Antioxidant and Anti-Inflammatory Activities of Buffalo Milk ?-Valerobetaine

Article abstract of DOI:10.1021/acs.jafc.8b07166

δ-Valerobetaine (δVB), a constitutive metabolite of ruminant milk, is produced in the rumen from free dietary N^e- trimethyllysine occurring ubiquitously in vegetable kingdom. The biological role of δVB is poorly known. Here, the antioxidant and anti-inflammatory potential of buffalo milk δVB was tested in vitro during high-glucose (HG)-induced endothelial damage. Results indicated that δVB (0.5 mM) ameliorated the HG cytotoxicity (0.57 ± 0.02 vs 0.41 ± 0.018 O.D. (P < 0.01). Interestingly, buffalo milk extracts enriched with δVB showed improved significant efficacy in decreasing reactive oxygen species, lipid peroxidation, and cytokine release during HG treatment compared to milk extracts alone (P < 0.05). It is noteworthy that δVB reduced the HG-activated inflammatory signal by modulating SIRT1 (0.96 ± 0.05 vs 0.85 ± 0.04 AU), SIRT6 (0.82 ± 0.04 vs 0.61 ± 0.03 AU), and NF-?°B (0.85 ± 0.03 vs 1.23 ± 0.03 AU) (P < 0.05). On the whole, our data show the first evidence of δVB efficacy in reducing endothelial oxidative stress and inflammation, suggesting a potential role of this betaine as a novel dietary compound with health-promoting properties.

Full text of DOI:10.1021/acs.jafc.8b07166

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Bioactive Constituents, Metabolites, and Functions
Antioxidant and Anti-Inflammatory Activities of Buffalo Milk #-Valerobetaine
Nunzia D'Onofrio, Anna Balestrieri, Gianluca Neglia, Alessandra Monaco, Marco Tatullo,
Rosario Casale, Antonio Limone, Maria Luisa Balestrieri, and Giuseppe Campanile
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b07166 • Publication Date (Web): 20 Jan 2019
Downloaded from http://pubs.acs.org on January 20, 2019
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J Agric Food Chem revision
Antioxidant and Anti-Inflammatory Activities of Buffalo
Milk δ-Valerobetaine
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Nunzia D’Onofrio , Anna Balestrieri , Gianluca Neglia , Alessandra Monaco , Marco Tatullo ,
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Rosario Casale , Antonio Limone , Maria Luisa Balestrieri *, Giuseppe Campanile
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§Department of Precision Medicine, University of Campania “L. Vanvitelli”, 80138 Naples, Italy.
‡Istituto Zooprofilattico Sperimentale del Mezzogiorno, 80055 Portici (Naples), Italy.
#Department of Veterinary Medicine and Animal Production, Federico II University, 80137 Naples,
Italy.
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†Tecnologica Research Institute, Biomedical Section, 88900 Crotone, Italy
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*Correspondence: Maria Luisa Balestrieri, Department of Precision Medicine, University of
Campania “L. Vanvitelli”, via L. De Crecchio 7, 80138, Naples, Italy. Tel.: +39 081 5665865; Fax:
+39 081 5665863. Email: marialuisa.balestrieri@unicampania.it
ORCID: 0000-0001-6001-1789
Key words: δ-Valerobetaine, Buffalo milk, SIRT1, SIRT6, Hyperglycaemia.
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Abstract
δ-Valerobetaine (δVB), a constitutive metabolite of ruminant milk, is produced in the rumen from
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free dietary N -trimethyllysine occurring ubiquitously in vegetable kingdom. Biological role of δVB
is poorly known. Here, the antioxidant and anti-inflammatory potential of buffalo milk δVB was
tested in vitro during high-glucose (HG)-induced endothelial damage. Results indicated that δVB
(0.5 mM) ameliorated the HG cytotoxicity (0.57±0.02 vs. 0.41±0.018 O.D. (P<0.01). Buffalo milk
extracts enriched with δVB showed significant efficacy in decreasing reactive oxygen species, lipid
peroxidation, and cytokine release during HG treatment compared to milk extracts alone (P<0.05).
δVB reduced the HG-activated inflammatory signal by modulating SIRT1 (0.96±0.05 vs. 0.85±0.04
AU), SIRT6 (0.82±0.04 vs. 0.61±0.03 AU), and NF-κB (0.85±0.03 vs. 1.23±0.03 AU) (P<0.05).
On the whole, our data show the first evidence of δVB efficacy in reducing endothelial oxidative
stress and inflammation, suggesting a potential role of this betaine as a novel dietary compound
with health-promoting properties.
