The Aggregated and Micellar Forms of ß-Casein Purified from Donkey and Bovine Milk Present Potential as Carriers for Bioactive Nutritional Compounds.

In recent years, there has been a growing interest in the pure casein fraction of milk protein, particularly ß-casein due to its physicochemical properties as well as its bio- and techno-functional properties. The utilization of self-assembled ß-caseins from bovine origin as nanocarriers for the delivery of nutraceutical compounds or drugs has increased dramatically. Concerning ß-caseins from other milk sources, the use of hypoallergenic donkey ß-caseins as a potential delivery vehicle for nutraceutical hydrophobic compounds is beginning to generate interest. The present review deals with casein micelles models, bovine and donkey ß-casein molecular structures, as well as their physical-chemical properties that account for their exploitation in nutraceutics and pharmaceutics. This review work suggests the possibility of developing delivery systems for hydrophobic bioactive compounds using ß-casein purified from hypoallergenic donkey milk, highlighting the potential of this protein as an innovative and promising vehicle for enhancing the enrichment and bioavailability of various bioactive substances in food products.

Effect of shape, gluten, and mastication effort on in vitro starch digestion and the predicted glycemic index of pasta.

Gluten-containing (GC) and gluten-free (GF) pasta consumption has been growing in recent years. The market offers a wide variety of pasta types, with differences in shape and formulation that influence the mastication process and, consequently, their nutritional behaviors (i.e. starch digestibility and glycemic response). This study investigated the effect of shape, gluten, and structural breakdown on in vitro starch digestibility and predicted the glycemic index (pGI) of GC and GF penne, spaghetti, and risoni. Pasta was cooked and minced to mimic short, intermediate, and long mastication efforts. Short mastication led to a higher number of big particles than intermediate and long mastications for all pasta samples, which was reflected in the different starch digestibility and pGI patterns. Multivariate analysis of variance showed that the three studied factors differently affected the in vitro starch digestion of pasta. Mastication effort, shape, and their interaction mainly affected the starch digestion rate and pGI. Gluten was the major factor in affecting the amount of digested starch. The results suggested that small shapes (i.e. risoni), the presence of gluten, and short mastication effort led to a lower pGI. The findings will be useful for the development of pasta products tailored to fulfill the needs of specific consumers following a rational food design approach.

Fecal Proteome Profile in Dogs Suffering from Different Hepatobiliary Disorders and Comparison with Controls.

In the present study, the fecal proteomes of clinically healthy dogs (HD = n. 10), of dogs showing clinical, ultrasonographic, and/or laboratory evidence of different hepatobiliary dysfunction (DHD = n. 10), and of dogs suffering from chronic hepatitis (CHD = n. 10) were investigated with an Ultimate 3000 nanoUPLC system, coupled to an Orbitrap Fusion Lumos Tribrid mass spectrometer. Fifty-two different proteins of canine origin were identified qualitatively in the three study groups, and quantitative differences were found in 55 proteins when comparing groups. Quantitatively, a total of 41 and 36 proteins were found differentially abundant in the DHD and CHD groups compared to the control HD, and 38 proteins resulted dysregulated in the CHD group as compared to the DHD group. Among the various proteins, differently abundant fecal fibronectin and haptoglobin were more present in the feces of healthy and DHD dogs than in chronic ones, leading us to hypothesize its possible diagnostic/monitoring role in canine chronic hepatitis. On the other hand, the trefoil factor 2 was increased in DHD dogs. Our results show that the analysis of the fecal proteome is a very promising field of study, and in the case of dogs suffering from different hepatobiliary disorders, it was able to highlight both qualitative and quantitative differences among the three groups included. Results need to be confirmed with western blotting and in further studies.

Effects of different steam injection conditions on cappuccino's nutritional profile.

The quality parameters of cappuccinos prepared with pasteurized milk or ultra-high-temperature milk steam-injected at different temperatures by a professional coffee machine have been assessed. In particular, the protein profile, the content of vitamins and lactose, the lipid peroxidation process, and the involvement of milk proteins in the foam formation were evaluated. The nutritional quality of milk seems not affected by the steam injection treatment carried out at a temperature of 60-65 °C, but at higher temperatures a decrement of lactoperoxidase, vitamin B6 and folic acid was observed. The milk used in cappuccino preparation is very important: pasteurized milk can form a more consistent and lasting foam with respect to ultra-high-temperature milk because of the presence of ß-lactoglobulin and lactoferrin, both playing an important role in the foam formation and stability. This work would provide additional information to the coffee industry for the preparation of high nutritional and organoleptic quality cappuccinos.

