This study aimed to determine the effects of sea buckthorn pulp oil treatment (800 mg/day for 60 days) on inflammatory, systemic oxidative/reductive status and endothelial function, in obese children. A total of 41 obese children (10–18 years old) and thirty controls were enrolled. The measurements were done before and after pulp oil administration. Ultrasounds were used for flow-mediated dilatation (FMD) and carotid artery intima-media thickness (IMT) measurements and colorimetric and ELISA methods for biochemical parameters. In the obese children versus the lean ones, increased oxidative stress (high malonyldialdehyde, high respiratory burst (RB)), low antioxidant defence (low blood glutathione, low TEAC), inflammatory status (high CRP, ceruloplasmin), adipocytokines disturbance (low adiponectin and high leptin), insulin resistance (high fasting C peptide), low FMD (
According to research, the number of children who are clinically obese has raised threefold in the past 30 years [
Obesity is associated with chronic low-grade inflammation and insulin resistance [
While leptin is considered a biomarker of vascular dysfunction, adiponectin improves endothelial cells function. Adiponectin may protect the endothelium, via its insulin-sensitizing, antiatherogenic, anti-inflammatory, and antioxidant properties [
Many sources of oxidative stress exist in obesity: hyperglycemia, hyperleptinemia, increased tissue lipid levels, inadequate antioxidant defenses, increased rates of free radical formation, enzymatic sources within the endothelium, and chronic inflammation [
The statements mentioned above are important to describe the complex metabolic misbalances present, both in childhood obesity and adulthood obesity, but in obese children there are still controversies about pathogenic mechanisms [
Endothelial dysfunction has been found to be present in the early stages of atherosclerosis. An indicator of subclinical atherosclerotic disease is endothelial vasodilator dysfunction, assessed by brachial artery flow-mediated dilation (FMD). Impaired brachial FMD is related to the prevalence and extent of coronary atherosclerosis and predicts cardiovascular events [
The common carotid artery intima-media thickness (C-IMT) measured by ultrasound imaging is also a marker of preclinical atherosclerosis. C-IMT values predict the likelihood of cardiovascular events in adults [
Because severe childhood obesity is associated with oxidative stress, providing antioxidant food supplements in addition to a hypocaloric diet is important for the treatment of obese children [
The pulp oil composition depends on the processing and origin of the berries [
The sea buckthorn pulp oil is rich in tocopherols and tocotrienols (up to 180 mg/100 g), vitamin K, carotenoids (up to 350 mg/100 g), fatty acids (67% unsaturated), and phytosterols (up to 2.9 g/100 g). The best natural source for palmitoleic acid, an essential fatty acid, is sea buckthorn pulp oil. Also, this oil provides a 1 : 1 ratio of omega-3/omega-6 and so has a lot of medical uses [
We designed this study to determine the effect of sea buckthorn (
A total of 41 overweight children (22 boys and 19 girls) with a mean age of
Anthropometric measurements, body mass index (BMI), waist circumference (WC), and hip circumference (HC), were assessed. BMI was calculated as body weight (kg) divided by square height (m2). Overweight is defined as a BMI at or above the 85th percentile and lower than the 95th percentile. Obesity is defined as a BMI at or above the 95th percentile for children of the same age and gender [
Blood samples were taken after an overnight fast. Standard enzymatic methods were used for measuring levels of serum total cholesterol, triglycerides, and high-density lipoprotein cholesterol (HDL-C). Total analytical variability, expressed as coefficient of variation (CV) was 2%, 1.9%, and 0.9%, respectively. Low-density lipoprotein-cholesterol (LDL-C) concentration was calculated by the Friedewald formula [
Oxidative stress status was evaluated by measuring monocyte respiratory burst (RB or NADPH oxidase activity), total plasma antioxidant capacity (TEAC), plasma malondialdehyde (MDA), erythrocyte glutathione peroxidase activity (GPx), and total blood glutathione (GSH).
Plasma total malonyldialdehyde (CV 13.8%) was analyzed by pretreating plasma samples with thiobarbituric acid in orthophosphoric acid containing butylhydroxytoluene as antioxidant. The pink-colored product was measured spectrophotometrically at 532 nm [
Reduced glutathione (GSH) in whole blood was measured by a colorimetric method using Ellman’s reagent, CV 9.8% [
Subjects were studied under identical conditions by the ultrasound systems equipped with vascular software for two-dimensional (2D) imaging, color and spectral Doppler Mindray DC 3, an internal electrocardiogram (ECG) monitor, and a high-frequency vascular transducer. A linear array transducer with a minimum frequency of 10 MHz, attached to a high-quality mainframe ultrasound system, is used to acquire images with sufficient resolution for subsequent analysis. On the study day, the subjects were asked to refrain from caffeine, anti-inflammatory drugs, and herbal supplements for ≥12 h before testing. All subjects gave their written, informed consent to participate. The brachial artery was identified at 5 cm proximal to the transient bifurcation. The brachial artery diameter was measured on longitudinal images, between lumen-intima interfaces of the near (anterior) wall and far (posterior) wall, at the same time in the cardiac cycle, during end diastole, which was identified at the onset of the R wave.