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1. Introduction
δ-Valerobetaine (δVB), a water-soluble trimethylated compound recently identified as
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constitutive betaine of ruminant milk and meat, is synthetized from free dietary N -trimethyl lysine
occurring in ruminant diet.1,2 The chemical structure of δVB, also named N,N,N,-trimethyl-5-
aminovaleric acid, 5-aminovaleric acid betaine, or 5-trimethylaminovaleric acid, shows similarities
with that of mildronate (meldonium). Mildronate, a clinically used cardioprotective drug, regulates
the energy metabolism pathways by reducing L-carnitine biosynthesis via γ-butyrobetaine
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hydroxylase and blocking the organic cation transporter type 2 (OCTN2) of L-carnitine. Indeed,
in silico docking simulations and OCTN2 silencing in cultured cardiomyocytes indicated δVB as a
possible inhibitor/substrate of L-carnitine uptake capable of reducing oxidation of fatty acids.5
Physiological concentrations of δVB, have been examined human heart tissue (3–22 μM) and
plasma by targeted mass spectrometry method. 5,6 In humans, increased plasma levels of betainized
compounds, including δVB, following dietary intervention with a diet rich in whole grains (without
modification of the habitual consumption of meat, dairy products, eggs, fish, fruit, vegetable, and
fat intake) correlates with improved insulin resistance and insulin secretion, suggesting a beneficial
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effects of this dietary pattern . The betaine profile of cereal flours shows uncommon betaines, such
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as valine betaine and glutamine betaine but not δVB. Indeed, the δVB peak at m/z 160.1 has been
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detected only in ruminant milk and meat. In particular, in the chromatographic conditions used for
the betaine profile of ruminant milk and meat, valine betaine and δVB eluted at different retention
times, corresponding to 24.8 min and 20.3 min, respectively.1
Among ruminant milk, Mediterranean buffalo milk (Bubalus bubalis), contains higher levels
of δVB, carnitine and short-chain acylcarnitine (acetylcarnitine, propionylcarnitine,
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butyrylcarnitine, isobutyrylcarnitine, and 3-methylbutyrylcarnitine) than bovine milk. These
metabolites, in addition to having an important physiological role in growth of suckling calf, confer
a high nutritional value to the Mediterranean buffalo milk, highly suitable for the production of
dairy products due to its peculiar composition and the high productivity standards of the Italian
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buffalo-breeding program. 9,10 Peptidomic and metabolomic profiling of the Mediterranean buffalo
milk and dairy products revealed the occurrence of a wide range of natural bioactive substances and
peptides endowed with recognized health benefits, including antimicrobial, anticancer
immunomodulatory, antidiabetic, antihypertensive, and antioxidant properties. 11-15
In light of the proven impact of lifestyle and nutrition on the molecular hallmarks of aging, the
employment of natural health-promoting compounds in the prevention of aging-related diseases,
including diabetes, cardiovascular disease, and cancer keeps gaining scientific interest.16,17
Of note, natural compounds mimicking calorie restriction regulate redox homeostasis by
modulating sirtuin 1 (SIRT1) signalling cascade.18-21 SIRT1 and SIRT6, nicotinamide adenine
dinucleotide (NAD+)-dependent deacetylates belonging to the class III histone deacetylases, are
enzymes controlling the transcription of genes involved in the cellular events regulating aging-
related disorder with multiple roles in the protection against vascular aging, inflammation, and
atherosclerosis.22,23 At vascular level, a strong interaction occurs between oxidative stress,
SIRT1/SIRT6 inhibition and nuclear factor-kappa B (NF-κB) activation during hyperglycaemia,
leading to the production of proinflammatory cytokines and activation of prothrombotic markers.23-
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The present study was designed to investigate the antioxidant and anti-inflammatory potential of
δVB during the endothelial damage induced by hyperglycaemia and the possible relationship with
SIRT1 and SIRT6. In vitro cultured endothelial cells are widely used to study vascular
inflammation during diabetes, since endothelial dysfunction is a key event in the onset and
development of vascular disease in diabetes. In this regard, the ability of pure δVB to counteract the
cytotoxicity induced by short-term treatment with high-glucose was evaluated in vitro as well as the
antioxidant and anti-inflammatory activities of buffalo milk extracts prepared by ultra-filtration
(cutoff of 3kDa molecular weight), focusing on the specific role of δVB.
2. Materials and Methods
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2.1 Chemicals
Minimum Essential Medium (MEM, GIBCO, 11095-080), foetal bovine serum (FBS), and Pen-
Strep were from Gibco, Life Technologies. NEAA (Non-Essential Amino Acid solution 100X) was
purchased from Lonza. Potassium ferricyanide, ferric chloride, 2’,7’-dichlorofluorescin diacetate
(DCFH-DA), α-amylase from porcine pancreas, starch, α-d-glucopyranoside, α-Glucosidase, and
MTT assay were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). TNF-α, IL-6 and IL-1β
ELISA assays (Cymax TNF-alpha ELISA, YIF-LF-EK0193; Cymax IL-6 ELISA, YIF-LF-
EK0260; Cymax IL-1beta ELISA, YIF-LF-EK0276) were from AbFrontier (Seoul, Korea).
