin A.

Aflatoxin M1 (AFM1) is the only toxin with the maximum residue limit in milk, and ochratoxin A (OTA) represents a common toxin in cereals foods. It is common to find the co-occurrence of these two toxins in the environment. However, the interactive effect of these toxins on hepatoxicity and underlying mechanisms is still unclear. The liver and serum metabolomics in mice exposed to individual AFM1 at 3.5 mg/kg b.w., OTA at 3.5 mg/kg b.w., and their combination for 35 days were conducted based on the UPLC-MS method in the present study. Subsequent metabolome on human hepatocellular liver carcinoma (Hep G2) cells was conducted to narrow down the key metabolites. The phenotypic results on liver weight and serum indicators, such as total bilirubin and glutamyltransferase, showed that the combined toxins had more serious adverse effects than an individual one, indicating that the combined AFM1 and OTA displayed synergistic effects on liver damage. Through the metabolic analysis in liver and serum, we found that (i) a synergistic effect was exerted in the combined toxins, because the number of differentially expressed metabolites on combination treatment was higher than the individual toxins, (ii) OTA played a dominant role in the hepatoxicity induced by the combination of AFM1, and OTA and (iii) lysophosphatidylcholines (LysoPCs), more especially, LysoPC (16:1), were identified as the metabolites most affected by AFM1 and OTA. These findings provided a new insight for identifying the potential biomarkers for the hepatoxicity of AFM1 and OTA.

Effect of rosiglitazone on 20-hydroxyeicosatetraenoic acid levels and CYP4F2 expression in HepG2 cells

&#x0D; &#x0D; &#x0D; &#x0D; Purpose: To determine the effect of rosiglitazone on the levels of the cardiotoxic arachidonic acid metabolite, 20-hydroxyeicosatetraenoic acid (20-HETE), in the human liver hepatocellular carcinoma cell line, HepG2.&#x0D; Methods: HepG2 cells were treated with thiazolidinedione rosiglitazone and the mRNA and protein expressions of cytochrome P450 4F2 (CYP4F2) responsible for synthesizing 20-HETE were measured using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. The levels of 20-HETE were evaluated using liquid chromatography/mass spectrometry (LC-MS).&#x0D; Results: Rosiglitazone significantly increased the levels of CYP4F2 mRNA and protein when compared with the control group (p &lt; 0.05). This was correlated with significantly increased 20-HETE levels in the culture medium of rosiglitazone-treated cells in a dose-dependent manner (p &lt; 0.05). The PPARγ antagonist, GW9662, significantly repressed the increased production of 20-HETE and CYP4F2 mRNA protein (p &lt; 0.05).&#x0D; Conclusion: Rosiglitazone increases the synthesis of 20-HETE via activation of PPARγ receptor and upregulation of CYP4F2. These findings may provide an additional explanation, at least in part, for the unwanted side effects of rosiglitazone on the cardiovascular system.&#x0D; &#x0D; &#x0D; &#x0D;

Synthesis and Critical View on the Structure-Activity Relationships of N-(Substituted phenyl)-/N-Diphenylmethyl-piperazine-Based Conjugates as Antimycobacterial Agents

This research focused on a three-step synthesis, analytical, physicochemical, and biological evaluation of hybrid molecules 6a–g, containing a lipophilic 3-trifluoromethylphenyl moiety, polar carbamoyloxy bridge, 2-hydroxypropan-1,3-diyl chain and 4-(substituted phenyl)-/4-diphenylmethylpiperazin-1-ium-1-yl fragment. The estimation of analytical and physicochemical descriptors (m/zmeasured via HPLC-UV/HR-MS, log ε2 (Ch–T) from UV/Vis spectrophotometry and log kw via RP-HPLC) as well as in vitro antimycobacterial and cytotoxic screening of given compounds were carried out (i.e., determination of MIC and IC50 values). These highly lipophilic molecules (log kw = 4.1170–5.2184) were tested against Mycobacterium tuberculosis H37Ra ATCC 25177 (Mtb H37Ra), M. kansasii DSM 44162 (MK), M. smegmatis ATCC 700084 (MS), and M. marinum CAMP 5644 (MM). The impact of the 6a–g set on the viability of human liver hepatocellular carcinoma (HepG2) cells was also investigated. 1-[2-Hydroxypropyl-{(3-trifluoromethyl)- phenyl}carbamoyloxy]-4-(3,4-dichlorophenyl)piperazin-1-ium chloride (6e) and 1-[2-hydroxy- propyl-{(3-trifluoromethyl)phenyl}carbamoyloxy]-4-(4-diphenylmethyl)piperazin-1-ium chloride (6g) most effectively inhibited the growth of Mtb H37Ra (MIC &lt; 3.80 μM). The substance 6g also showed interesting activity against MM (MIC = 8.09 μM). All obtained data served as input values for structure-activity relationship evaluations using statistical principal component analysis. In fact, the toxicity of both 6e (IC50 = 29.39 μM) and 6g (IC50 = 22.18 μM) in HepG2 cells as well as selectivity index (SI) values (SI &lt; 10.00) prevented to consider these promising antimycobacterials safe.