Fecal Protein Profile in Eight Dogs Suffering from Acute Uncomplicated Diarrhea before and after Treatment.

Acute diarrhea is a very frequent condition affecting dogs; nevertheless, little is known about what happens in the GI tract during such conditions. Proteomics allows the study of proteins present in a specific biologic substrate, and fecal proteomic investigations have been recently implemented to study GI diseases in dogs. In the present study, the fecal protein profiles of eight dogs suffering from acute uncomplicated diarrhea at the time of inclusion was investigated for the first time, and then the same patients were followed, replicating two further evaluations at two subsequent time points (after 2 and 14 days from the first presentation), with the aim of gaining possible new insights regarding the pathologic changes in the gastrointestinal environment during such conditions. Two-dimensional gel electrophoresis (2-DE) was performed, followed by mass spectrometry. Nine spots, corresponding to four (groups of) proteins (i.e., albumin, alkaline phosphatase, chymotrypsin-C-like, and some immunoglobulins), showed significant differences at two or more of the three time points investigated, almost all behaving similarly and decreasing at T1 (2 days after the onset of the condition) and significantly increasing at T2 (14 days after the onset), mainly evidencing a reaction of the organism. Further studies including a greater number of patients and possibly different techniques are needed to confirm the present findings.

The Research Field of Meat Preservation: A Scientometric and Visualization Analysis Based on the Web of Science.

Meat plays a significant role in human diets, providing a rich source of high-quality protein. With advancements in technology, research in the field of meat preservation has been undergoing dynamic evolution. To gain insights into the development of this discipline, the study conducted an analysis and knowledge structure mapping of 1672 papers related to meat preservation research within the Web of Science Core Collection (WOSCC) spanning from 2001 to 2023. And using software tools such as VOSviewer 1.6.18 and CiteSpace 5.8.R3c allowed for the convenient analysis of the literature by strictly following the software operation manuals. Moreover, the knowledge structure of research in the field of meat preservation was synthesized within the framework of "basic research-technological application-integration of technology with fundamental research," aligning with the research content. Co-cited literature analysis indicated that meat preservation research could be further categorized into seven collections, as well as highlighting the prominent role of the antibacterial and antioxidant properties of plant essential oils in ongoing research. Subsequently, the future research direction and focus of the meat preservation field were predicted and prospected. The findings of this study could offer valuable assistance to researchers in swiftly comprehending the discipline's development and identifying prominent research areas, thus providing valuable guidance for shaping research topics.

Changes in the Physical-Chemical Properties and Volatile Flavor Components of Dry-Cured Donkey Leg during Processing.

In order to explore the quality variation and flavor formation of dry-cured donkey leg, the changes in physical−chemical composition, lipolytic, free amino acids content and volatile flavor compounds were investigated in this study. Six fresh, trimmed hind legs with average weight of 8.12 ± 0.8 kg were taken from male Dezhou donkeys slaughtered at the age of 24 months with the average live weight of 240 kg. The entire processing time was eight months long including six stages, specifically: cooling, salting, air-drying, fermenting and aging. Samples were collected at 0 d, 10 d, 20 d, 30 d, 65 d, 105 d and 165 d of processing. The results showed that the pH value remained stable in the range of 6.2~6.6. The moisture and water activity significantly decreased (p < 0.05) during processing. The chloride content, ash, total volatile basic nitrogen (TVB-N) and peroxide value (POV) significantly increased (p < 0.05), from 0.45% to 12.39%, from 3% to 17%, from 1.43 mg/kg to 8.98 mg/kg and from 1.39 g/100 g to 5.26 g/100 g, respectively. The thiobarbituric acid (TBARS) value reached its highest value of 0.39 mg MDA/kg at the end of the salting stage and then decreased to 0.34 mg MDA/kg. Eighteen free amino acids and fifteen free fatty acids were detected, and their contents were significantly increased during processing (p < 0.05). Volatile compounds were analyzed using solid-phase microextraction (SPME) and gas chromatography−mass spectrometry (GC−MS). Among 114 volatile compounds detected in dry-cured donkey leg, aldehydes, esters, alkane and alcohols were more abundant in the final products, with relative concentrations of 41.88%, 5.72%, 5.35% and 5.25%, respectively. Processing significantly affected the physical−chemical properties, which could contribute to the formation of flavor substances of dry-cured donkey leg.