Antibodies against SIRT1 (rabbit monoclonal, ab32441), SIRT6 (rabbit polyclonal, ab62738), total
antioxidant capacity assay, α-amylase activity assay, and lipid peroxidation assay were obtained
from Abcam (Cambridge, UK). Antibody against NF-κB (rabbit monoclonal, C22B4) was purchase
from Cell Signaling Technology (Danvers, MA). Antibody against γ-tubulin (mouse monoclonal,
GTU-88) was obtained from Sigma (St. Louis, MO, USA). Secondary antibodies Alexa Fluor® 488
and Alexa Fluor® 633 were obtained from Life Technologies Italia (Monza, Italy). Cyclooxygenase
and radical scavenging activity DPPH assays (1898-66-4) were from Cayman Chemical (Ann
Arbor, MI, USA). All other solvents and reagents used were of analytical grade.
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2.2 Animals management and milk sampling
Animals were from Southern Italy (between 40.5° N and 41.5° N). Buffalo, maintained in open
yards, were fed with50% forage, 0.93 MFU/kg of dry matter (DM), 14% crude protein/DM, 42%
NDF/DM and 26% ADF/DM. Bulk milk was collected from a total of 250 Italian Mediterranean
buffaloes (130.7±4.4 days in milk) of 129 kg metabolic weight. The average milk yield in buffalo
was 14.6 equivalent corrected milk (ECM = 730 Kcal).
2.3 Preparation of milk extracts
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Water-soluble extracts (WSE) of buffalo milk were prepared as described by Servillo et al. with
minor modifications. 1,8 Briefly, after the removal of the fat globules by centrifugation of the bulk
milk at 3000xg for 15 min a 4°C, the pH was adjusted to to 7.2, and then the central aqueous phase
was filtered through Amicon Ultra 0.5 mL centrifugal filters a (3kDa molecular weight cut-off).
Finally, the WSE were filtered through a 0.22 µm Millipore filters before begin used.
2.4 Synthesis and purification δVB
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The preparation of δVB was performed as previously described , using a heterogeneous phase
reaction in the presence of KHCO
of 5-aminovaleric acid were dissolved in 20 mL of methanol, added with 1 g of KHCO
iodomethane, and stirred 12 h at room temperature. The addition of KHCO (1 g) and iodomethane
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with iodomethane as methylating agent. Briefly, about 100 mg
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(10 mL) was repeated twice more. Finally, the mixture was centrifuged and the supernatant was
evaporated to dryness at 40 °C in a rotavapor. The residue, containing the product, was dissolved in
10 mL of Milli Q grade water and applied on 10 cm column filled with a mixed-bed resin of
Dowex-1-OH- and Biorex-70-H+ (1:1 v/v). The aqueous wash from this column was then applied
to a 10×2 cm column with AG50WX8-H+ resin, and washed with 20 mL of water. Finally, the
product was eluted with 30 mL of 6 M NH OH and evaporated to dryness under a stream of air. The
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reaction yield from various preparations was near 90%. The purity of the product was assessed by
full scan HPLC-mass spectrometry on a 25 cm Supelco Discovery C8 column eluted isocratically
with water containing 0.1% formic acid. The chromatography of the purified product, obtained from
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different preparations, showed in each case only one peak at m/z 160.1 whose MS fragmentation
pattern, obtained with analyses conducted by HPLC-MS/MS spectrometry in the same
chromatographic conditions, was that expected for δVB (Supplementary Figure A).
2.5 Total antioxidant capacity
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The total antioxidant capacity was determined by colorimetric assay (Abcam, ab65329) following
manufacturer’s instructions. Specifically, buffalo milk extracts were diluted 1:1 (v/v) with protein
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mask before adding 100 µL of Cu working solution. Samples were incubated at room temperature
for 90 min protected from light. Total antioxidant activity was expressed as mmol/L of Trolox
equivalents.
2.6 Radical scavenging activity by DPPH
The free radical scavenging activity of the buffalo milk extracts was examined in vitro using 1,1-
diphenyl-2-picrylhydrazyl (DPPH) radical assay (Cayman Chemical). DPPH free radical
scavenging activity was performed by adding 20 μL of buffalo milk extracts to 80 μL of 2,2-
diphenyl-1-picrylhydrazyl (DPPH, 100 μM in ethanol) in a 96-well microplate. Samples were
incubated for 20 min in dark room at room temperature. Controls contained PBS instead of the milk
extract samples. The change in absorbance value was measured at 517 nm by Tecan Infinite 2000
and DPPH free radical scavenging activity was reported in percentage and calculated using the
equation: % Radical scavenging = (A517 Control – A517 Test) / A517 Control × 100.
2.7 Reducing power
Reducing power of buffalo milk extracts was determined according to the method described by
Adesegun et al..26 Buffalo milk extracts (2 mL) were mixed with 2 mL of 1% potassium
ferricyanide, followed by incubation at 50 °C for 20 min. After incubation, 2 mL of trichloroacetic
acid solution (10%) were added and samples were centrifuged 1000 ×g for 10 min. Supernatants (2
mL) were mixed with same volume of deionized water. Finally, 0.4 mL of ferric chloride (0.1%)
were added and absorbance was measured at 700 nm (Tecan Infinite 2000). The increase in
reducing power is indicated by the increased absorbance. Ascorbic acid was used as standard.