In Vitro Cytotoxicity and Oxidative Stress Evaluation of Valerian (Valeriana officinalis) Methanolic Extract in Hepg2 and Caco2 Cells

Traditional treatment methods are becoming popular and commonly used in many societies and have become the first treatment option for most people. While some of these methods are helpful, they can interact with medications the patient is taking for another disease and cause a variety of life-threatening risks. Valerian (catweed) plant is used in traditional medicine as a sleep aid due to its sedative effects. Valerian may also exert anticancer effect in vitro. In this study, the cytotoxicty and oxidative stress effects of valerian root extract were evaluated in human liver hepatocellular carcinoma (Hepg2) and human colorectal adenocarcinoma (Caco2) cell lines. The cytotoxicity was evaluated via the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide test. Total reactive oxygen species analysis was performed via a 2',7'-dichlorodihydrofluorescein diacetate assay in flow cytometry. Inhibition concentration 50 values were calculated as 936.6 and 1097.5 µg/mL in the Hepg2 and Caco2 cell lines, respectively. It was observed that valerian root extract did not induce oxidative stress in HepG2 and Caco2 cell lines. These results indicate that the use of valerian root extract as an alternative method in cancer treatment may not be effective and may cause a risk for public health. On the other hand, it may be safe at recommended tolerated concentrations since it does not cause oxidative stress.

Cytotoxicity and apoptosis response of hexagonal zinc oxide nanorods against human hepatocellular liver carcinoma cell line

Hexagonal zinc oxide nanorods (ZnO-NRs) were prepared via solution process using zinc nitrate hexahydrate (Zn(NO3)2·6H2O, 0.25 M), hexamethylenetetramine (HMT) and sodium hydroxide at low concentration (4 × 10-3 M in 100 ml of distilled water) and refluxed at 100 °C for an hour. The HMT was used because its act as a template for the nucleation and growth of zinc oxide nanorods. The X-ray diffraction patterns (XRD) clearly reveal that the grown product is pure zinc oxide. The diameters and lengths of the synthesized nanorods lie between 200 and 250 nm and 2–3 μm respectively as observed from the field emission scanning electron microscopy (FESEM). The chemical functional properties was analyzed by Fourier Transform infra-red (FTIR) spectroscopy. This study was designed to show the possible effect of ZnO-NRs in human liver cancer (HepG2) cells. It induces cytotoxicity via reactive oxygen species (ROS), mitochondrial membrane potential. For gene expression analysis, it induces apoptotic gene marker P53, Bax, caspase3, Bcl2 genes. Based on analysis and observations, it concludes that the ZnO-NRs were utilized for toxicity at dose dependent via ROS generation against human hepato cellular liver carcinoma cell line.

Influence of vitamin K2 on lipid precursors of inflammation and fatty acids pathway activities in HepG2 cells.

Vitamin K2 (VK2) is one of the two types of vitamin K present most in the human diet. VK2 seems to have a beneficial effect on inflammation related to type 2 diabetes mellitus. The aim of this study was to evaluate the influence of VK2 on lipid precursors of inflammation in lipid-overloaded human liver hepatocellular carcinoma cells. Cells were incubated with VK2 and/or palmitic acid (PA). The concentrations of lipid fractions and their fatty acid compositions were measured by gas-liquid chromatography. The expression of proteins involved in the inflammatory process was detected using western blotting. The concentration of triacylglycerols (TAGs), activities of the n-3 pathway in TAGs, and lipooxygenase 15 expression were significantly elevated in cells incubated with PA and VK2. In the same group, a marked elevation in diacylglycerol (DAG) 20:4 was observed. VK2 supplementation lowered the expression of tumour necrosis factor-alpha and interleukin-6 compared to that in the PA group. The data indicate that VK2 redirects fatty acid metabolism into the deposition of a safe TAG fraction by increasing the concentration of anti-inflammatory n-3 polyunsaturated fatty acids in this fraction. Moreover, VK2 stimulates the synthesis of anti-inflammatory factors and has anti-inflammatory effects by reducing DAG 20:4.