Evaluation of the Fecal Proteome in Healthy and Diseased Cheetahs (Acinonyx jubatus) Suffering from Gastrointestinal Disorders.

Fecal proteomics allows for the identification of proteins and peptides present in stools and is useful in finding possible new biomarkers for diagnosing and/or monitoring gastrointestinal (GI) disorders. In the present study, we investigated the fecal proteome in healthy and diseased cheetahs (Acinonyx jubatus). Captive individuals of this species frequently show gastrointestinal disorders characterized by recurrent episodes of diarrhea, rare episodes of vomiting and weight loss, associated with Helicobacter spp. infection. Fecal proteomic evaluation has been performed by two-dimensional electrophoresis followed by liquid chromatography-tandem mass spectrometry. In healthy cheetahs, the results showed the presence of the following proteins: collagen alpha-1 (II) chain, transthyretin, IgG Fc-binding protein, titin, dystonin, isopentenyl-diphosphate Delta-isomerase 1, sodium/potassium-transporting ATPase subunit alpha-1 and protein disulfide-isomerase A6. The presence of albumin isoforms was found only in diseased cheetahs. The present paper reports the study of the fecal proteome in the cheetah, evidences some differences between healthy and diseased patients and confirms, once again, the potential of fecal proteomics for the study of the GI environment, with promising developments regarding the identification of new diagnostic/monitoring markers.

Effects of Intra-Articular Autologous Adipose Micrograft for the Treatment of Osteoarthritis in Dogs: A Prospective, Randomized, Controlled Study.

The purpose of this study was to estimate the safety, feasibility, and efficacy of the intra-articular treatment of autologous microfragmented adipose tissue in dogs with spontaneous osteoarthritis (OA) in comparison with hyaluronic acid (HA), the standard intra-articular treatment. Specifically, it clinically evaluated pain and lameness, the radiographic progression of osteoarthritis, and synovial fluid inflammation. This was a prospective, single-center, parallel-group, randomized, controlled, in vivo clinical study. Participants (n = 40) received either a single intra-articular injection of microfragmented adipose tissue or a single intra-articular injection of HA (1:1). Clinical outcomes were determined using a specialistic clinician assessment obtained by the completion of a specific clinical form based on the Vesseur modified lameness classification system, a pain evaluation using the Visual Analogue Scale (VAS), the measurement of the range of motion (ROM) of the affected joint, limb circumference, and the owners' score evaluation using the Canine Brief Pain Inventory (CBPI) for up to 6 months after the time of injection. Patients underwent a radiographic examination to establish the degree of OA in the affected joint, and synovial fluid samples were collected to assess the biochemical environment of the joint and evaluate and quantify the cellular population and the presence of three specific inflammation biomarkers for up to 60 days. The results of this study suggest that microfragmented autologous adipose tissue is safe and can effectively relieve pain and improve function in dogs with spontaneous articular OA. This one-step procedure is simple, timesaving, cost-effective, minimally invasive, and eliminates the need for complex and time-intensive cell culture processing. Furthermore, the clinical evidence and cytological results suggest better long-term pain control, resulting in an improvement in joint function, compared to HA treatment. The canine spontaneous OA model could play a key role in developing successful treatments for human medicine.

Effects of Ageing on Donkey Meat Chemical Composition, Fatty Acid Profile and Volatile Compounds.

Donkey meat samples obtained from muscle Longissimus Thoracis Lumborum (LTL) taken from 14 entire donkey males slaughtered at 20 months and aged for 1, 8 and 15 days were analysed with the aim of determining the chemical composition, physical attributes, fatty acid profile and volatile compounds. Ageing did not significantly affect the chemical composition and colour parameters, while cooking loss was significantly (p < 0.05) higher at 8 and 15 days of ageing. Thiobarbituric acid reactive substances (TBARS) content significantly (p < 0.01) increased during ageing, while shear force values significantly (p < 0.01) decreased. Ageing significantly (p < 0.05) increased polyunsaturated fatty acids (PUFAs) determined both at 8 and 15 days after slaughter. Volatile compounds were analysed using solid-phase microextraction (SPME) and gas chromatography−mass spectrometry (GC−MS). Among 109 volatile compounds determined in donkey meat, hydrocarbons were the most common molecules detected. Ageing affected 21 of the detected volatile compounds; both total aldehydes and total ketones contents were significantly (p < 0.05) higher 15 days after slaughter. Total furans and total alcohols were significantly (p < 0.01) higher 15 days after slaughter, as well. Significant modifications of donkey meat volatile compounds can be attributed to ageing periods longer than 7 days.