2.8 In vitro α-amylase inhibition assay
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The antidiabetic activities of milk extracts were investigated using a spectrophotometric assay.
Briefly, 500 μL of α-amylase from porcine pancreas (1.0 unit/mL), was premixed with 500 μL of
samples. After preincubation at 37°C for 10 min, 500 μL of 1% starch was used as a substrate in
phosphate buffer solution (pH 6.9) to start the reaction. The reaction was performed at 37°C for 15
min and terminated by the addition of 400 μL of DNS reagent (1% 3,5-dinitrosalicylic acid and
12% sodium potassium tartrate in 0.4 M NaOH). The reaction mixture was heated for 15 min at
100°C and diluted with 10 mL of distilled water in an ice bath. Inhibition of α-amylase activity was
determined by measuring absorbance at 540 nm by using a Tecan Infinite 2000. Reducing sugar
was estimated by interpolation of obtained absorbance value of milk samples at 540 nm in the
standard graph. Acarbose is used as positive control. The percentage of enzyme inhibition was
calculated as follows: % Enzyme inhibition = (A540 Control – A540 Test) / A540 Control × 100.
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2.9 α-Glucosidase inhibition assay.
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α-Glucosidase inhibition was determined by spectrophotometric assay. α-Glucosidase (1 unit/mL)
was dissolved in 100 μL of 0.1 M potassium phosphate buffer (pH 6.8) and mixed with 50 μL of
buffalo WSE with or without δVB. After preincubation at 37°C for 20 min, 50 μL of 5 mM p-
nitrophenyl α-d-glucopyranoside (pNPG) was added as the substrate. The enzymatic reaction was
performed at 37°C for 40 min and stopped by the addition of 1 mL of 0.1 M sodium carbonate. α-
Glucosidase activity was determined by measuring the release of p-nitrophenol from pNPG at 400
nm. Acarbose was used as positive control and the inhibition percentage of the α-glucosidase was
calculated as follows: % Enzyme inhibition = (A400 Control – A400 Test) / A400 Control × 100.
2.10 COX inhibition assay
Cyclooxygenase (COX) 1 and 2 activities were determined by COX inhibitor screening assay kit
(Cayman, 701050) according to the manufacturer’s instructions. Briefly, 160 μL assay buffer and
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10 μL heme were added to the background wells, while 150 μL assay buffer, 10 μL heme and 10 μL
COX1/COX2 enzyme were added to the 100% initial activity wells. Buffalo milk extracts (10 μL)
were added to the sample wells whereas 10 μL DMSO was added to the background wells. The
plate was carefully shaken for a few seconds and incubated for five min at 25°C. The colorimetric
substrate solution (20 μL of N,N,N,N’-tetramethyl-p-phenylenediamine) was added to each well,
followed by the addition of arachidonic acid (20 μL). The plate was carefully shaken for a few
seconds and incubated for 5 min at 25°C. The absorbance at 590 nm was read by a microplate
reader and the inhibition ratio of COX1/COX2 enzymatic activity was calculated according to the
manufacturer’s instructions. The COX inhibitory activity (%) was represented as inhibition % of
control (pure enzyme).
2.11 Endothelial cell culture and treatments
Endothelial cells (EC) (ATCC, CCL 209) were maintained in minimum essential medium (MEM,
Gibco, 11095-080) supplemented with 20% heat-inactivated fetal bovine serum, penicillin (100
U/mL), streptomycin (0.1 mg/mL) and non-essential amino acid (1%) solution at 37°C in a
humidified atmosphere (95% air, 5% CO ). Cells were kept sub-confluent and media were changed
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every 2 days. Cells used in experiments were between passage 19 and 25. As for treatments, buffalo
milk extracts were added to EC before starting short-term treatment with normal glucose (NG, 5.5
mM) or high-glucose (HG, 30 mM) and incubated for 48 and 72 h at 37°C in a humidified
atmosphere (95% air and 5% CO ). To evaluate the contribution of δVB to the antioxidant power of
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milk extracts, the pure substance was tested alone or added to milk extracts to a final concentration
of 0.5 mM.
2.12 MTT cell viability
Cell viability was assessed using the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide
(MTT) (Sigma-Aldrich) assay, according to the manufacturer’s instructions. Briefly, EC (4×103
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cells/well) were seeded in a 96-well microplate and incubated in an appropriate medium overnight
at 37°C in a humidified atmosphere (95% air and 5% CO ). The effect of buffalo milk extracts on
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EC viability was evaluated by adding different volumes (up to 20 μL) to a final volume of 100 μL
of culture medium 3 h before starting the short-treatment with HG. The absorbance was measured at
570 and 605 nm according to the manufacturer’s protocol, using a microplate reader Model 680
Bio-Rad.