Preparation and characterization of sulfated inulin-type fructans from Jerusalem artichoke tubers and their antitumor activity

The modification of polysaccharides is important for enhancing their biological activities. In this study, a pure inulin-type fructan, denoted as Jerusalem artichoke polysaccharide (P-JAP), was purified from Jerusalem artichoke tubers and modified by sulfation via treatment with a sulfur trioxide-pyridine complex to produce its sulfated derivative (S-JAP). Fourier-transform infrared spectroscopic analysis confirmed the successful introduction of sulfate groups. The inhibitory effects of S-JAP on the proliferation of human liver hepatocellular carcinoma (HepG2) cells was evaluated via a CCK-8 assay, and the pro-apoptotic effects were assessed using annexin V-FITC/PI double staining. The inhibition rates of various concentrations of S-JAP on HepG2 cells after 24, 48, and 72 h were significantly higher than those of P-JAP; moreover, S-JAP succeeded in promoting cell apoptosis. Thus, the sulfate-modified polysaccharide extracted from Jerusalem artichoke tubers was shown to exhibit effective antitumor activity with potential for further development.

Evaluation of in-vitro bactericidal activity and anticancer effects of venom fractions of Pseudocerastes persicus snake

Introduction: It was reported that snake venom contains a wide mixture of proteins and peptides with various toxicological and pharmacological effects. In recent years, the isolation of effective molecules from snake venom as a drug to treatment of some incurable diseases is considered by many biological research centers. This investigation aimed to determine electrophoretic and chromatographic patterns of Pseudocerastes persicus snake venom and in-vitro study of bactericidal and anti-cancer properties of its fractions in a human hepatocellular liver carcinoma cell line (HepG2). Materials and methods: The fractions of venom were isolated and collected using preparative RP-HPLC. The Bactericidal activity of fractions in 20 μg/ml of protein concentration was investigated toward Gram-negative and Gram-positive bacteria. Antibacterial susceptibility tests were done using MTT and MIC assays. Furthermore, cell viability was measured by MTT reduction assay and confirmed with Neutral red uptake assay following exposure of HepG2 cancer cells to 20 and 40 μg/ml of protein concentrations of fractions having more antibacterial properties. Apoptotic effects were investigated using the comet assay. Results: SDS-PAGE pattern of the crude venom revealed 10 major bands with molecular weight ranging from 13.2 to 99.25 kDa and the single-step separation of different fractions from the venom by HPLC resulted in isolation and collection of 11 fractions. The results of this study showed that two fractions of snake venom have significant antibacterial activity against Gram-positive as compared to Gram-negative bacteria. On the other hand, our findings indicated that one of them is not toxic to the HepG2 cells. Discussion: We for the first time reported bactericidal and anticancer effects of isolated fractions of Persian horned viper venom. These properties make components of these fractions a favorable source for isolation of molecule(s) with antibacterial and antitumor activities.

Construction and antitumor activity of selenium nanoparticles decorated with the polysaccharide extracted from Citrus limon (L.) Burm. f. (Rutaceae)

Selenium nanoparticles (SeNPs), a potential cancer therapeutic agent, have attracted widespread attention owing to their high bioavailability and remarkable anticancer activity. Nevertheless, the poor water solubility and dispersibility of SeNPs seriously limit their applications. In the present study, we synthesized stable and individual spherical selenium nanoparticles (CL90-Tw-SeNP2) with an average diameter of approximately 79 nm using a polysaccharide extracted from Citrus limon (CL90) and Tween-80 as the decorator and stabilizers. The proportion of selenium in CL90-Tw-SeNP2 was 10.6%. CL90-Tw-SeNP2 possessed high stability and good dispersion in water for more than three months. The subsequent biological assay revealed that CL90-Tw-SeNP2 showed remarkable antitumor effects against HepG2 cells, with an IC50 value of 49.13 μg/mL, by inducing cell apoptosis. Furthermore, an in vivo zebrafish assay to explore possible applications indicated that CL90-Tw-SeNP2 could inhibit the proliferation and migration of tumors and the zebrafish angiogenesis. These results indicated that CL90-Tw-SeNP2 could be a potential agent for cancer treatment, especially against human liver hepatoma cancer.