To obtain maximum diameter, we stimulated blood flow in the brachial artery with the use of a sphygmomanometer, which was placed antecubital, at the forearm. Its arm cuff was inflated to more 50 mmHg above systolic blood pressure, for 5 minutes. After the cuff was deflated, the maximal increase in diameter occurred approximately 45–60 seconds after release of the occlusive cuff.
The children were allowed at least 10 minutes of rest after reactive hyperemia. We measured again the baseline diameter. Then we administrated one tablet, sublingual, of nitroglycerin of 0.4 mg. Peak vasodilation occurred 3 to 4 minutes after nitroglycerin administration. Images were continuously recorded during this time. The maximum diameter was the maximum mean diameter observed at 3 to 4 minutes after nitroglycerin administration [
Carotid artery IMT measurements were done according to a predetermined, standardized scanning protocol [
Results were expressed as mean ± standard deviation (SD)/standard error of the mean (SEM) for quantitative variables. Data were analyzed using the statistical package SPSS version 16.0. Differences between groups were analyzed using Student’s
Body mass index (BMI), waist circumference (WC), hip circumference (HC), systolic blood pressure (SBP), and diastolic blood pressure (DBP) were higher in the obese children versus the lean ones (Table
Clinical parameters.
Parameters | Control group | Obese before treatment | Obese after treatment |
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---|---|---|---|---|---|
Age (years) |
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Ns | Ns |
Sex (F/M) |
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|
Ns | Ns |
Weight (kg) |
|
|
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<0.001 | Ns |
Height (m) |
|
|
|
Ns | Ns |
BMI (kg/m2) |
|
|
|
<0.001 | Ns |
WC (cm) |
|
|
|
<0.001 |
|
HC (cm) |
|
|
|
<0.001 | Ns |
SBP mmHg |
|
|
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<0.001 | <0.01 |
DBP mmHg |
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<0.001 | <0.01 |
BMI: body mass index; WC: waist circumference; HC: hip circumference; SBP: systolic blood pressure; DBP: diastolic blood pressure;
There were statistically significant differences between control and obese groups regarding plasma levels of triglycerides, total cholesterol, uric acid, albumin/globulin ratio, albumin, adiponectin, leptin, and C-peptide (Table
Plasma variables.
Parameters | Control group | Obese before treatment | Obese after treatment |
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---|---|---|---|---|---|
Cholesterol mg/dL |
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<0.02 | <0.03 |
Triglycerides mg/dL |
|
|
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<0.001 | <0.01 |
HDL-C mg/dL |
|
|
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<0.05 | Ns |
LDL-C mg/dL |
|
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|
Ns | Ns |
apoB/apoAI |
|
|
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<0.01 | Ns |
Uric Acid mg/dL |
|
|
|
<0.002 | Ns |
GGT (UI/L) |
|
|
|
Ns | Ns |
Glycemia mg/dL |
|
|
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Ns | Ns |
ALT (UI/L) |
|
|
|
<0.05 | Ns |
Creatinine mg/dL |
|
|
|
Ns | Ns |
Bilirubin mg/dL |
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|
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Ns | Ns |
Leptin ng/mL |
|
|
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<0.001 | <0.01 |
Adiponectin ng/mL |
|
|
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<0.03 | Ns |
C peptide ng/mL |
|
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<0.03 | <0.049 |
Fibrinogen g/L |
|
|
|
Ns | Ns |
CRP mg/dL |
|
|
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<0.04 | Ns |
Ceruloplasmin mg/dL |
|
|
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<0.02 | <0.01 |
Albumin g/dL |
|
|
|
<0.002 | <0.008 |
HDL-C: high-density lipoprotein-cholesterol; LDL-C: low-density lipoprotein-cholesterol; TG: triglycerides; CRP: C reactive protein; ALT: alanine aminotransferase;
The markers of subclinical atherosclerosis were modified in the obese children versus control (IMT was higher, and FMD was lower). After treatment, IMT was decreased (Table
Markers of preatherosclerosis.
Parameters | Control group | Obese before treatment | Obese after treatment |
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---|---|---|---|---|---|
IMT (mm) |
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<0.01 |
|
FMD % | 18.64 ± 0.81 |
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<0.0001 | Ns |
IMT: carotid artery intima-media thickness; FMD: flow-mediated dilation;
Correlations (
Correlations for IMT and FMT.
Correlations | Weight |
BMI |
Waist circumference |
Fasting C peptide |
CRP |
Leptin |
Adiponectin |
apoB/apoA | DBP |
---|---|---|---|---|---|---|---|---|---|
IMT (mm) |
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|
FMD % |
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— | — |
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|
Relations (
Correlations for adiponectin and leptin.
Correlations | Weight |
BMI |
Waist circumference |
Fasting C peptide |
HDL-C |
DBP |
apoB/apoAI | apoA |
---|---|---|---|---|---|---|---|---|
Leptin (ng/mL) |
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— | — | 0.40 | — | — |
Adiponectin (ng/mL) |
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|
In the obese children, the antioxidants: total blood glutathione, and total plasma antioxidant activity (TEAC), were decreased, while oxidative stress markers: MDA (malondialdehyde) and monocyte NADPH oxidase (RB), were increased (Table
Redox plasmatic profiles in the studied groups.