Breast Cancer Treatment: The Case of Gold(I)-Based Compounds as a Promising Class of Bioactive Molecules.

Breast cancers (BCs) may present dramatic diagnoses, both for ineffective therapies and for the limited outcomes in terms of lifespan. For these types of tumors, the search for new drugs is a primary necessity. It is widely recognized that gold compounds are highly active and extremely potent as anticancer agents against many cancer cell lines. The presence of the metal plays an essential role in the activation of the cytotoxicity of these coordination compounds, whose activity, if restricted to the ligands alone, would be non-existent. On the other hand, gold exhibits a complex biochemistry, substantially variable depending on the chemical environments around the central metal. In this review, the scientific findings of the last 6-7 years on two classes of gold(I) compounds, containing phosphane or carbene ligands, are reviewed. In addition to this class of Au(I) compounds, the recent developments in the application of Auranofin in regards to BCs are reported. Auranofin is a triethylphosphine-thiosugar compound that, being a drug approved by the FDA-therefore extensively studied-is an interesting lead gold compound and a good comparison to understand the activities of structurally related Au(I) compounds.

Effects of Donkeys Rearing System on Performance Indices, Carcass, and Meat Quality.

This study compared growth rates, carcass, and meat quality obtained from 24 male crossbred donkey foals reared for meat production under an intensive (I) or extensive (E) feeding system. Donkeys were slaughtered at 16 months of age; the average final body weight, hot and cold carcass weight, and hot and cold dressing percentage were significantly higher (p < 0.05) in the I group. Samples of Longissimus Thoracis et Lumborum (LTL) were taken from each foal for chemical and physical analysis. Group I showed significant (p < 0.05) higher intramuscular fat, while the E group showed significantly (p < 0.05) higher protein and unsaturated fatty acids (UFA) contents, including n-3 essential fatty acids. Saturated fatty acids (SFA) and glycogen were significantly higher (p < 0.05) in the intensive system, monounsaturated fatty acids (MUFA) were significantly higher in the extensive system. The ratio PUFA/SFA was significantly higher (p < 0.05) in group E. The Thrombogenic Index (TI) was significantly higher (p < 0.05) in the I group. Meat tenderness was significantly higher (p < 0.05) in group I. The feeding system had no effect on cholesterol content and meat color characteristics. Donkeys average daily gain, carcass weight, and some meat quality parameters were significantly affected by the rearing system.

Clinicopathological and Fecal Proteome Evaluations in 16 Dogs Presenting Chronic Diarrhea Associated with Lymphangiectasia.

Canine intestinal lymphangiectasia (IL) is a condition characterized by variably severe gastrointestinal signs, frequently associated with laboratory abnormalities; the research for markers allowing a better understanding of the severity degree and/or obtaining an early diagnosis and/or monitoring is continuously progressing. In the present study, we investigated possible new diagnostic/follow-up markers in IL dogs, namely, serum C-reactive protein, serum bacterial lipopolysaccharide, serum cleaved cytokeratin 18, serum citrulline, and zonulin (in both serum and feces). A fecal proteomic study looking for possible confirmation and/or new marker candidates was also performed. All markers in both substrates, with the exception of serum citrulline, significantly differed between diseased and control dogs. Fecal proteomics allowed the retrieval of three proteins in IL dogs (Fc fragment of IgG-binding protein; transthyretin; proproteinase E) that were not previously found in clinically healthy subjects. Although further studies are needed, C-reactive protein, bacterial lipopolysaccharide, cleaved cytokeratin 18, and zonulin (in both serum and feces) resulted as promising markers for canine IL; similarly, fecal proteomics represents a road worthy of being pursued in the search for candidate biomarkers.

Vitamins in Human and Donkey Milk: Functional and Nutritional Role.

BACKGROUND: Whole milk is a good source of all the nutrients, and it also contains a sufficient number of vitamins to permit regular the growth of the neonate. Dairy cow milk can create allergy in infants less than 12 months old because of the high caseins and ß-lactoglobulin content. In these circumstances, donkey milk can represent a good replacement for dairy cows' milk in children affected by Cow Milk Protein Allergy (CMPA) because of its close chemical composition with human milk, mainly due to its low protein and low mineral content. Milk vitamin content is highly variable among mammalian species and it is strictly correlated with the vitamin status and the diet administered to the mother. Fat-soluble vitamins content in donkey milk is, on average, lower compared to ruminants' milk, while vitamin C content determined in donkey milk is higher compared to dairy cows' milk, showing a great similarity with human milk. In donkey milk, the content of vitamins of the B-complex such as thiamine, riboflavin, niacin, pyridoxine, and folic acid is higher compared to human milk. The use of donkey milk as a new functional food must be further evaluated in interdisciplinary clinical trials in which pediatricians, dietitians, and food scientists must be involved to deepen the knowledge about the positive health impact of donkey milk in different sensitive people, especially children and the elderly.