2.13 Intracellular reactive oxygen specie detection
Intracellular ROS levels were determined by using the cellular reactive orange fluorescence oxygen
species detection assay kit (Abcam, ab186028) according to the manufacturer’s instructions. EC
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(5x10 cells/well) were seeded in 96-well microplate in 20% FBS-supplemented media. After 12h
of sub-culturing, EC were pre-treated with 10 μL of buffalo milk extracts for 12 h before starting
high-glucose treatment for 48 h. Control cells were incubated only with NG (5.5 mM). After
treatment, the cells were incubated with ROS orange working solution (100 µL/well) at 37°C and
5% CO , for 60 min. The fluorescence intensity at 540 nm excitation wavelength and 570 nm
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emission wavelength was measured by using a Tecan Infinite 2000.
2.14 Cytokine level measurement
TNF-α, IL-6 and IL-1β detection was performed by ELISA assays (Cymax TNF-alpha ELISA,
YIF-LF-EK0193; Cymax IL-6 ELISA, YIF-LF-EK0260; Cymax IL-1beta ELISA, YIF-LF-
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EK0276), according to the manufacturer’s instructions. Briefly, EC (4x10 cells/well) were seeded
in 6-well plate and treated under HG condition for 48 h. At the end of treatment, EC lysates (100
μL) were added to pre-coated wells. Non-bound cytokines and other components of the sample
were removed by washing following by addition of polyclonal antibody against TNF-α, IL-6, and
IL-1β. At the end of incubation, anti-rabbit IgG horseradish peroxidase (HRP) was added to each
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well. Quantitative analysis was performed by measuring absorbance at 450 nm by using a Tecan
Infinite 2000.
2.15 Lipid peroxidation assay
Malondialdehyde (MDA) measurement was carried out with lipid peroxidation assay kit (Abcam,
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ab118970), according to the manufacturer’s instructions. Specifically, EC (2 x 10 ) were harvest to
the end of treatment and homogenized in 303 µL of MDA lysis solution. To generate MDA-TBA
adduct, 600 µL of TBA reagent were added into each well containing 200 µL of sample, incubated
at 95°C for 60 min and then cooled to room temperature in an ice bath for 10 min. Finally, 200 µL
of supernatant (containing MDA-TBA adduct) were added into a 96-well microplate for analysis.
Absorbance was measure immediately on a microplate reader Model 680 Bio-Rad at 532 nm. Total
MDA levels (nmol/µg of protein) were calculated based on the standard curve and normalized to
protein levels.
2.16 Western blot analysis
EC monolayers were washed twice with 1X PBS and immediately scraped-off the plate in lysis
buffer (50 mM Tris, pH 7.4, 150 mM sodium chloride, 1% Nonidet P-40, 1mM EDTA, 1mM
sodium orthovanadate, 1mM sodium fluoride, 1 mg/mL leupeptin, 1 mg/mL aprotinin, 1 mg/mL
pepstatin A, 1mM PMSF). Scraped cells were transferred to a microcentrifuge tube, kept on ice, and
sonicated for 10–15 s to complete cell lysis. After 30 min of gently shaking at 4°C, lysates were
centrifuged at 12000 x g for 10 min and supernatants were collected. Protein extracts (50-70 μg)
were separated by 7-10% SDS-polyacrylamide gel electrophoresis and transferred onto
nitrocellulose membranes by Trans-Blot Turbo Transfer System (Bio-Rad). Nitrocellulose
membranes were incubated with 5% w/v milk, 1X TBS, and 0.1% Tween-20 at 25 °C for 2h
followed by overnight incubation at 4 °C with primary antibodies against SIRT1 (1:1000) (rabbit
monoclonal, Abcam, ab32441), SIRT6 (1:2000) (rabbit polyclonal, Abcam, ab62738), NF-κB
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(1:3000) (rabbit monoclonal, Cell Signaling, C22B4), or α-tubulin (1:10000) (mouse monoclonal,
Sigma, GTU-88). After incubation with secondary antibodies (1:10000), membranes were washed
three times and bands were detected by the enhanced chemoluminescence kit (ECL, Amersham).
Semiquantitative densitometry of Western blots was performed using ImageJ software.
2.17 Confocal laser scanning microscopy analysis
EC cultured in 24-well plates containing microscope glass (12 mm) (Thermo Scientific) were fixed
with 4% (v/v) paraformaldehyde for 20 min and then permeabilized with 0.1% (v/v) Triton X-100
in PBS for 10 min at room temperature. Immunofluorescence detection of SIRT1, SIRT6 and NF-
κB was performed by using specific antibodies against vimentin (1:1000) (mouse monoclonal,
Sigma, V6630), SIRT1 (1:200) (rabbit monoclonal, Abcam, ab32441), SIRT6 (1:500) (rabbit
polyclonal, Abcam, ab62738), and NF-κB (1:500) (rabbit monoclonal, Cell Signaling, C22B4).
Secondary antibodies were Alexa Fluor® 488 (1:1000) or Alexa Fluor® 633 (1:1000) (Life
Technologies). Microscopy analyses were performed using Zeiss LSM 700 confocal microscope
equipped with a plan apochromat X63 (NA1.4) oil immersion objective. The fluorescence intensity
was evaluated with ImageJ software and expressed as arbitrary fluorescence units (AFU).