Biomimetic Recognition-Based Bioorthogonal Host–Guest Pairs for Cell Targeting and Tissue Imaging in Living Animals

Biomimetic Recognition-Based Bioorthogonal Host–Guest Pairs for Cell Targeting and Tissue Imaging in Living Animals

Pyrroloquinoline quinone alleviates oxidative damage induced by high glucose in HepG2 cells.

Hyperglycemia as a common metabolic disorder in diabetes led to oxidative stress, inflammation and other complications. Natural and manufactured antioxidants alleviates the side effects of diabetes. The purpose of current study is to investigate the effect of pyrroloquinoline quinine (PQQ) as an antioxidant on the content of glucose-induced oxidative stress generation in the cells of the human hepatocellular liver carcinoma (HepG2) by inhibiting advanced glycation end products (AGEs) formation. The HepG2 cells were exposed to high dose (50 mM) of glucose (HG) only and with PQQ (HG + PQQ). Treatment with high dose increased AGEs formation, expression of receptor for advanced glycation endproducts (RAGE), reactive oxygen species ROS production, and oxidative stress markers in treated HepG2 cells. Interestingly, PQQ significantly reduced AGEs formation and (RAGE) expression, ROS formation, and inflammation induced by glucose. In conclusion, PQQ has a potentiail role as an antioxidant to reduce the oxidative damage during hyperglycemia by AGEs inhibition.

4N donor atoms moiety of transition metal complexes of a Schiff base ligand: Synthesis, characterization and biological activities study

Prepared new Schiff base ligand [H2L] with 4N donor atoms moiety by reacting amine derivative of o-phenylenediamine hydrazide moiety with benzaldehyde. Co(II), Ni(II), Cu(II) and Zn(II) of the new Schiff base were prepared by using a molar ratio of ligand: metal as (1:1). The structure of the Schiff base and the prepared complex was determined by 1H NMR, UV–Vis, IR, Mass spectrometry and molar conductivity. The thermal stability of the complexes had been studied by TGA. The spectral statement displays that the ligand behaves as bidentate ligand. On the basis of the spectral studies, reveal that an octahedral geometry had been assigned for all complexes. The Anti-proliferative effect was screened against human hepatocellular liver carcinoma cell line (HepG2) and antimicrobial activity had been inspected. Result points that complexes demonstrate the increase cytotoxicity in proliferation to cell lines as compared to free ligand.

Efficient synthesis and molecular docking studies of new pyrimidine-chromeno hybrid derivatives as potential antiproliferative agents

Various novel heterocyclic compounds containing pyrimidine nuclei 5H-chromeno[4,3-d]pyrimidine (4a–c, e–h, l–r, t) and pyrimidine-5-yl-(2-hydroxyphenyl)methanone (5a, c, d, f–k, m–o, r, s, u) were synthesized from the reaction of guanylhydrazones (2a–u) and 3-formylchromone (3). These compounds were tested against human liver hepatocellular carcinoma cell line (HepG2) and human breast adenocarcinoma cell line (MDA-MB-231) using the MTT assay method. Furthermore, molecular docking calculations were performed to compare the biological activities of various novel heterocyclic compounds against cancer proteins. In these calculations, the protein used are crystal structure of the BRCT repeat region from the breast cancer associated protein, 1JNX, crystal structure of VEGFR kinase (liver cancer) protein, 3WZE, and crystal structure of an allosteric Eya2 phosphates inhibitor (lung cancer) protein, 5ZMA, respectively. After molecular docking calculations, absorption, distribution, metabolism, and excretion/toxicity analysis was performed to examine the properties of various novel heterocyclic compounds for their future use as drugs.

Effect of emulsification on the antioxidant capacity of beef myofibrillar protein-derived bioactive peptides during in vitro human digestion and on the hepatoprotective activity using HepG2 cells

This study aims to determine the effect of emulsification on the antioxidant capacity of bioactive peptide extracts during in vitro human digestion and on their hepatoprotective activity in human liver hepatoma (HepG2) cells. Bioactive peptide extracts are obtained from myofibrillar proteins in beef by alkaline-AK and papain hydrolysis. In addition, emulsions are prepared using lard oil. The antioxidant activity of emulsified bioactive peptide extracts during in vitro human digestion was higher than that of non-emulsified bioactive peptide extracts. Furthermore, both emulsified bioactive peptide extracts and non-emulsified bioactive peptide extracts increased cell viability and decreased cytotoxicity and aspartate aminotransferase activity in HepG2 cells. Hence, emulsification treatment did not influence the hepatoprotective effect. The effect of the emulsification of bioactive peptides used as drug delivery substances can thus be considered to be limited only to the antioxidant activity of the peptides.