Faecal proteome in clinically healthy dogs and cats: Findings in pooled faeces from 10 cats and 10 dogs.

BACKGROUND: In the scientific literature, there are only a few manuscripts available on small animal faecal proteomics. METHODS: In the present pilot study, this evaluation was performed using pooled faecal samples from 10 clinically healthy dogs and, for the first time, in 10 clinically healthy cats by mean of two-dimensional electrophoresis followed by liquid chromatography-tandem mass spectrometry. RESULTS: Our results showed the presence of nine (albumin, alkaline phosphatase, chymotrypsin-C-like, cytosol aminopeptidase, elastase-3B/proteinase E, immunoglobulins and nuclear pore membrane glycoprotein 210) and 14 (albumin, caspase recruitment domain-containing protein, chymotrypsin-like, deleted in malignant brain tumours 1 protein-like, hypothetical protein LOC107375, immunoglobulin, kallikrein-1, superoxide dismutase, transthyretin precursor, interstitial collagenase-like) different proteins in canine and feline faeces, respectively. CONCLUSION: These preliminary findings document the presence of a range of proteins in the faeces of apparently healthy dogs and cats and may serve as a basis for larger, prospective studies to establish reference proteomic data against which diseased populations can be compared.

Rheological Properties and Growth Factors Content of Platelet-Rich Plasma: Relevance in Veterinary Biomedical Treatments.

Platelet-rich plasma (PRP) is a nontransfusional hemocomponent, considered as a powerful concentrate of growth factors (GFs) therapeutically used to stimulate tissue regeneration. The use of autologous PRP, as the patient's own biological material, for therapeutic purposes represents a safe and effective alternative to conventional treatments in both human and veterinary medicine. The aim of this study was the characterization of canine PRP from rheological and biological points of view. Thus, a characterization of the viscoelastic properties of the PRP systems was performed in order to clarify the influence of different calcium concentrations, in the presence of autologous thrombin-rich solution, on the PRP gels' mechanical properties, from which the applicability of these systems in biomedical treatments is strongly dependent. Then, an evaluation of the content of GFs in PRP, activated or not with thrombin, and stored at different temperatures (37 °C and -20 °C) was performed over time, outlining, for the first time, the importance of the effect of physiological temperature (37 °C) on the production of GFs. A clinical case study conducted in a dog with a complete rupture of the common calcaneal tendon (Achilles tendon) confirmed the relevance of this hemocomponent in the daily veterinary clinical activity and the potential translational value for human health.

Comparison of Carcass and Meat Quality Obtained from Mule and Donkey.

The aim of this study was to compare the carcass characteristics and the chemical and physical parameters of the meat produced by 10 male crossbred donkeys and 10 male mules slaughtered at 16 ± 1 years of age. The carcass weight and dressing percentage were significantly (p < 0.05) higher in mules. Samples of the muscle Longissimus thoracis (LT) were analyzed. Donkey meat showed a higher fat content, while the glycogen content was higher in the mule LT. The total collagen content was higher in the mule LT; in this muscle, the shear force values were higher compared to the donkey LT. The lightness parameter (L*) was lower in the mule LT, while the redness index (a*) was higher in the mule LT; this muscle showed a higher content of iron, while zinc was higher in the donkey muscle LT. The donkey LT muscle showed a higher content of essential amino acids (52.2%) compared to the mule LT (50.1%). The results obtained demonstrated that the chemical and physical traits of mule and donkey meat were similar to those of other kinds of equid meat.

Proteomic characterization of kefir milk by two-dimensional electrophoresis followed by mass spectrometry.