2.18 Statistical analysis
Data were presented as group mean±SD. One-way analysis of variance (ANOVA) was used to
compare data, followed by Scheffe’s test for pair wise comparisons. Statistical analysis was
performed using the SPSS software package for Windows 17.0 (SPSS Inc., Chicago Illinois). A
statistically significant difference was accepted at P<0.05.
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3. Results
3.1 Effect of δVB on high-glucose cytotoxicity
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In order to investigate the effect of δVB on the HG-induced cytotoxicity, we first evaluated its dose-
dependent effect on EC proliferative capacity. Results showed that cell viability was not affected by
treatment with δVB for 72 h up to 1 mM (Figure 1A). The efficiency in reducing the HG effects
started at 0.1 mM with the highest potency at 0.5 mM (P<0.01vs. HG) (Figure 1B), the
concentration chosen for this study.
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3.2 Cytoprotective effect of buffalo milk during high-glucose treatment
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The effect of water-soluble buffalo milk extracts on EC viability was tested by incubating cells for
48 h and 72 h with increasing volumes of milk extracts (up to 20 μL) to a final volume of 100 μL of
incubation media. Dose-dependency results showed the absence of cytotoxicity up to 72 h (Figure
2 A,C). When the cytoprotective effect of milk extracts was tested during short-term treatment with
high-glucose (HG) (30 mM), a noticeable increase of cell viability was observed at 48 h (Figure
2B) and 72 h (Figure 2D) of co-incubation with milk extract, with the highest effect observed when
cells were co-treated with 10 μL of buffalo milk extract (WSE+HG) for 48 h (p<0.01). This effect
lasted up to 72 h, even if to a less extent (p<0.05). Based on these results, the incubation time of 48
h and the milk extract volume of 10 μL were chosen for this study.
3.3 Antioxidant anti-inflammatory and anti-diabetic activities of buffalo milk
Water-soluble milk extracts of buffalo milk show a high total antioxidant capacity (TAC),
expressed as mM of Trolox equivalent capacity, (P<0.01 vs. Ctr) (Figure 3A). Similar results were
observed when the ferric reducing power and the DPPH radical scavenging rate were determined
(Figure 3B,C), as well as COX1/2 inhibitory activity (Figure 3D). In particular, the COX1 and
COX2 inhibition was 132 % and 119% of control, respectively. Water-soluble milk extracts of
buffalo milk show also a significant antidiabetic activity, expressed as percentage of α-amylase
inhibition activity (P<0.01 vs. Ctr) (Figure 3E). Similar results were observed when α-glucosidase
inhibition assay was performed (P<0.01 vs. Ctr) (Figure 3F). When milk extracts containing about
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100 µmol/L of δVB were enriched to a final concentration of 0.5 mM, a significant increase of the
antioxidant and anti-inflammatory activities was observed (Figure 3), suggesting the involvement
of this betaine to the antioxidant and anti-inflammatory potential of the buffalo milk extracts.
3.4 In vitro antioxidant and anti-inflammatory effects of buffalo milk δVB
Next, we evaluated the efficacy of buffalo milk-δVB to counteract oxidative stress and cytokine
release induced by hyperglycaemia. Results indicated that co-exposure of HG with buffalo milk
extract (WSE+HG) decreased ROS production (P<0.01) (Figure 4A) and malondialdehyde (MDA)
levels (Figure 4B). The antioxidant activity of buffalo milk was paralleled by a consistent decrease
of cytokine levels (Figure 4C) (P<0.01). These effects were potentiated by enrichment of buffalo
milk extracts with δVB (0.5 mM). Indeed, a further decrease of ROS, MDA and cytokines levels
was observed (Figure 4) (P<0.05). Specifically, δVB addition to WSE (WSE+δVB+HG) reduced
intracellular ROS levels to a higher extent compared to WSE alone (WSE+HG) (144.1± 8.6 in
WSE+δVB+HG vs. 181.3±9.01 in WSE+HG) (P<0.05) (Figure 4A). Consistently, δVB addition
also potentiated the effect of buffalo milk extracts on MDA and cytokine levels (Figure 4B, C)
(P<0.05).
3.5 Modulation of SIRT1/SIRT6-NF-κB axis by δVB
Results indicated that δVB reduced SIRT1 and SIRT6 downregulation during short-term exposure
to HG. Specifically, confocal-laser microscopy and Western blot analysis indicated that SIRT1
downregulation in cells treated with HG in the presence of δVB (δVB+HG) was 27.8±3.5 AFU
compared to 37.86±1.8 AFU in cell treaded with HG alone (P<0.01) (Figure 5). Of note, δVB
positively modulated SIRT6 expression during HG treatment. Indeed, following co-treatment with
δVB+HG an increase of SIRT6 fluorescence intensity was observed (70.1±4.2 vs. 47.04±5.46 AFU
in HG) (Figure 6). At the same time, δVB blocked the HG-increased expression of NF-κB protein
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(Figure 7). These effects are even more pronounced when the δVB (0.5 mM) was added to buffalo
milk extracts supporting a synergistic or additive effect with other milk components.