Parameters | Control group | Obese children | Obese children after treatment |
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---|---|---|---|---|---|
TEAC (mM Trolox) |
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<0.02 | Ns |
NADPH oxidase (RLU) |
|
|
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<0.01 | <0.03 |
Total blood glutathione |
|
|
|
<0.05 | Ns |
GPx (UI/L) |
|
|
|
<0.03 | Ns |
MDA (mmol/liter) |
|
|
|
<0.05 | Ns |
Puberty influences the plasma values of the parameters measured in this study. A possible limitation of our study is that most of the subjects are at puberty, and gender is important.
Obesity in children has been independently correlated to endothelial dysfunction, inflammation, and oxidative stress markers, major risk factors for atherosclerosis [
Sea buckthorn flavonoids are the berry part most studied for the beneficial effects in preventing cardiovascular diseases. The clinical and animal studies on cardiovascular effects of sea buckthorn seed or pulp oil are few [
In hypercholesterolemic rabbits, CO2 extracted sea buckthorn seed oil had significant antiatherogenic and cardioprotective activity, improving the plasma lipid profile [
In our study, treatment with sea buckthorn pulp oil reduced the total plasma cholesterol, the apoB/apoAI ratio, and plasma triglycerides. An improvement should be noted of HDL-C and LDL-C levels, but the effect was weak. The composition of sea buckthorn pulp oil, rich in monounsaturated fatty acids (MUFA), can justify this result. Kris-Etherton et al. demonstrated that a high-MUFA, cholesterol-lowering diet may be preferable to a low-fat diet because of more favorable effects on the cardiovascular disease risk profile [
Fasting C peptide is known as a surrogate marker of insulin resistance and reflects the endocrine secretory reserve of the pancreas. In this study, sea buckthorn pulp oil treatment, by improving the plasma fasting C peptide level, may have beneficial effects, reducing insulin resistance. This effect may be due both to palmitoleic acid and to antioxidant vitamins in which sea buckthorn pulp oil is rich. There are some arguments. It was demonstrated that palmitoleic acid reduces muscle insulin resistance and prevents beta-cell apoptosis [
Fasting insulin levels are generally increased in obese adolescents. It is known that insulin stimulates vascular smooth muscle growth [
In our study, the obese children had much lower values for FMD (flow-mediated dilation), a marker for early atherosclerosis. FMD was correlated negatively with anthropometric parameters, with apoB/apoAI ratio, with fasting C peptide, and with diastolic blood pressure and positively with adiponectin. Although sea buckthorn treatment reduced blood pressure and fasting C peptide level, the FMD values were not improved significantly after treatment.
The other marker of early atherosclerosis, the IMT, was higher in the obese children
In our study, the plasma inflammatory markers (CRP, leptin, and ceruloplasmin) were significantly increased in the obese children, while fibrinogen was not modified compared to the levels in the lean children. Treatment had a weak anti-inflammatory effect by reducing leptin and ceruloplasmin. In the literature, a few studies on obese children have shown increased plasma levels of inflammatory markers, such as leptin, IL-6, CRP, and fibrinogen [
As we mentioned, leptin was high and adiponectin was low in the obese children
Even though there are only few studies on systemic oxidative stress in childhood obesity, most of them demonstrated increased lipid peroxidation, estimated by MDA [
The low total blood glutathione concentration and high GPx activity in the obese children
Albumin represents a very abundant and important circulating antioxidant known to protect endothelial cell from oxidative injury [
It is thought that the evaluation of oxidative status may allow for the identification of patients at an increased risk of complications. Decreasing the levels of chronic inflammation and oxidative stress in childhood may decrease cardiovascular morbidity and mortality in adulthood [
Providing foods with high antioxidant/anti-inflammatory capacity, in addition to a hypocaloric diet, is crucial for the treatment of obese children. Because of its unique composition, [
In this study, treatment with sea buckthorn pulp oil (800 mg/day, 60 days) did not modify significantly the markers of antioxidant defence (TEAC, GPx, and total blood GSH) or the level of lipid peroxidation (MDA) in the obese children but reduced respiratory burst. By lowering NADPH oxidase activity in monocytes, sea buckthorn pulp oil treatment reduces the formation of the free oxygen radicals (the anion superoxide) and so may improve atherosclerosis by reducing macrophage-mediated inflammation.
In conclusion, treatment with sea buckthorn pulp oil (800 mg/day, 60 days) influences the blood levels of different variables, known as markers of different pathogenic pathways of atherosclerosis. The treatment lowered inflammation, blood pressure levels, improved plasma lipid profile, and reduced respiratory burst. The strong argument that sea buckthorn pulp oil prevents atherosclerosis is by lowering IMT values.
None of the authors of the paper have any direct financial relation with the commercial identities mentioned in the paper.
Bogdana Vîrgolici and Daniela Lixandru had equal contribution to this paper, and they acknowledge the Postdoctoral Program POSDRU/89/1.5/S/60746, from European Social Fund.