Kefir is a type of fermented milk obtained thanks to the introduction of "kefir grains" in mammalian milk. Kefir grains consist of lactic and acetic acid bacteria and yeasts in alternative proportions that are held together by a matrix of complex sugars known as "kefiran." Thanks to the fermentative process, the kefir milk is rich in nutraceutical substances such as amino acids, vitamins, and mineral salts. The most valuable compounds of kefir fermentation are mainly lactic acid, exopolysaccharides, and bioactive peptides, the resulting products of proteolytic release from milk proteins (caseins and whey proteins). Among the nutraceutical properties of kefir are antimicrobial and antitumor activity, immunomodulating effect, and cholesterol-lowering effect. Therefore, in light of these intriguing properties of kefir milk, in this work, a proteomic analysis, by two-dimensional electrophoresis followed by mass spectrometry, has been performed. As a result, milk-derived polypeptides were identified in commercial kefir milk from organic farming. In particular, polypeptides deriving from κ-, αs1 -, and αs2 -caseins that may have potentially beneficial effects on human health have been detected.

Dietary Intake of Vitamin D from Dairy Products Reduces the Risk of Osteoporosis.

BACKGROUND: Vitamin D and calcium are important dietary compounds that affect bone mass, even if other minerals (potassium, zinc, etc.) and vitamins (A, C and K) are also involved. Vitamin D and certain minerals, in fact, play an important role in calcium homeostasis and calcium absorption. Hip fracture incidence is higher in Europe and the United States, where calcium is frequently included in the human diet; while the occurrence of these fractures is lower in developing countries, where diets are often poor in calcium. This condition is named the "calcium paradox", and may be partially explained by phosphate toxicity, which can negatively affect mineral metabolism. It is important to maintain correct dietary calcium-phosphate balance in order to have a healthy life, reducing the risk of osteoporotic fractures in older people. Vitamin D can also act as a hormone; vitamin D2 (ergocalciferol) is derived from the UV-B radiation of ergosterol, the natural vitamin D precursor detected in plants, fungi, and invertebrates. Vitamin D3 (cholecalciferol) is synthesized by sunlight exposure from 7-dehydrocholesterol, a precursor of cholesterol that can also act as provitamin D3. Dietary intake of vitamin D3 is essential when the skin is exposed for short periods to ultraviolet B light (UV-B), a category of invisible light rays such as UV-A and UV-C. This can be considered the usual situation in northern latitudes during the winter season, or the typical lifestyle for older people and/or for people with very white delicate skin. The actual recommended daily intake of dietary vitamin D is strictly correlated with age, ranging from 5 µg for infants, children, teenagers, and adults-including pregnant and lactating women-to 15 µg for people over 65 years.

Multi-Targeted Anticancer Activity of Imidazolate Phosphane Gold(I) Compounds by Inhibition of DHFR and TrxR in Breast Cancer Cells.

A class of phosphane gold(I) compounds, made of azoles and phosphane ligands, was evaluated for a screening on the regards of Breast Cancer cell panels (BC). The compounds possess N-Au-P or Cl-Au-P bonds around the central metal, and they differ for the presence of aprotic or protic polar groups in the azoles and/or the phosphane moieties to tune their hydrophilicity. Among the six candidates, only the compounds having the P-Au-N environment and not displaying neither the hydroxyl nor carboxyl groups in the ligands were found active. The compounds were screened by MTT tests in SKBR3, A17, and MDA-MB231 cancer cells, and two compounds (namely the 4,5-dicyano-imidazolate-1yl-gold(I)-(triphenylphosphane, 5, and 4,5-dichloro-imidazolate-1yl-gold(I)-triphenylphosphane, 6) were found very cytotoxic, with the most active with an IC50 value of 3.46 µM in MDA-MB231 cells. By performing enzymatic assays in the treated cells lysates, the residual enzymatic activity of dihydrofolate reductase (DHFR) has been measured after cell treatment for 4 or 12 h in comparison with control cells. Upon 12 h of treatment, the activity of DHFR was significantly reduced in both SKBR3 and A17 cells by compounds 5 and 6, but not in human MDA-MB231 cells; interestingly, it was found remarkably high after 4 h of treatment, revealing a time dependence for the DHFR enzymatic assays. The DHFR inhibition data have been compared to those for the thioredoxin reductase (TrxR), the most recognized molecular target for gold compounds. For this latter, similar residual activities (i.e., 37 and 49% for the match of SKBR3 cells and compound 5 or 6, respectively) were found. Binding studies on the regards of ct-DNA (calf-thymus-DNA) and of plasma transporters proteins, such as BSA (bovine serum albumin) and ATF (apo transferrin), were performed. As expected for gold compounds, the data support strong binding to proteins (Ksv values range: 1.51 ÷ 2.46 × 104 M-1) and a weaker interaction with ct-DNA's minor groove (Ksv values range: 1.55 ÷ 6.12 × 103 M-1).