4. Discussion
The results of this study represent the first evidence on the antioxidant and anti-
inflammatory activities of milk δVB. We provide evidence that δVB and buffalo milk extracts
protect endothelial cells against oxidative stress and cytokine release during short-term treatment
with high-glucose. Recently, δVB, detected in human and mouse heart tissue at micromolar range
(3–22 μM and 38–78 μM, respectively), has been shown to regulate energy metabolism by causing
inhibition of β-oxidation of fatty acids at physiological concentration (100 μM) with an effect
5
similar to that of mildronate. Accordingly, we found that δVB, which showed no cytotoxicity at
the concentration up to 1 mM, dose-dependently reduced the high-glucose cytotoxicity starting
from 0.1 mM and with highest efficiency at 0.5 mM.
Foods rich in natural activators of SIRT1 have the potential to be harnessed as “anti-aging
foods” in a heathier nutritional pattern. SIRT1 activators interfere with histone acetylation, shorter
telomere length and lower global DNA methylation levels associated with risk factors for
cardiovascular disease, including oxidative stress and inflammation.24,25,27 Notably, activation of
SIRT1 by resveratrol has been shown to be beneficial for the regulation of endothelial senescence
and in the protection against oxidized low density lipoproteins-induced oxidative damage.23,28
Among natural dietary compounds, betaines take part to the modulation of cardiovascular
epigenome by modulating methylation or acetylation of target genes.17,29,30 Nowadays, the
beneficial properties of glycine betaine, proline betaine (N,N‐dimethyl‐L‐proline or stachydrine),
and ergothioneine (betaine of 2-mercapto-L-histidine) in lipid and carbohydrate metabolism-related
disorders are well recognized.17,29,30 Instead, data about γ-butyrobetaine (γBB) (γ-aminobutyric acid
betaine), are still somewhat conflicting. Based on its relationship with carnitine metabolism, it has
been hypothesized that γBB relates to carotid atherosclerosis and its complications linked to high
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levels of TMAO. 31-33 On the other hand, it has been shown that decreased L-carnitine levels and
increased γBB levels following administration of mildronate correlates with reduced atherosclerosis
in apoE/LDLR−/− mice. Moreover, increased levels of plasma γBB attenuated the development of
endothelial dysfunction during hypertension and hyperglycaemia. 35 In this context, our in vitro
results indicate the ability of δVB to interfere with processes linked altered glucose homeostasis via
multiple pathways orchestrated by SIRT1 and SIRT6 conferring to this betaine a potential role as
dietary bioactive component. It is worth to highlight that the role of δVB as a substrate for the
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OCTN2, able to block or slow down L-carnitine intake, opens up new questions about its cellular
fate and possible relationships with L-carnitine and γBB metabolism.
It is well recognized that the effects of food bioactive compounds in reducing the risk of
developing chronic diseases depends on their bioavailability and the additive/synergistic
interactions with other food bioactive components. 36,37 In this regard, results from pre-clinical and
clinical studies indicated that natural antioxidants taken alone did not show significant preventive
effects, suggesting that health benefits are achieved by the combination of multiple compounds
through the whole-food consumption.36,38 For this reason, to study the biological properties of δVB,
a particular attention was paid to evaluate the antioxidant and anti-inflammatory activities of the
milk extracts, where this betaine has been recently identified. Of note, ruminant milk, particularly
buffalo milk, contains higher levels of δVB compared to γBB (about 106 µmol/L and 32 µmol/L,
respectively) along with short-chain acylcarnitines, healthy peptides, and other natural bioactive
components. 1,8,10,11,15 However, although in vitro results showed that the effects of milk extracts
were improved by the addition of pure δVB, it cannot be ruled out that their effectiveness can result
from the combined action of δVB with other bioactive components of milk.
The findings of this study uncover the health-promoting potential of δVB and buffalo milk-δVB
against oxidative stress and low-grade inflammation associated with type 2 diabetes mellitus. The in
vitro activities of δVB and buffalo milk extracts, consistent with reports on the cardiometabolic and
vascular benefits of dairy milk in adults with prediabetes,39,40 open up new perspectives for natural
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compounds and dietary-based approaches effective against hyperglycemia-induced endothelial
function impairments.41-43 Despite the comprehensive information concerning the biological
functions of naturally occurring food-derived trimethylated compounds, elucidation of specific
targets and mechanism of action of δVB, most likely common to L-carnitine metabolism, will
require further investigations. It will be equally important to gain understanding of the tissue-
specificity of δVB to unveil possible other biological roles of this betaine, until recently considered
rare but, instead, occurring at consistent levels in ruminant milk.
Abbreviations
COX, cyclooxygenase; DM, dry matter; EC, Endothelial cells; HG, high-glucose; MDA,
malondialdehyde; NF-κB, nuclear factor-kappa B; OCTN2, carnitine/organic cation transporter type
2; ROS, reactive oxygen species; SIRT1, sirtuin 1; SIRT6, sirtuin6; WSE, Water-soluble extracts;
γBB, γ-butyrobetaine; δVB, δ-valerobetaine.
Acknowledgement
Work in our laboratory is supported by the cofinancing PONI&C 2014-2020-Horizon 2020-
RAZIONALE (N° 129) F/050129/01-02-03/X32.
Conflicts of Interest
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The authors declare that there are no conflicts of interest in the present work.
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Figure 1. Dose-response effect of δVB on endothelial cell viability. (A) EC viability determined by
MTT assay on cells treated under normal glucose conditions for 72 h with different concentration
(up to 1mM) of δ-Valerobetaine (δVB); insert chemical structure of δVB. (B) Cell viability was
determined after 72 h of treatment with high-glucose (HG, 30 mM) alone or in presence of δVB,
(HG+δVB) at different concentrations. EC control were grown in media containing normal-glucose
(NG). Results are the mean±SD of six independent experiments with *P<0.001 vs. NG; ** P<0.05
vs. HG; §P<0.01vs HG.
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Figure 2. Effect of buffalo milk extracts containing δVB on endothelial cell viability during high-
glucose treatment. EC viability determined by MTT assay on cells treated for (A) 48 h and (C) 72 h
with different volumes (5, 10, 15, 20 μL) of water-soluble extracts (WSE) of buffalo milk under
normal glucose conditions (NG). Cell viability determined after (B) 48h and (D) 72 h treatment
with high-glucose (HG), HG in presence of WSE (WSE+HG) and HG in presence of WSE enriched
with δVB (0.5 mM) (WSE+δVB+HG). Results are the mean ± SD of six independent experiments
with *P<0.001 vs. NG, §P<0.05 vs. HG, **P<0.01 vs. HG.
Figure 3. In vitro antioxidant anti-inflammatory and anti-diabetic capacity of buffalo milk. (A)
Antioxidant capacity was determined by total antioxidant activity, (B) ferric reducing power, and
(C) DPPH free radical scavenging activity assays. (D) COX1/COX2 inhibition assay was used to
determine the in vitro anti-inflammatory activity of buffalo. Anti-diabetic capacity was assessed by
(E) α-amylase inhibition assay and (F) by evaluating the inhibition of α-glucosidase activity.
Results are the mean±SD of five independent experiments with *P<0.01 vs. Ctr; §P<0.001 vs. Ctr,
†P<0.05 vs. WSE.
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Figure 4. Cellular antioxidant and anti-inflammatory activities of buffalo milk enriched with δVB.
(A) Intracellular ROS production, (B) malondialdehyde (MDA), and (C) cytokine levels measured
after 48 h treatment of endothelial cells with normal glucose (NG), high-glucose (HG), high-
glucose in presence of buffalo (WSE+HG) milk extracts, δVB (0.5 mM) and WSE enriched with
δVB (0.5mM) (WSE+δVB+HG). Data are the mean±SD of five independent experiments.
*P<0.001 vs. NG, **P<0.05 vs. HG, §P<0.05vs. WSE+HG.
Figure 5. Buffalo milk effect on endothelial SIRT1 protein expression. (A,B) Confocal laser
scanning microscopy with representative confocal images of SIRT1 (red) and vimentin (green).
Secondary antibodies were Alexa Fluor 488 or Alexa Fluor 633. The fluorescence intensity,
expressed as arbitrary fluorescent units (AFU), was calculated with ImageJ software analysis. The
mean of the unspecific fluorescence of the secondary antibodies was calculated with ImageJ
software and subtracted. (C) Western blot analysis of SIRT1 expression levels. Inset, representative
image of Western blot analysis. Lane 1=NG; lane 2=HG, lane 3=WSE, lane 4=δVB, lane
5=δVB+HG; lane 6= WSE+δVB+HG. Values are the mean±SD of four independent experiments.
*P<0.001 vs. NG, **P<0.05 vs. HG, §P<0.01vs. HG.
Figure 6. Buffalo milk effect on endothelial SIRT6 protein expression. (A) Representative confocal
images of SIRT6 (red) against vimentin (green). (B) Confocal laser scanning microscopy
fluorescence intensity, expressed as arbitrary fluorescent units (AFU), of secondary antibodies. (C)
Western blot analysis of SIRT6 expression levels. Inset, representative image of Western blot
analysis. Lane 1=NG; lane 2=HG, lane 3=WSE, lane 4=δVB, lane 5=δVB+HG; lane 6= WSE+
δVB+HG. Values are the mean±SD of four independent experiments, with *P<0.001 vs. NG,
**P<0.05 vs. HG, §P<0.01vs. HG.
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Figure 7. Buffalo milk effect on endothelial NF-κB protein expression. (A) Representative confocal
images of NF-κB (red) and vimentin (green) and (B) fluorescence intensity determination by
ImageJ software analysis. (C) Western blot analysis of NF-κB expression levels. Inset,
representative image of Western blot analysis. Lane 1=NG; lane 2=HG, lane 3=WSE, lane 4=δVB,
lane 5=δVB +HG; lane 6=WSE+δVB+HG. Values are the mean±SD of four independent
experiments. *P<0.001 vs. NG, **P<0.05 vs. HG, §P<0.01vs. HG.
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